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Thesis presented for the. Dereo of Doctor of Philosophy of the
NOJLDG'EI rS
• I am moat grateful to the'Chemiatry Department and University
of Edinburgi for provision of the faoilitjOs tihich enabled this
research to be caied out. .lidtrice and asistance received from
Professor-Itoll Campbell., O..L, has been much appreciated. and I
must thank my supervisor, 'Dr. R o A. Wall, for his patience and •
guidance throughout. I should also like to thank my colleagues,
• past and present, fOr. making the past three years such a worthwhile
and en3o3rabie experience. In. particular, I should like to record
the ansistahoo received from 1,T. SalEon,.B.Sc., in• amino acid.
• analsio work 'and P. !dinton, .Sc., for writing tha computer
program used. Finally,. I should like to. thank the Science
Ilesearch Council for the provision of a Research Studentship award
which iado this $ork,possible, • • •
CONTENTS
GERAL INPRODtJCTIO 1
SCION 42
(i) General 'echnique 42
Culture of Porphyridium. Cruontum
Barvesting of the Algae; E*traction and Purification 58
(,.) Idntificatjon o.f the 17-terina1 Amino Acid of . B-phycoèr7thrin . 63
DissOciation of Bphyooethrin in .
.Lqueous SOlutoü . . 68
Dissociation Studies using Iterourial Compounds . . ., 73
() Paraiierourich1orobonzoate 73 • . . (b) rercuioh1or6honr1su1phonic
Acid 76
(C) tercuric Ion 79
Dissociation Studies using Guanidine Buffer Solution 81
Gusnidine Solution c1ono 81 • • . Guanidirie So1utibn With • .
f3-zercaptoethanol 85 Guanidino Solution with
• . ., Dithióthieito1 • . 93 (a) Frontal ond Zonal Analysos 105
(9) R-phycoorvthrin 312
(io) Bio-Gla3a 115
DISCUSSION 117
.BIDLIOGR1 •
• . • . . . 128 •.
S
!TUDIES OL! THE ALGAL flILIPR0TflI1, 13-PHYQERYTHPIrT
1,BSPRk('T
The Rod alga Porphyrl.dium cruentum uas grown in an artificial
sea-water medi.in and the biliprotein B-phycoerythrin extracted from -,
the'alga after breaking down the, cellular structure of the latter using I ' -
high froquenóy sound. The biliprotèin was separated froD cell residues
by oentrifugation and filtration; thea purifiea by absorption
chromatography on columns of tricalciuri phosphate gel, phycooyaninbeing
the main impurity romoved by this treatment. The prqtein was then ,
precipitated by addition of solid amonium Bulphtead stored in the
precipitated state at 000. After this purification visible and
ultraviolet absorption characteristico 'of biliprotein solution were
-consistent With those preioualy reported for:B-phycoerythrin.
?urther purification could ho achieved by fractional Irecipitation or
molecular siovo chromatography.
The first experizontal study carried : the phyooerythrin
was jdontiication of th.Nterminal amino acid by preparation of.a
dinitrOphenyl dorivativO. ' Using paper chromatography and standard
derivatives for comparison this amino acid was shown to be inethionino.
The nain work of the project was devoted: to studying the
dissociation of B-phycoerythrin, the principal techniques used being
uoléoulàr sieve chromatography (preparative-and analytioal), absorption
epeotrophotomotry and amino- acid analysisi Two aspects of dissociation
were studied - dissociation in aqueous', solution (i.e., natural
dissociation) ad dissociation brought about by che-ical iieanz.
Dissociation jn aqueous solutIon was -first observed whon a sample
of L)-jhycoerythrin zaa being furthar purified by preparative molecular
sieve chromatography - throo bands of phycoeythrin separated of which
two uoro isolated. Phoos were found to difoi in visible absorption
characteristics and the systxa was then studied fuz'hor by analytical
rolecii1ar sieve chromatography. ContthuozsspcctrophotOviotric
monitoring of column effluent showed that there was separation into
threo.subunits and by calibraticn of the gel colutm with proteins of
known molecular treight it was possible to estimate the molecular weights
of these. i certain decree of roassOoiation of the smallesi subunit
was also demonstrated. - On the basis of those results together with
some earlier work in this laboratory the presence of a dissociating-
asociatizig equilibrium syB ten in aqueOus solutions of the -phycoerythrin
was postulated and the tzorL Was published in the forinof a short
comnunicatjon, (riorao azidUaU, 1967), a copy of which is appended.
This equilibrium system wee further studied by the molecular sieve
chromatography technique of frontal. amalysis Using practical
toohniquoo , devoloped by '7ir or and Sheraga (1S63) and theory developed
by Cilbort ( 1955 ) the presence Of a disocitin-assooiatin
equilibrium systom iivolving at least one polymeric species (i.e. greater
than dimer) was established, confirming the previous postulate.
For dissociation by ohomical teacs various bond-breaking reagents
and media wero tried, both covalent and zion-covalent bonds requirina to
be broken. The aim. was to dissociate the B-phycoerythrin as much as
posoible into ito emallest oubunit and to estimate the molecular weight
of-the sauo, i,o., the minIaa1 m1ecular weight of the biliprotein.
orcurial compounds and niercurie ion wore tried with some success although
they did rot lead to compléto breakdown. t!et a strongly ionic i.*iedium
(6r guanidine hydrochlOrdo) was tried but, it was found nocessry to
add a disuiphide bond-breaking reagent to. complete breskdot:rn. To start
with the thiol .ercaptoethano1 was used in conjunction with the
strongly Ionic medium for this purpose; but iot successful was
Cle].an&'s reagent, d±thiothrol.tol, a compound having sovoral
athantaoo over other thiols vs ed for this purpose. Use of this
compound in conjUnction with the 611 guanidine solution led to
what appeared to be complete breakdom of the protein and the
miniinil molecular weight of the subunit produced was estimated
as 36 000, A escond, saller fraotioi was also detected but after
isolation and analysis foz'amino acid content the figures were
compared to those for the other fraction and native B-ph3rcoerythrin
• and it proved to be some impurity or artifact formed dvtring reaction.
The above Dinimal molecular weight was not in complete agreement wIth
that fovndfrom the work on.dissciation in aqueouo solutionbut this
can probably be explained by the kinotics of the euilibriuia process
loading, to false etiiates of noleau.lar weightQ Further frontal
aualyoia work should resolve this problem and the same approach might
• load to defin.te identification of the species involved in the
equilibrium.
Finally, a little work. was done on the closely related biliprotoin,,
• R-phycoerythrin. Chenicel breakdown wac tried and siuilar spectral
oho.nes to those observed for ,fl-phycoerythin indicated that similar
breakdown had probably taken place, although no separation or molecular
weight estimation was attempted. The fzontal analysi tethnique was
also applied and the results indicated the presenOe of an equilibrium
systen again involving, at least one polyeric apeàies as for
•
B-phyooerythrin. R-phycoerythiin would therefore appear to behave in
a very similar Panner to Ba.phycoorythrin with respect to dissociation,
EWERAL INTRODUCTION
Description and Occurrence of the Biliprotei ng
The biliproteinc are photosynthetically active red and blue
proteins called phycoerythrinc and phycocyanins respectively'. They
occur naturally only in groups of algao be1oning to the Thallophyta,
being of general occurrence in plants of the divisions Rhodophyta (Red
algae), Cyonophyta (Blue-green algae), Cryptophyta (Cryptomonad algae)
and in one -or two mmbers of the Chiorophyta. Table 1 gives a list of
come of the algae whOo biliprotainc have boOn otudicd.
The biliprotoins are related to Phytoohromo, a chromoprotoin of
higher plants, in particular by their siziIar. prosthetic gmupaj as
Confirmed by Siog'olrne. (1965) , ThesO prosthetic groups, or
chromophoros, are totrapyrroles Imown as phycobilins which differ from
the chiorophyllo tn not Ong readily released from the associated
protoin, thus the complete biliprotoina have been studied more
intensively than the separate phycobilins. The biliprotoinc are
located in the lamellas of oh.orop1ats of Red elgao (Brody and
Vatter, 1959); electron miôrograph studios of Blue-green algae
indicate that they also contain ch].oropiaat-like structures (EThers
ot ci. 1957), with the bilproteine located in ti0ir lomcllao rather
than in free cbrofratophoroe or in the cytoplasm (Thomas Ond do Rover,
1955). The chromophorosarO fully discussed later in this
introduction (See pages 31-35).
NopncJaturo
Haxoot ci (1955) originally proposed "bilicbromoprotoin" as a
general none for these protOins but this was modified to "biliprotein"
by O'hoch (1950) and this is now the most widely used term. The
prefix "bill" indicates the relationship between tho prosthetic group
TABLE 1. DISTRIBUTION OF ME ALGAL BILIPROTEINS
ALGAL GROUP AITh SPECIES BILIPROTEIIIS REFERENCES
RHODOPHYTA
CLASS:BANGIOPHYCEAE
ORDER: PORPHYRIDIALES
Porphyridium cruentuni B-PS; R-PC: Allo-PC. Haxo et al (1955) O'hEocha (1955)
ORDER: BANGIALES
Porphyra tenera R-PE;' C-PC; Allo-PC Hattori & Fujita (1959) Porphyra perforata R-PE; R-PC; Ailo-PC. Jones & Blinks (1957) Smithoranaiadum B-PS; C-PC; Allo-PC. Airth and Blinks (1956)
CLASS: FLORIDEOPHYCEAE .
ORDER: NEMALIONALES
Rhodochorton rothii R-PE; PC. O'hEocha (igss) Rhodochorton floriduluni' B-PE; PC. O'hEocha & O'Carra (1961)
ORDER: GIGARTINALES'
Plocamium pacificum R-PE. O'hEocha (1958)
ORDER: CRYPTONENIALES
Gratel'oupia sp. R-PE; C-PC; Allo-PC. Hattori & Fuji'ta (1959)
ORDER: RHODOMENIALES
Rhodomenia palmata R-PE; R-PC; Allo-PC. C'hEocha (1960)
ORDER: CERAMIALES . .
Ceramium rubrum R-PE; R-PC;Allo-PC. Svedberg & Katsürai (1929 Pôiysiphonia uceolata R-PE: C-PC; Allo-PC. Hattori & Fujita (1959)
CYANOPHYTA
Toly-pothrix tenuis C-PS; C-PC; Allo-PC. Hattori &'Fujita (1959) Arthrospira maxima C-PC Allo-PC. O'hEocha (1958) Phormidium ectocarpi C-PE. . O'hEocha (1955, 1960) Anabaena cylindrica C-PC; Allo-PO.. Haildal (1958)
CRYPTO?HYTA
Hemiselmis virescens PC. Allen et al (1959) Cryptomonas ovata PS. Haxo & Fork (1959) Senniasp. PS; PC . O'Eoc}a.&,Raftery (1959) Cyanidium caldariuni C-PC Lllen (1959)
Phycoerythrin : PC Phycocyanin)
(chroniophoro) and the "bile pigment&' (Leinberg and Legge, 1949).
Other terms that have been used include "phycochromoproteids"
(Iy1in, 1937), "tetrapyrryl proteins" (r!aurowit.z, 19)0 and
"phycobiliprotein&' (Bogorad, 1965). The naming of the individual.
biliproteins themselves depends on their colour - the red
• biliprotoins are called phyooerythrino and the blue-green phycocyanins.
Further subdivision is p9asible on the basis Of visible absorption
• spectra • spectra of the phy000rythrins may have one, two or three
maxima in the visible regiàn and thoso are distinguished by the
prefixec C-, B.' and It- r.eepectively, (the letters were originally '
selected to distinguish phycoerythrina in the classea Cyanophyta,
angialoa and.other Rhodophyta but now refer sOlely to' spectral
differences). The colour and spectral properties of the proteins
are fully disOusSed later in the introduction (pages
The, phycocyanins 'are distinguished in a similar manner with C- having
one inaxium'in the visible region and,R- having two., In addition to
these there is allo-phycocyanin, formerly knpwn as p-phycooyanln,
hich' is widely iiotributed in .thö algae., Its spectrum has one
maximum' and a shoulder in the visible region.
,roith of Alfrao (Naturai and Artificial): Factors Affecting Growth
Various factors affect the bi]iprotein content in algae, e.g.,
in Iarine Littoral Red algae the biliprotein content varies with
season; ' in Cerámium rubrurn it a000unte4. for 1.9 of the dry weight
in December and January but only for about half of this in T4aroh
(Lemborg, 1928) • For phycocyanin in the alga the reverse ,.was the
case. Also phycocyanin content, is less in deep growing'algae than in
species growing in intertidal or upper sublittoral levels (Icylin, 1937),
(Jones and Blinks, 1957).,' • • •
3
Several factors can affect the artificial culture of algae
iücluding:-
the intensity and nature of the light usod for illumination
the temperature
the composition of the culture medium.
There are many examples of these effects: A natural biliprotein content
in, Mie.:Red algae of 2 is about the highest recorded but a 24 yield
(dry weight) of phyqooyanin from Anacystis nidulans grown artificially
at 3900 and under low inten3ity white light was recorde4 (flyers and
Kratø, 19501. Light intenèity can'a1so affect the relative
proportions of different :biiproteins in the same alga,. e.g., in
Anabaenasp. which forms phyoodyanin only under high intensity light
but somephycoerythrinas well, at lower intensities. In contrast,
however, AnaCystis nidulane never forms any pbycoerythrin whatever
light Condition is used. The type or quality of light used can also
have an effect, e,g., the ratio of phycoorythrmn to phycocyanin in
1olypothriz tenuis depends on whether the light source is fluorescent
or incande8cent (Flatten and Fujita, 1959a)4. Similarly in a
chlorophyll-less mutant of Cyanidinium caldarium phycooyanin forms
under light of 450nin or 600nm but not under light of 550nm, 650nrn r
7QOnm (Nichols and Bogorad, 1960)6. The growth of phycoerythnin in
Pohidim centum (the alga used in this research) is stimulated
more by green light (546nm) than by blue (436nm) at low intensities
but at. highintensities the oppositeis true (Brody and Emerson, 1959).
These workers, also showed that coinp1eientary chromatic adaptation
(einnced foni,ation of the pigmit that nost. strongly absorbs the
indicent light) is effective only at low inteity.
4.
Thore are fôt';er eiainp1ee of tho effoct of temperature on
• culture although Gamier (1959) studied this on Oseillatoria
subbrevis. 'Generally best growth is achieved at or a].ittle above
room temperature (e,ga, culture of Porphyridium cruentum in this
• laboratory was carried out at 220.4
Ceran chemicals are essentIal to growth, nitrogen bing the
ôat iiportantof those. Foationof algae is,limitod in nitrogen
deficient media (Pog, 1952; HattOri and Fujita, 1959o), A good
example of the importanóe of nitrogen Occurs with Tolypothrix tenuis
fot''whiOh,pred1luminated nitrogen-deficient cultures formed
biIprotOin in the dark when nitrate was added tO the medium. In
•hio case the ratio of the pigments was affected by the charactor
of the light used during the pre-illumination period, green light
avouring synthesis of phycoerythrin and red light favouring phycocyanin.
In culture8 grown heterophioafly in dar1oss phycocyanin was still
-f6mod but not phycoerythrin (Hattori and F.ijita, 1959; 1960).
Phosphorus is also a major nutritional element required for normal
growth of algae but variation in quantity does not seem to affect the
relative amounts of the biliproteina formed. Liowever, defioiencès of
sodium or molybdonuin in modia supporting Blue-green algae both seem
to have an effect, , the amount of phyoOcyànin produced decreaming as
the amount of either element decreases (Fogg, 1952; Allen and Amnon,
• . 1955). Similarly iron deficiency will reduce the concentration of
both phycoerythriñ and phycocyanin (Borosoh, 1921)4 r1aoroquantities
of calcium are required by Blue-green alaae but only mioroquontities
by Red. algae. Various other elements are required in trace amounts
including manganese, vanadium cobalt, zinc, copper and' boron but all
• in such small amounts as not to affect the yields Of biliproteins if
• . . 5.
any are prosent in exceas or, deficiency cotpared to their optiruiii
value, o potassium appears to be reçuired in any medium.
Generally, preparation of a culturo medium with the optimum amount
of each of the wny trace elomens ia more important to the rate of
grouth than to the yide or the gelative poztiona of bil±proteina
foiec1, e.., in !nabena oylindrica opticvm contontrations of the
various mèronutriento can leaI to a tio4iuridred'fold incroaso In
the growth rote.
Othor compounds van also have sorn effect, e.g., in the grouth of
PorphyriUum cruentum (see öxporimentai. section (2) ) itwas found
• that the •prcience of.a. vitamin in trace amounts improved grotzth.
Also agitation ok the medium by paaoing through air enriched with 5
carbon dioxide instead of air clone considerably apeedôd the rate Of
growth. ' . • .
Fr'oa all the foregoing it is thCrefóre clear that growth, . in
terEs of the relatIve amounts of the bilipoteIns formed,' theiryloldo
and the rate of growth, varies considerably according to the nitrients
• present in the mediue as well as depending on' the type and 'intensity
• of the light used for illut3ination and the tempraturo of c1ture,.
Careful variation of all these factoro La essential to ochiovo
desired rocults. , .. . • .
Itractipn and Purification of the 3iliproteine
•
To isolate the biliprotoias'it is nocescary to break down the
• cellular structure of the alga. . '1any methodo havo been used to
• achieve this broakdoin including macoration, grinding with an abrasive •
agent such as Oaid or glana wooli ultrasonic osculation cnd repeated
frooing and thawing.. A combination of some. of these methods can also
6
be us'ed, of qourse, as was done in this project (ultrasonic.
disintegrati.on with freezing and thawing, see experimental
section (3) ). The resultant aqueous extract is then centrifuged
to remove the bulk of the dIsintegrated cell matter, the biliproteina
remaining in solution,. These operations are always ca'ried Out in
the cold and dark.
Fractional separation of thó biliproteins and removal of other
watez'-soluble algal constituents was Originally achieved by fractional
precipitation f011owed by crystaUisation frOm a ionium. sulphate
olution but this isa lông and tedious method, particulerly if the
protein required is present in sraU amounts only. It. is most
useful when crystalline proteins are required, i,e 0 , protein of
highest purity. This method has now been replaced in most instances
by the more efficient process of absorption ..chromatograph,
originally developed by Swingle and Tieliva (1951),. The crude
biliprotein solution is pásood through a column of tricaloium
phosphatégel,oeiite acting as a support and by eluting with
iucreaeingly concentrated phosphatu buffer solutions the various -
prot*an be separated., The phycoerythrins are usuaily found to
preeede the phycooyaninn. ?any proteins have been purified in thi
way. and it is now a fairly. standard ,tocuiiue (Icrasnovekil ot al,
1952; Raxo, ot al, 1955; Jones and Blinks, 1957; O'hEooha and
Raxo, 1960).. Direct abaorption on tricalcium phosphate has been
used for large sOale preparations Of biliproteins (Tlselius, 1954)1.
Another method of purification developed more recently is extraction
of the biliprotein into a suitable 'sOlvent, n-butanol be one
example (Fujimori. and Pecci, 1967b). A more detailed description of
methods used is given in experimental sectIon (3).'
7
For still ft*rther purification the technique of gel
filtration (or molecular sieve chromatography) can be used. The
pro teins or protein and unwanted material, are separated on the
basis 'of differing molecular weights, the largest molecules 'passing
through the gel column first and the smallst last (see bxperimeita1
section (iXe)). The method is also of great use in removing any low
molecular weight contaminating entities and for the desalting of
eolutions .(Raftery and O'hEocha, 1965;. iriksSon and Halidal, 1965).
The latter , two workers also tried dietIy1aininoethyl cellulose
(DEAr Celluloso) in the purification of biliproteina from several Red
algae and found the method fast, efficient and reasonably
• straightforward (Eriksson and Ralidal, 1965).
Other methods for purification of the biliprotins used with some
success have included disc eleotrophoresis (Hjerten and 1oebach, 1962);
zone eleotrophoresis (lijerten 1958; 1963) ion exchange resins, e.g.,
'Bothan and Westlund (1956) had some success 'with the strong anion
exchange resin DoWex-2 (Ci); rivanol (2-ethoxy-6., 9-diaminozioridine
'lactate) can precipitate the protein out from mucous substances
(Fujiwara, 1955) but there is sorne difficulty in remo'ing all traces
of the rivanol, '
Ciyeta11isation of the Bili'i,roteina
The biliproteins in their state of highest purity are crystalline.
Crystallinity is beat achieved by fractional precipitation with
'ammonium sulphate as mentioned (See page 6); e.g., Svedborg and Lewis
in 1928, Lemborg (930) published the first photographa of crystals.
• ' •eny ro1]ers have since,suooeeded in crystaiiisihg and photographing
the crystals of various of the biliproteths including Fujiwara (1955);
[1
liattori and Pujita, (1959), using a combination of direct
absorption and fractional precipitation; Salmon (1967), who
oystal1ieod both D-phrcoorythrin and C-phycocyanin, cloar
photographs being obtained.
An interesting feature o± orstallination is that the 'shapo Of
the crystals àbtainod can vary and depends on the pH of the solution
in which they are formed For example, B'a.phycoorythrjn can foru
either needle-lute or prism-shaped crystals 8Ad C-phyoocynin can
form needles or platolots depending on thè pH (Ftajiwara, 1955;
Salmon, 1967). This offset was originally noticed by Bouillone
Ualrand and Delarge in 1937 and is found to be the case for several
of the bilipr'oteino,
yaicl Properties of the Bilirotoina
(a) Colour Puorsoence:
It has already been mentioned that the algae containing the
bjliproteins are either Red or Bluegreon iith the biliproteins
tho,se1ves, contained in the ehloro1asts, contributing partly to this
colour. The algae show very little fluorescence but when the
biliprotejns are releaed from the algae into aqueous solution they
are brilliantly fluorescent (toLndon and 1inio* 1952). Tho
phycoorythrino form orange'.red solutiona yhich ozhibit bright orengo
fluorescence whilst the phycocyanins fore blue-green solutions which
ohibit a duller red fluorescence. Fluore000ne spectra produced
Under ultraviolet irradiation have boon studiOd and it is found that
the fl- and B- phycoerythrina have maiima at 570-580im;
allo.i.phycocysnin has a maximum at 663nm (French and Young, 1956;
Fzonch ot al, 1956) C-phyeooyanin from the Rod alga Porphyra
naiadun (now called Smithora risiadum) has a xaimun at 637mm
(French ot al, 1956) tThilst the semé biliprotoin from Oscil].atoria ap.
has a maximum at 650-680nm. Various maxima between 637nm and 680nzn
have been reported for_the phycocyan1no (Borne, Cre.opi and Katz, 1963)0
PH has some effect on the intensity of the flucrecence observed;
the most intense znazinium for R-hycoerythrin is obServed at pH
7.5 (Krasnovskii et a]., 1952) and that for 0-phycocyanin. at pH 6-605,
(Lavore]. and 1Oniot, 1962). ,•
(b) Visible and U1traiolt./bsorDtion: .
The 'absorption spectra Of the biliproteins are their most
characteristic physical property.. As explained earlier (aoo page 2)
absorption maxima are sodistinotjve that 'they are used as the basis
for distinguishing the various bilipr.oteint3, C-, B.- and H-
phycoerythrins having one, two and three maxima in the visible region
respectively. The aene is the ease, at least in part, for the
phycooyanin., The absorption spectra of the biliproteins can be
observed prior to extraction from the algae (Haxo and Blinks, 1950)
and thøre is found to be vory little difference after extraction
although some maxima are occasiOnally shifted to slightly shorter
wavelengths (Emerson and Lewis, 1942; Ualldal, 1958).
For the. phycoeryt}iriris the wavelengths of the maxima and the
rétativo extinction coefficientS deond on 'the species the
biliprotoin is extracted from and also to a small extent on the method
Of preparation for spectral analysis (which is alwaye cax'ried out in
aqttooua solution); e.g.., some R-pbycoerythrins do not have the
characteristic 540nm peak rhioh Others have ($edberg and Pirikps6n.,
1932; Haxo ot al, 1955; O'h1ooha, 1960) and the 560-565n side .
peak normally present in B-phycoerythrin disappears if the protein
10.
'is twice rocryatallised (Airth and B1incs, 1956). If extraction
is very prolonged there may also be some change, e.g., C-phycoerythrin
from ?horrnidium develops a second. maximum if oxtrotion is
continued over a period of months rather than days (O'hEocha and Haxo.,
1960). This is due to the action of proteolytic enzymes which are
'most active in the extraCts Of cells from four month old oulture
(Othooha and 'Curley, 1961,)6
pical visible absorption maxima for the phycoerythrins are
as follows:a.
• DilLprotein ' iave1enth(s) '
C.phycoerthrin. ' 565nin
• , B-phyqoerythzin 565nni; 545nm; • shoulder 'at 495-500nm
R-pbycoorythrin •.. •. 565nmo, 545nni;. 495zui
In ad4ition. to these there are the phycoerythrins from the'
Cryptomonads but they have slightly differing properties including the
visible spectra which show only one inaximiiii, uàualiy in the 545-568nm
•
region (iiion et al, 1959; Oth1ocha and Raftery, 199; Baxo and
Fork, 1959),
The phrcooyanino vary in the sane way as the phycoerythrins, e.g.
• p11 can affect the intensity and iiavelength of the visible absorption
to some extent, ' In addition there is often some suspio±on that
samples of ,phyoocysnins aro. contaninated with phyooerythrins
(Eattori. and Fujita, 1959;' Blinke, 1954; French ot al, 1956, and
others), it being much more difficult to separte small amounts of
pbycoerythrins from a phycocyanin than the other way round.
Typical maxitia for the phycocyanins are as f011ows, however:-
11
33i1iprgtein Uavelen±ths
CphyoOoyenin 615n
R-phycocyanin 615nm; 553nni
Allo-phycocyani.n 650im; shoulder at 620thn
As with the phrooerythriris the properties of the Cryptoronnd *
phycocyciine are found to differ from, the others (Allen et al, 1959)0
The biliproteins also have oharacteriatio. ultraviolet absorptions
but with less distinctions botween the phyooerythrina and the
pb-eOoyanins than in tho viable region. All have naxixmia in the
regions 365nm eM 278.300nm with the phycoerythrins having an
additional maximum at 305-316nm (flxb et al, 1955; Battori and
FujLta 1959; 0 1 hEooha 1960). '
Typical absorption spectra Of some -biliproteins are shown
diagrarnatically In figure 1..
In the U.V. region most colourless proteins also absorb at
278.-280nin and the ratIo of the absorption at this wavelength to that
of the princIpal visible absorption maximum (visible :' u.v.) gives a
good idcation Of the biliprotein parity, i.e., the lower the U.V.
absorption at this wavelength the lees ivmpurlties present and this
shows up as a higher ratio value, This 4ll be frequently mentioned
In the experimental àoction whore, for B-pycoerythrin, the ratIo of
the 545nm peak to the 280nrn peek was taken for this purpose, a value
of 4 or more being considered indicative of high purity
The visible absorption of the biliproteins is attributed to the
interaction between the prosthetic group (phycobi.u.n) and the
apoprotein and this will be more fully discussed later. (see pages 36-37).
It was originally thought that there was some relation between the
absorption and the taxOnoinic positions of the algal' sourceS of the
6•S 4a
i o 0•1
• r'J\
A
I I •,(I
0*
Wovetg(
!
P5j Abo& Cvts
— — f Ph 0trwi,n (cvo,sis ubrw4).
• • CPord
—. —. -.• C- P% orbhrt4% (9 g tniIn..)
Cvo P .sjtPvY% Rtfcsc.e.$)
e.l
'0:
AF
I -H
J 01
.I 01
0.
I I
1' I...' Ii\
p
• 'If % Ii P
Lj
I., •• .It\
'I
I4UV .3UY
• Wove ((4%3%I. ()
• [((ftL R10 G1' Cvcs •
• -
• •• — — -• -. — - - C - 6W% (Nogoc.. rtuscori") •,
— • —— A((.- Ph a.ntr (ooc ttu,c.ori''3
(esoic( iwA (ic
12
biliproteins but this hae since been dioproved (e.g oj Allen ot alp
1959; O'bEocha and Raftery,. 1959; OYhEóoha and Haxo, .1960).
TheU.V. and visible spectra are very useful in determining the
number and type of the different biliproteins in any alga in addition
to providing the above criterion for purity. For example, Red algae
usually contain phycoerythrin and one or two; phycocyanina which can
be separated from the phycoerythrin byco1umn chromatography or br
fractional precipitation (see pages 5-7). Comparing spectra before
and after such a purification indicates ho'ti siiocessful it has boOn.
Spectral changes are also used. for many othorpurposos, o.g,, they
can act as a guide in telling whether donatur&tion has occurred,
Whether reagents have affected the chromophoro-apoprotein 1inkage
whether bonds have been bràken as desired After a treatmónt. The
use of the absorption spectra in such trays (in conjunction with oher
evidence) will be fraqueit1y observed and rtentioned in the
experimental Section. . .
(a) Poleoular Ueirhts of the jliproteins:
The rto1ecular weights of the bi1ipro1eino, a. for most
macromoleculeS, are subject to quite a large degree of error due to
the limitations of the methodd used to determine them and are
therefore only approximate. The situation is further confused. as
their moiooular weights are pH dependent, i.e., the moIeu1es can
split into subunits at certain pH values, This can also be brought
about in Other tays and is fully discussed in the next subsection..
This subsection will therefore deal àn.y uith the.moleoular
weights of the biliproteins in their native, und.iesociatod states in
vhich they, are likeliest to existat or near to their isoelectric
13
points. Tho fo1loing table lists some of;tie.mo1cu1ar weights
reportod, specifying the pH ranges over which theae hold or have
been measured.
Bjliprotejn. Lpl. lit • 2H_Lanve , ronc es.
R-phycoerythrin 291 000 3,0-10,Q Eriksepn-uenso1, 1938. NolSfl and O'hEocha, 1967,
B-phycoerythrin' 290 000 4.34.5 Airth and flunks, 1956. Brody and Brody, 1961
C-phycoorythrin 226 000 5,2.7.2 Hattori and Fujita, 1959.
R..phyeoo3in 273 000 2.5-6.0 riksson-Quonoel, 1938.
C-phyoocyzaniu 276 000 47 Hattori end 1?ujita, 1959.
Alló-phycooyanin 134 000 72 Hattori and lijita, 1959.
An average error in these estimetins in about 1 5 000. The technique.
noot uidely used for determination is u1traentrfuatjon; an
alternative method is to use the results of mino-acid analysis
(see pages 26-28).
Some confliøts arose aver the mO1ern1ar weights when they were
firat inveetigato. . For exaDplo, edber and Katnurai (1929)
orina11y reported 0-phycocyanin as having a rolecular weight of
208 000 5 000 but ErikssonQuenzo1 (1938) found that they, and
other workers, had not corrected the sedimentation constants for the
density and visoosity of the açlvent. This meant that their figure
of 208 000 was too low and after applying the corrections should have
been in the region 270 000'290 000 (Erikcson-uonsej made the
correction for H- and B.i.phycoerythrins but it was also applicable to
the 0-phycocyanin result).
Finally, Nolan and 0!)ooha (1967) detorninéd the molecular
weights of some of the Cryptononad biliproteins and found them to be
much lower than those of the biLiproteins from othor algal sources,
141-
i.o., their molecular weights were like other properties in
differing markedly from the rest 'àfte biliprotoins. A typ±cal
• Cryptomonnd phycoerytlu'in bolecular weight is abOut 27 800 and that
of a phycocynnin about 37 300.
(ci) flffec
The diosociation of the biliproteins is very olosoly tied in with
estimates of their'roiecular weight3 and was the subject of much 'of
• this projoct Dissociation 'can be brouht' about in several ways
including varying the pH of oolutione end bond breating by choirtcal
• rethod3a Separation of the, diSsoc ted species (subunits)'' •'
prodt'cod is best achieved by molecular sieve chromatography (gel
filtaation) (see experiirental section Ci) (o) ).
The table of molecular weights given in the'last"aubeotion has
• the pH range for which the estimate is valid specified - this is
uully fairly close to 'the isoelectric point. Outside •those
ranges there is fraquontly dissociation of the blliprotoin and many"
• ozainples of this have bean "reportOd, some of which folioti:-,
• ' tYhOn Svedberg ,ñd IatGurai (1929) iepoited C-phoocyanin as
having a toleculsa' weight Of 208 000 near its ieoe1eotio point
(pH 4.7) they also noticed and mentioned. that at pH 6jO one-third' of
the biliprotein existed as half niolecul@s and 'at pH 12 it all
• consiSted of molecules one-sixth of the original size, ike.,' as the
• pH bocamô iore alkaline and further :away from the isoelectric point
(4.7-+6,8-+12) there was a corresponding change from undiemsooiated-
ha1f-dissociated46no-Sixth sized moleOuies. This was also'
' found by Rattori and. Fujita (1959) tiho '.stimated the molecular weight
of C-ph-cocyanin at pH 7.2 as being 133 000. By applying the above
iR
dissociation to this a molecular weight for undiOsociated apoolos
of 276 000 was indicated and thie Is the appoziiriate value since
found aftor applying corrected sedimentation constant measurements.
Borne et al (1963) found that 0-phycocyanin from Plectonema
oalothricoides dissociated at the iGoeleotric point itself to some
degree in the presence of urea nd sodium dodecyl sulphate -
centrifugal analysis of the biliprotein indicated that 'appreciable
amounts of low molecular weight cOmponents were present and the
minimum iOleou1ar weight of the3e was later estimated to be 30 000
(Borne et al, 1964). There has been some argument regarding this
minimum molecular weight figure for C-phycocyanin but a series of
three different experimental detorminations byKao and Borne (1968)
has strongly reaffirmed the above figure as has work by Neufold end
RiCgs still more recently (1969).
- Further evidenceof dissociation comes from the visible
absorption of the proteins, óog.., for C-phycooyanin from .Anacystie
nidulans the absorption and fluorescence spectra changed in some
reSpects as the p11 varied - the spectrum at pE 5.7 was taben to
represent undiosociated species. (Io1eou1ar weight 276 000) and that
at pH 7.5 half.dissoclated species (i1o1eou1ar weight 338 000)
(l3ergeron, 1963). Berns and Edwards (1965) studied this further on
C-phycocyanin from Pleotonernaccalotbriooides by using an electron
microscope. They observed structures with a central hole which they • interpreted as the hexamer ctr.áturo previously postulated - this at
• higher magnification was later demonstrated to consist of six
globular monomer unite which were arrayed approximately at the
vertices of a regular hexagon. ,. . .•
1ore recent woz: on Q-phycocyanin has confirmed the pH dependence
16
of the association-dissociation effect and it has also beon shorn that
other factora can bring about the same - these inólude the ionic
strength of the aolut{on,. biliprotein concentration and tempez1atur
(Hattori et al, 1965). The ring4ike hexanier idea has received
urthor. suort from the ;iOrkof Scott and Borne (1965) who studied
sedioontation velocity with varying pH;, ionic st'onths, temperatures
and bfforo and then postulated in6n6nior(-trimer44 hezaxner 49
dodocarner equilibrium system on the basis of all the evidence. Both
of these groups workOd on deuterated phycocyanin (produced by
oti1tu'ing the Alga 'n heavy water) in which all the hydrogen is
rOplaced by dOtiteriuin,: The only difference in standard physical
properties was a shift of about 7nm in the visible absorption maximum.
As far as the dissociation effects were concerned they were very
similar but oocurrad to a much less marked degree than for normal
phycoc3ranii. This was slightly unexpected as thodioóooiation effects
can be éxplainod on the basis of e1ootrostaticintoraction, i.e,,
the phyoocyenin xnolôOule@ are negatively Ohargod 'in these pH regIons
• where dissociation Occurs and as the hydrogen ion concentration
increases this charge will reduce, therefore association rather than
dssociatjon will be Observed.. Similarly increasing the ionic
strength of the :oqlution will reduce interactions. However, if this
was the only explanation doizteriophy000yenjn and normal phycocyanin
trould be expected to exhibit associationadissooiation to the sale
extent and this is not in faCt the obaorvéd Case, The postulated
explanation is thitin addition to electrostatic intoractione
hydrophobic side-chains at involved in an important and specific tray
in the union of subunits; deuteration reduces the extent of this
interaction between the hydrophobic side-chains of individual. units
17
and therefore deutoriophycocyanin shows loss association-dissociation.
• than rorma1 phycocyanin. Finally, some recent work, by Neufeld and
• Riggs (1969) on C-phyoocynin .f'pm, Anacystis, r dulans has indicated
the equilibrium oystem to be monomer +-dimer4.+ hexaner++ do4ocamer,
j',o, diner instead of trier,, with. the hexazuor Predominating . at, low
pH v1ues end high protein concentration but ailution leading to
monomer at low pH values and.'dimer at high pH values. This was alsd
indicated during work on the same. protein by Craig and Carr (1968)
and It seems now as if two equilibriun systems might exist, depending
on the algal source of the C-phyoocyanin,
From C-phycooyanin to R-phydocyanin Which was for. some time
thouh't to be C-phycocyanin contaminated with sOme phycoeryhrin
(Hattori and Fjita, 1959).: This, has now been di.aproed, however,
(Albez'tcon and Nyns, 1959; O'Carra and 0'hocha, 1965) and it has.
since been shqwn' to behave as A homogeneous solution. Spectral
differences botween it and' pure C.phyo,oyanin have also ruled out the
contamination posobility (O',Carra and,0'hEooha, 1966).. As a
distinct bliprotein, therefore b it was also 'investigated for
dependence of rnoleàuThr weight on pH and it was found to be stable in
the ph range 2e5 to 6, but disqciatod into particles about half the
normal' size at pH 7.O-.8,5 ('as originally reported by Eriksson-Quensel
inl98). . .
Allo-phycooyox4n is the other distinot Blue-green biliprotoin..
• (although "allo" means diffei'ont frOm, nOrmsl) having been orystallisod
as thin platelets and its molecular weight estinatd as 134 000 at pH
7,2. At pH 11.6 it is fOund to dissociate but in an irreversible
mariner (Rattori and Fujita, 1959),,
The phycoërythrine behave in a similar manner depending on the
pH but rost work on dissociation in aqueous solution hao bei' done
Ton the phycocyanine. Noltn and O'hEocha (1967) reported the
• . sopration of a ibuer molecular weight component from R-phydoorthrin
• ' uin mólocular : 50V0 ohronatoFaphy and in this laboatoiy '
-phycoerthrin trao studiod in a oiuilar fashion. The result was
observation of a definite dissociation of theprotein in aqueous
solution into threo components of differing colocular weight.
Spoctr]. difteroncos bettieen these wore also apparent and a certain
dogroo of roassociation after 'precipitation was alco proved. The
.dioeocjatiOn-assooiation 's.tom' poetniated was conoOr4.dimer4-Ppo1ymer4
All of this urL is fully described in the oxpericontal section
(cection(4) (also Iliores and tTall, 1967).
• ' . C-pbôoorythria, stable in the pH rango 542-7.2, was found to
• , dissociato at p1l C (Hator and2uith, 1959). R-phycoorythrin
has now also boon proired to dissooiate to a cortain extOntD. 'a subunit
hp_vina colocular weight around 43 000 having been isolated (Nolan and
O'bEooha, 1961); 'this'sipportod the work of Hjerton (1963) who
roport4 that fl-hycoerrthrin gave two zones on polyacrylaulde gel
oloctrophoresis and on ultracentrifugtion.
Per all the biliprotoins a lot of work has boøn done on
diasoo.ation by chemical broakdotm of bonding, both covalent and
non-covalent. Vuoh evidence has coe from the use of p-ohloromereuri-
bonsoto, (P.c.h.)., a eulph3rdryl blocking reagent. Treatment of a
bi1protein with this compound results In spootral changes and it
is usually possible to separate coveral cubunito (Jones and
i?ujimori, 1961; Pujiniori and Quinlan, 1963). ' Pujimori '(1964) also
chotyod treatnent of soco of these isolated. 'subunits with -
glutathiono could lead to at least partial reassooiation, restoration . -
19
of Spectral features being the main evidence. With co-workers
he followed this up 'and showed that R-phycoerythrin from Ceramium
rubruin on treatment with P.C.NB 4 could be separated by gel
filtration into four distinct subunits having spectral differences
and that treatment âf 'these with glutathione could lead to quite a
high degree Of reassociation for at least one of them (Fujitnori and
Peoci, 1961a)., Similarly they showed that B-phycoerythrin from
Porphyridium cruontum could be split into two subuflite which also
underwent partial reaaeooiation (Fujinioriend. Peccio 1967b; see also
experimental section (6)(a) ). They also tried another mercurial
compound, p-merouriu-phenylaulpbonjc acid (P...P,.s,A.) on
C-phycocjranin from Anaoystis .nidulans and showed that there was an
almost identical eplitting effect to that hrougt about by P,M.C.B.
with some degree of t'eaasocjation again being possible (Fujimori and
Pecci, 1966); sijni]ar reu1ts using this compound on B-phycoerythrin
were found in this laboratory (Be0 experimental section (6) (b)).
From all the foregoing, it is clear that '.breakdown of the
biliproteine into subunits by chemical methods is quite possible and
many other resgents have been tried with some success. Two types of
bond exist which require to be broken; the non-covalent bonds
between adjacent chains and covalent bonds between and within chains.
The non-covajent bonds are considered to be bdrogen bonds between
the ohaine plus electrostatic interactions betwean and COO-
residuSa which can be broken in eeera1 ways. Varying the pH of
solution, the ionic strength of buffer.,, the biliprotein concentration
and the temperature have all been reported as bringing about some
dissociation 0 Other Chemicals in addition to the mercurial
compounds can also be used including sodium dodecyl sulphate and
20
• euôcinic anhydride which trors quite successfully by introducing
a negative charge as ±'ollotrs -
-COIm-protein
Ca2 -
+ +I%N..protein ) + 2R
.CH2 -COO o • ..
• • This resuith in arepulciiOn bettg set up bettreen chains where previously
there tias an attraction as is replaced by 000 9 therefore
breakdown occurs. Suocinie anhydride can also be used at various
p11 values with success (Deal et al, 1964; Hass, 1964).
Disuiphide bonds at4§ tho only covalent bonds Icnown to definitely
oxict between polypeptide obajne although ester-tyi,e linkages are
theoretically possible. Those diaulphide cross-lin!ages may be
either intaror intra oiaii. There aro.several methods of breaking
them such as cleavage by oxidation using.performtc acid (Toennies end
Iamil1es, 1942; Sanger, 1945) which worke as fol1owo:.
U12-3R2 (0)) R1S03 3 +
major disadvantage 'of this, however, is that individual amino-acids
and residues (in particular tryptophan) can be affected by the method.
There Ic also reductive oleavage which can be• brought about by
• several reagents including sodium borohydride (whicI iaa the
disadvantage of being a general reducing aget and therefore lacke
epeoificity) cyanide and su1phitei6ed in conjunction with a
non-covalent bond brker both of.which are quito effectivo; thiols
such as cystoine, reduced g1utathion, thiogylyoollio acid, •
-morc&ptoethy1cigne and -rnercaptoethano1. The course of reaction.
ucing,thiols is as,follot'75:- • .
21
R1S-SR 1 + '2R2SH " 2RSH' + R2875R2
An ezoessof thiol is obviously required' to drive reaoton,'to the
right. Since thiol (-Sn) groups are formed by this rOaotión there
is the possibility that they might recombine,ie. disuiphide exchange
with fornation of new Inter or intra chain dieu1ph.tde bonds may oCcur
(e.g., as reported by Kereeztes-Nagy and Klotz, 1963). To prevent
this it is necessary to add a 'blocking' reagent which cOmbines with
the SN groups. Several cómpounde are suitable including the
mercurial compounds,::; ifl), 'maleinttde,
N-ethyl inaleimide and iodoaCotic acid. The lator is about the
most sucCessful of these reagents, provided an excess is not used as iodine may be produced WhiOh can óau5e oxidatIon.
In this laboratory it has been found, apart from studieS using
morurial Compounds alàne, beet to use. a Cornbinatiion of a
nOn-covalent bond-breaking' med.tzm ouOh 'as strong guanidine buffer'
solutjon in conjunction with a covalent bond breaking reagent to
achieve maximum dissociation. P '—meroaptoethanol Is effective as
the latter but a better reagent is dithiothreitol (Ôie1ác'e reent).
Full descriptions are in the ezperljnental àeotion (aeotioii (7) ).
It is clear therefore that diosociation'of the biliproteina can
be brought abokt in a variety of ways and iek an important property
doáerving Close study. No molecular weight estimatIons in
particular âan be carried out Without bearing in n4nd the possible
dissociation effects. Some degree of reassociatiOn brought about
bychemioal methods and varying physical conditions has also been
proved. Of rnost 'interest is the undoubted presence of equilibrium
systems (monorner...dimer or trin1er4hexamer49dodeoamer) in
aqueous solution, Finally, in addition to the isolation of subunis
22
by gel filtration and spectral evidence of dissociation a
combination of the two on a larger scale, in which a !naxinaum
concentration of eluant from a column equal to the concentration
of loaded sample is obtained, cafled frontal analysis allows
observation of the presence of the equi1brium system. directly.
Some basic theory has been derived and it is po8aibie to obtain
more quantitative infOrmation about the System. Some work along
these lines is described in the experimental section ($etion (8))).
(e) Denatiiratjn
When the structure Of a protein is fully described there are
three aspects which must be taken into account., The first is the
primary structure which is the arrangement of the covalent bonds in
the molectile as indicated by the usual structural formulae. This
is determined by chemical analysis (see pages 24-26). Next comes
the secondary structure of the protein which is the spatial
relationship of neighbours along peptide chain, i1e,, the
geometry of the bonds. Combined with this to deecribe the
conformation of the protein is the tertiary structure whioh is the
gross folding of the chain as a whole whoh may bring close to each
other parts of themolSoule otherwise widely separated along its
backbone.. Determination of the secondary and tertiary structure is
much more oomplicated.than that for.the primary structure
(crystallography being a main tool) and is beyond the scope of this
review.
However, the chemical, physical and biologioalpropertjes of a.
protein depend just as much on the secondary and tertiary structures
as on the primary structure, When the conformation of a protein is
23
altered from that in its native state partial ortotal loss of
biological function occurs and this is the effect or phenenon.
knovn as dônaturation.: S
• The biliprotoinsdenatue quite readily. Obviously denaturation
will oöctu' whq,noVer.th6y.aresubjected to cheinial attaok but in
addition heat and bright light can canoe denaturation. For this
reaeon the eztraotion and purification procedures as well as sany
onporirzionto are orriec1 out on the proteins' in their native. otate in
the cold (0-500 and in the dark or at least shielded from bright
light.
Proof that donaturation has occurred can come fron the absorption
spectra (see figure ?) - this is tho case for most proteins,, the U.V
absorption in particular usually differing in Intenitr and tmvelength
bettosa native end denatured protOin (e.g. 9 Xumars0h, 1955). For
the biliprotains characteristic peaks in the.'ieible region are
roplacOd by a general lorer ntenoity áborption over the Tyhol&
region, although cextai±k maxima may iso be removed, and there is an
increase in the UV,, absorption. The spectral criterIon for purity5
no longer holds# Diroot evidence is also immediately apparent as the
• 'oharacteristio fluorescence of the solution is lost, being rlaoed,
• in the OaCG of pbycoérythrins, by a purple ãolour. In the
precipitated state there is a similar chance in colour from red to
purple, O'hocha and O'Carra (1961) studied the donaturation of
some pbycoerythrins using dilute acid and strong urea solution.
They observed quenching of fluorescence, decreasing intensity of
visible sbsórpton.tiith corresponding inoreaeodU o.V,: absorption and
'they also reported increased chromophore activity. •
'Change in the bicloginal function of the bi1iprtèins caised by' •
24
denatu'ation will - be mentioned in the relevant section (see pages
31-35).
Analysep Of the Biliproteina
(a) Elementary
The elementary compOsitions of several biliprotiena (i.e., the
total relative amOunts of all the elements in them) have been Iiown
for some tine • Crystalline sap1es of C-hycoeyain,
allo-phycocyanin and C-phycoerythrin, all from the alga Tolypothrix
tenuis, gave fairly similar analyées results which were on average
40P carbon 1 7 hydrogen and 15 nitrogen,. The two phycocyanins
also contained 0.6 sulphur and no ash whilst the C-phyooerythrin
had Onl7.0015eulphurand 007 ash (Hattoriand Pujita, 1959). The
sulphur content for R-phycoer3rthrin and R-phycocynin haa been
reported as high as 1,6 (Akabori and Pujiwara, 1958; Raftery and
O'bEooha, 1965),. Most of this sulphur Oorues from suiphurocontaining
amino-acids - Rat tery and O'hEooha recovered about two-thirds of the
l.6 sulphur in R-physoerythrin from Ceramium rubruni as this but
could not explain the origin of the remaining one-third, and they had
not used any ammonium sulphate during purification of the biliprotein.
Similarly, Ithnmel and Smith (1958) had failed to account - for the
total sulphur content of R-phycooyanin and C-phy-cooyanin from
Porphyra tenera but in contrast total recovery of sulphur as
su1phurcontaining 5inio-aoida was obtained by Rat tory and O'hEooba
(1965) on C-phycooyanin froni Noatoo muscoruni..
1enentary composition also includes the amount of chrOniophore
present in the biliprOtein, these being tetrapyrz'oles (sea pages
38-39). A 455 yield of chroniophoro from C.'phycocyanin was obtained
by Clondenning (1954) which he Calculated to indicate about sixteen
25
chromophore groups in each Eloleoule, total molecular weight being
about 273 000. This was supported by Bx'od and BrOdy (1961) who
used a non o3trictive assay method based on partiolo voight.,
spocific extinction coefficient, fluorescence iifet.mo end
flubrecconco yield.. If an avorago chromopliore group molecular
weight is tokon as 590 thon the protein should have about 3.4
chrOmophoro by weight, Different prteins bind different numbers
of chromophore groua (which thay be duo to the availability of thO
chromophoro grOupa in the cell, according to Brody and Brody).
1.'-,oGt of the weight of the. biliprotoina is aocountäd or by the
amino-acids plus the chromophoro groups 1 The remainder is made up
by carbohydrate content 1 the amount. 9f which vaies from prötoin to
protoin, Fujiwara (1961) was the first to observe that
R-phycoorythrin is a glycoprotein and she found that this protein,
extracted from Pôrphyra tenera, contained 47 carbohydrate, a
valuo con ±rmod by Raftery (1965) for R-phycoorythrin from
Ceramiwn rubrum. Chromopoptidos obtainad by peptic digests of
R-phycoorythrin have also boon shown to contain carbohydrate
(]Püjiwara, 1960); oho also reported sOme in C-phyoocyanin from
Porpha. tenora (Ftzjittara, 1961). and Sasaki and Tsuchiya (1961)
followed this up by showing the presence of at loact seven sugars
(inc1udig galactoso, glucose, mennose end zyloae) after hydrolysis
of the'samb protein. Also reported hab boon about 5 carbohydrate
bound in R-phyàoorythrin from Rhodoinonia palmata (Heard, 1966);
less than 21 in B-phycoerythrin from Porhyridiui cruontuin
(Vaughan, 1963; Paterson, 1967) and loss than ~ in C-phycocyanin
from Anabaona cylindrlca (Lang, 1968),. All those figures indicate
that some of the bi1iproteixat least are genuine glycoprotoino
26
(e.g.., R-phycoerythrin) whilst some of the others are not
(e.g,, R-phycocyanin), their carbohydrate content probably coming
from ext'ace1lular material not removed during purification.
Finally, it has been Suggested that if the carbohydrate is present
in the biliproteins as sulphate eater (as with certain marine algae)
the missing sulphur content of the proteins would be accounted for,
but this has still to be conclusively prove4.. .
(b) 'Amino-acid Analyses
.Awinoaoid residues account for 75.89$ of the tOtal biliprotein
weight; . tabe 2 gives amino-acid sn3lysia results for some Of the
bi1prot6in The method Of determination nowadays is by, use of
the ion exchange method (NoOre and Stein, 1954) in automatic
an1yes (see also experimental section (.)() ) which has superseded
the oldor methods of two.'dimensional paper Chromatography and
thin-.láyer chromatOgraphr (e.g.., 0 'hEocha and Raf tory,. 1959; Levy
• and Chuñg, 1953 Airth and Blinks, 1956; Raftery and O'hEooha, 1965).
The general picture of,amino-acia content in the biliproteina
diecornable from these results is that the dicarboxylliO acid
• amino-acids are present in greater amounts than the basic amino-acids
• (the reverse is usually the case for plant proteins) and that there
is a . high content of' amino-acids which contain hydrophobic aide-
chains (e.g., valine and leucine).
The figures can be used, to calculate the expootfd isoelectric
point of the proteins and a159 to give an estimate of the biliprotein
molecular weights, including the minimal molecular weight, An
example of the former is the good agreement obtainOd in isoelootric
point value for C-phycooyanin as calculated from the amino-acid
analysis figures and as found by eleotrophoretic atudiea (pH 4.76)
TAS LE a Tme A,tiwo Ac's CoMc'osrr 10W5 OF SOPIE tLIP1OTE INS (RIIEtEiV,'4 O'kIoc..IA, I6S)
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27
(Iimme1 and Smith,. 1958); similarly good aëreeient was obtained
fOi' R-phycoerythrin from Qera.mium rubrum using amino-acid and.
ammonia rOajdues for oa1cu1aion and comparing to the figure from
e1ootrophors1s (pH 4.3) (Raftery and O'hEocha, 1965), mis did
not work for R-phyooerythrinfrom Porphyi'a teneM, however, sn mus,t
therefore be treated with some cáuton. '
There are several examples of biliprotein molöcular weht
estimation using the athinoacid' analysis figzrss; o.; BGXIS, Soott.
and 0 'Reilly (1964) who based theIr calculations on the amounts of
the least abuidant:amino-.ao1d (yetI.ne and hintIdne) tmd estimated
• a minimlini molecular weight for C-phycooanin from several algal
sources of .30 000, a va].uo in good agreoment with that determined
• ueig ultraoeritrifugation sth other methods (Borne et al, 1964; Kao
and Borne, 1968; Neufold.an&Riggs, 1969). The'sanie jrotein from
the Red alga Porphyra teneza: as pt'e'ü-iously calculated to have a
minimum molecular woght of 38 100 (Iuinxnel and Smith, 1958), Using
the N-terminal aiiino-aoit'(see next subsection),. 0'Carrá (1965)
estimated the total moleCular weight of C-phyoocyanin as being
138 00 whióh, after allowing for the dissOciation effect', is in
very good agreement with os timates using other methods., The
C-phcoerythrjn minimum moleCular weight was calcu].atéd to bó 61 500
• . (Raftery and O'hBooha, 1965) assuming one cystine resi4uè per
integral unit, This may well be high i however, as both eye tine and
cysteiné 'tend to give low yields on protein hydrolysis, For
B-phycoerythn fm. Pophyridiu :aruentum aperometri.a. t.tration
indicated eight suiphydryl groups per molecular weight of290 00
(Fujiori and Quinlan, 1963), For R-phycoerythrin niinir' zuoleCular
weights of 14 600 (Iinurol and Smith 1958) and 19 100 (Raftery and
Oi't rejtSue hu 4mtc.5mt u'E cLs3uw.4 ' IfAd case. • '
• 28
O'hEocha 1965) woro found for the protein from dfforent a1a1
aourceo, These values D however, based on one histidine roiduo
per integral unit, wore lower than the ultracontx'ifugal estimates
(sos pages 14-200
(c) Chain' germinal 3ia]ys .
Thio requiros two analysO, one for N-torniinal amino-acids and
the other for C-terminal amino-acids. The former are usually
determined by the rnethod of derivative preparation óithor a
dinitrophenyl derivative (mothod of Sanger, see experirnontal aect±on.
or a phenylisotijoeyanatg do±'ivative (othod of Edman developed
by Fraonko1Conrat ot al s 1955) fol1oed by aOid hyOoiysis to
release the Nterninal amino..aoid derivative which ,oan then be
doterjnod qualitatively (and quantitatively if dosirea).,. i newer
method whoh has the advontage of roquiring.1es3 drastic hydro1yia
conditione after preparation is to Oke a f1uoronitropyidine
derivative (Signor at al,. 1969). In all cases identification is
usually by papOr chromatography or thin layer chromatography using
Standards prepared from ptro amino-acids,
For R-, B- and, C phycoerythrino inethionine has always been found
to bo the only -torrninO1 amino-acid (O'Carr a and O'h2ocha, 1962;
also confirmed on B-phycoerythrin in this 1aboraory) apart from a
small amount Of N-terminal aspartic acid in R-phycoerythrin from
Coramiuin rubrum reported by Vaughan (1963)0 Quant±tctive results
vary - Vaughan estimated nine meth3.onine residues per molecular weight
unit of 290 000 for R-phycoerythrin using the Edzuan method whilSt
0'Carra (1965) estimated fourteen methionino residues from the same
protin using the Sanger method. The 1atter value is in better
29
• agreement with the results obtained from total amino-àci4 analysis
and C-terminal analysis. 0 4 Carra also found eight niethioz4ne
roaidues per molecule for C-phy000rythrin.
For the phyoocyanins the situation is not quito so straightforward
C-phycocyanin from Nostoo muscorum appears to contain two
N-torminalniolècules of throonine (OCarra', 1965; O'hEotha and
1aftory 1959) but the minimum: molecular weight, that this leads to is
• much .highex' than that calculated in Eevoral other ways. It. is
therefore thought possiblø that sono 'masked 3 terminal acids, which
might even hO. nonnino-acid, are present, However, Crospi ot al,
(1967). found N-terminal methiônino for C'phycócyantn as uel]ae
threonine iii 'côntr st to 0' Caa'o results. Fox' R-phycocyaniu
threonine and methionino have both been found so N-torminal
• •amino-aôlds (0'Carra, 1965). Ho Ouggostod that R-phycooyanin, might,
therefore' contain two tyi,es of subunit, one related to C-phy000yania
and the other to the phy-ooerythrixis
C-terminal analyseo have also been carx'1e4 øut - for R- and B-
• phycoorythrino and -phycooyanin alanine appears to be the only
C-rminaI amino-acid (Raftery and'O'hEooha,. 1965), being present to
the extent of twelve residues per molecular weight unit of 290000 for
each whilst for C-phycocyaiin only serino had been roported
(O'hEocha and Raftory, 1959; O'Carz'a, 1965) to the extant of four.
residues per molecular weght unit of 276 000.
Generally,' theso analyses results (total 'amino-acid and
terminal amino-acid) when considored quantitatively are found to. be
in quite good agreement with molecular woight estimations (miiimal
and total) calculated in other ways (see pages 12-22) although there
are one or tv anomolouo rOsults.
30
(d) Overall 31ruoture of the Bilij,ro1eins
The foregoing subsections have dealt with the olezentary
otructuró of the biliproteine including the carbohydrate content
the roltive amounts of all the einio-oic1e present and the
identification and amounts of the oh&in-terininal amino-acids,
Tho final overall structure of the proteins can then be found by
determining the exact amino-acid aequnCo in tho chains and studying
the secondary and tertiary structures of the moleoule (seo pages
22-24),,
Determination of the ainó-acid soquénôe in proteins is done. by
breaid1ng down the chain. into sl1er, more manageable unitS called
peptidoso To do tiiie it is first nooessary to break down the
overall folding of the ohano and to split any intechain disuiphide
bonds, usually ,,by oxidation oi reduotion (see pages 14-22), The
chain can then be split into poptidés by specific cleavage using
proteolytic enzymes such as trypsin and pepsin or'by chemical means,
usually oxi4aton, eoa., by N-broso auooinicdo or bromine. Once
the rlet &iIiant peptidos have been isolated their amino-acid sequences
Oan be established using a cOmbination of total amino-acid analysis,
• end group determinations a4 the extra weapon o partial hydrolysis,
i.e., further breakdown of the peptide into a number of smaller unite
which can be purified aM examined further, flydrochioric acid is
often used for this purpose' (e.g,, 12 for 48 hours at 25 00) and
the resultant oer1apping units may contain up to about six
axnino-.acjdc, The amino-acids sequence of those fragments can be
established chemically a little more oasily and then they can all
be built up to give the sequence of. the larger unit from which they
• were dertd. This procoss is then cOntinued until the anino-acd
3].
s.equene of the whole chain is determined. It is, hottovor, a
1ong tedioua and complex business, Nowadays physical methods are
being usM a lot more to. establish the squence of. the small ...
fragrnoflts released by totalbydrolysis, mass spec troaetry being the
most iaeful (o,., Agarwal-et al, 1969, who also lists other examples).
In the specific case of the biliprotens he Structure is
complicated f±ther by the presence of the chromophore groups and
the carbohydrate content. Cleavage of chains results in release
of Ohrbmopeptidee, i,o,, peptide& with ôhromophore group3 still
attached,,, Further discussion of theo with reference to the linkage
is uoI in thó next aedtifto, .
The Phroobilins : ..
* () Description, Nomenclature and Sruoture
The proathetic groups of the algal biliproteine wer first
investigated by gitsato (1925) and Kylin (1931) as well as by Lornberg
(1930) who showed thorn to be related to the animal bile pients
from which the terms "biliprotoin" afld. 'phyoobilin" were then derived,
These bile pigments are totrapyrrolic structures, some examples of
which are shown in figure 3. The phycobilins have been found to
have very similar structures to these. The chemistry of linear
t6trapyrro1es has been revieWed by Lemberg (1930), Qray (1953) and
Stevens (1959).. . . .. . . . .
Fairly drastic conditions of hydrolysis are. required to release
• the phycobilinë in a peptide-free otate from the biliproteirLd and for
this reaeón ost of the work done on them has been on Chromopeptides.
instead of on the free phycobilins, As a result of this, and
purification difficulties, the exact structures of these compàuda
FLGUt3. 5otE -
T'(eIcrL GLLe Ptis
32
has,been the subject of come coritroversy.because the 4rastic
conditions of hydrolysiG used in particular have been tIought. to
caucoalteratiozt : in structure. 1 Vore recently newer methods of
=104siria the phycobilins invoiing less. drastic conditions have
boon tound which has helpod.to ola up. such; difficulties r this
is mórè fUlly 11ccussod latQr 4.n.the sotione. !oi!c of. the
• postulated structures with their original refórences are shown in
• . figure 5,
Lemberg (1950) namdihat he c6ncidero4 to be the ntive
prosthetic, groups of C-phycocyanin and Rphy000rrthrin ,hycocyanobilin
and phycoerythrobilin rocpective13r With Logge (193) ho followed
this up and concluded that those compoundo which trOt'o obtained after
hydrolycie of the biliprotoino (with 30 methanol'- hydrochloric
acid at eo°c) wore identical with nesóbiiiv±oiin and mecobilirhodin
rospcotio1y (see figure 3), They also considered phycocyanobilin
to be an ozidiood.foryn of phycoorytbrobilin but after Siódei (1935)
showed that the compOunds when oynthosiaod wore isomoriö Lomborg (1949)
otiod this view, to the étracted phycobilino.. it is possible that
the pigments isolated earlier , wore 4rtiftetti.. formed during hydrolysis.
O'hEooha (1958), however, failed to find any indication of a simple
reItionehip between the tuo,phydobilina and also found no evidence.
of relationship between the phycobilins and the bile pigments as
postulated. byLonberg (1930) apart from obvious simi'ar structures,
Thö phy-cobilins have eoEiC characteristic proortioo like the
intact biliprotoins - they, do not fluoresce when Isolated but
combinô with zinc ions to fOrm br±liiantly fluorescing comp1oes
whicha1co have characteristic absorption spectra (o'bEocKa, 1958).
The low ash, content of purified .biliproteina (Lomborg, 1928;
- 33
}attori and Fujita, 1939a) ±ndicatd that the phycobiline are not
coip1ezod with !otaXs in thoir native etato hotievôr0 They also
have charaOteristic absorption opectra come of which are ohown in
figure 4 Viib10 absorption has Qhoi.m up that appeara to be
iorneriea1ion of phycobilins from some of the bil±protoiris e,g
bttroen two phycobtlin extMoted fro (-phyoocyaxthi (from Anabaena
cylindrica) end the tro different - ferris of phyoobilin shown
(figure 5) for the phycoorythrii piiicnt in ooncOntrated hydrochloric
.oid when the principal abcorption maximun changes travo1bnth from
576nx to 500niii, The socoild. of these structures is the loss
doijugat6d6 hence the deoroae• In wave1ngth (Lembor and Leggo j.
• 1949) and was termed a urobilinoid conjuatod system (OhEocha, 1969;
0'hEoolm and O'Carz'a, 1961), Imu1ar compound is.reieaso1 from
• R-phyooôrythrin in 4ddition to the onpeotod phyeoorythrobilin.
Howover careful cork by O'hEách. (1958) using ieee drastic bydrolysis
conaiions to release the phycobilino had indicated that all the
phycoerythrine contained the oào chromophoro group on the baoio of
opoctral Gvidenc. It then appeared that the pients obtained
depended to quite en .extont On the conditions ithed in the treatment
to release thotn the phycohilin isolatod by O?hEocha (1958)
after mild hydrolyeis of C-phycoc3ranin differed from thit isolated,
by Lomberg (1930) using rore vigoouo brdro1ycio but the former
•
could bo converted to the lattor undOr stronger cóndltioñe.. These
pigments clearly had. to bG o1ated and: wore distinguiehed In terms
of the wao1engths of their respective visible riaxima o,g. the
• phycooyanobi1n obtained by 0thOoba was referred to as phycobilin
630 ihi1st that obtained hytembergwao phycobilin 608. Similarly
there was phycobilin 655 from C-phycocyanin from a dfforent algal
FIUL L
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34
source (O'hEociia and Lambs, 1961), However, it has now be5n proved
that these are all identical and the small spectral differences are
duo only, to the methods of iolat1on. .•
The hydrolysis process usua11 requires acid, concentrated
hydrochloric acid at room. temperature for thirty minutes being the
least borore conditions required o This Is etill fairly drastic
and, as nentioned, led to doubts as to whether or not the phycobilina
released arc identical to those in the intact biliproteins . Some
spoetral differonos seemed to indicate that this time not the case,
e,,, phycobilin 608 could not be reconciled with the visible
aborptiorx of intact C-phycocyenin (Rabinot'iits, 1951), To try and
recolve such anomalies it was necessary to develop less drastic
motheds of hydr1yoie and Hattori and Ftij±ta(1963)were auoeossftzl
in thic by ref luxing phyoocyaniñ (or Intact Blue-green algae) with
methanol in the presence dfsscorbic acid (since found not to be
necessary 0 1 03rra and 0 1 hIoo}a, 1964), a good yisld of phyoobilin
being obtained,. Crespi et al (1967) usod.a Similar method thobtain
a good yield of phycocyanobilin and subjected it. to N.R1 and Pass
cpectral studios which led, to the triicture shom infiiro 5? (the
distribution of aidechaina shown being somewhat arbitrary, however)
Cole ot al (1967) denatured come biliprotein with trlchioroacotic
acid, refluxod with methanol and then eterified the product by
treatment with boron trifluoride. The re3ultant crystalline pigment
was inVestigated., by N1.6R. and mass apectrometry.And shown to have an
alriot identical structure to that put forward by Crespi ot al (figure
Such deeradativo atudies were hanpered by the difficulty of
isolating and purifying enough material and this has only recently
L L:0 cn)
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p
C64
Siucrut
WAKE ._ I uc-rutE
PC
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fROTEicki FF1 L. T 0
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coix II
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n
35
been overcome by the iniorodegradation technique evolved by
Ridiger (1967). This method applied tophyôooyanin resulted in the
structure shown in figure 5 being put forward.
Enzymic cleavage has also been used - Lemberg (1928) used pepsin
• V on Bu.phycoorythrin and obtained a coloured material soluble in amyl
alcohol (now 1own to be a ohromopeptide, however.) but this can
release phyoobilins, e,go, Siegelman at al (1967) isolated
V crystalline phycocyanobilin diethylostor after enzy4o cleavage; also
Paterson (1967) in this laboratory had some success, V
Overall there was stlI argument as to the exact stiuctures of
the piycobi1ina but recent work has cleared up the rob1em-that V
mezt±oned above and that by Chapman Ot al (1968). They showed that
othanolio hydrolysis and acid hydrolysis resulted in the formation V
of id.ont±cal products and eggesVted, that slight structural differences
as found by Croapi et al and Cab. et al were simply due to individual
interpretations of experimontal date and slight differences in methods.
The structure proposed by all this york (figure 5) has since been V
further confirmed by studies using douteration and NJ.R, on the
exchangeable hydrogon in phycoerythrobilin (Crospi and Iats,, 1969). V
Chapman ot al (1968a, 1968b) found some other interesting features -
the rocovory of chromophore is neVer complete which means that some
of the groups in. the biliprotein are shielded and the s000nd
V obromophore thought to be present in phycoerythrins (called V
V phycourobilin) oeems in fact to. be. a protein-phyooerythrobilin complex
instead. They examined all the phycoorythrins and found only one
V ldnd of ohromophore group to be present,and isolablo. The same
is truO for the phycocyanins except for R-phrcooyanin which they
found to contain both phycocyanobilin and phycoerythrobilin.
36
(b) Linkage to the Apóprotein
The bonds between 'chromophore and apoprotein are very., stable
as mentioned and as a result the exact nature of the linkages has
been difficult to determine with any degree of certainty, although
many, theories have been put forward.:
Denaturation of the biliroteinu af'ects the fluorescence
(quenching it) and the, aborption (intensity in visible region
decreased, U.V. inoreased) and can be brought about in many ways
(pages 22-24). None of these methods or reagents will release
amino-acid free pigments, however, concentrated acid usually being
required for this which auggeeted that the bonds are strong, i.e.,'
covalent. Lemberg (1930) suggested that for R-phycoerythrin the
linkage was through the free.amino group of an N-teminal acid; or
through the £-amino group in lysine to the free acidic groups on.
the propionic acid side-chains of the tetrapyrrolic groupe. This
was disproved by O'hEocha (1960), however, when he found that all the
t-NR2 groups and N-ter'minal groups were free to react with
dinitrofluorobenzene al'though O'Reilly and Berna (1963).did md an
unaltered lysine residue from C-ph cooyahin, They suggested a
peptid.e-ype linkage, but the relatively high rate of hrdrolysis of
the linkage compared to that of known peptide bonds seemed to
disprove, this (Q'hocha, 1960; '.O'Carra, 1962)4" The presence of a
suiphydryl blocking reagent to prevent recombination of ôhroniophore.
with protein -fragments,was found necessary by O'Carra (1962)4
Working from the tetrapyrrole, O'Carra ot a (1964) pointed out that
none of the groups in the molecule seemed capable of forming a stable
but acid-hydrolysable linkages alsO if any of the oxygen or nitrogen
containing groups were participating in the linkage zinc Oomplex
37
formation (with bonding to the pyrrole nitrogons - O'hEooha and
O'Carra, 1961) would not be the same for free and bound pgmenta.
Since there are no spectral differences between the two types of
complex this would not 000m to be the case. This left the carboxyl
groups on the propionic acid, side-Ohains which coul4 form ester-type
linkages • Acid lability supported the idôa of these ihioh. could be
from one or both of the propionic acid side-chains to one or more
hijdroxyl groups on the. protein chain, e.g., those of sorine, thréOnino
or tyrosine. Also poosible was an eSter-type linage between a
carboxyl group on capartic or glutanic acids to one of the SaEe on
the chromophore fOred by enolifation of a ring keto group,
Uooently more definite evidence has been put forward for
phyooerythrobilin thioh,• in addttion to confirming the structure as
proposed by Chapman et al (1968a, see figure 5) showed the presence
of a double link to the apoprotin. The first is an ester-typo
linkage between aerine in the protein chain and one of the propionic
acid side-chains in the ohroiophoro whilOt the other is between
glutamic acid in the cháin which can bond throuh its -carboxy1
group, to the lactim grouping in the ohromophore (see figure 6).
• This work was done using less drastic and carefully ôontrolled
degradative Oxidation to yield, small chrornopoptides which have the
• ohromophoro group intact (Riiger and O'Carra, 1969) and by digestion
with proteolytic onymes(ri1lilea and O'Carra, 1968). Similar
linkages have been found for all the typos of pbycoorythrin and the
• problem now sCorns to have been resolved for the phy000rythrobilins at
least, with the phycocyanobili,ns no doubt following shortly.
F%GvU( 6. LtncGEs
ç KIIL IZAA tus0
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38
Bioloipa1 Role of the Biliproteins
Although most algae (and higher: plants) Involved, in
photosynthesis az'e 'found to be those containing chlOrophyll a,
other pigments can 'also contribute to the photosynthotiO process,
a fact first reported by Engelmann (1883, 1884), He showed that
the phyoobilins of Red and Blue-green algae 'wexe as effective as
chlorophyll a in sensitizing photosynthesis. This was later
confirmed by Emersoi and Lewie (1942) using Chroococous turgidus, a
Blue-green algap: for which more than half of the absorption in the
yellow region is due to phycooyanin and the ovoraU yie14 is ao high
as that for ohlorophyll..only sensitized photosynthesis in the' Green
alga Chiorella pyrenoidesa. These' etperiinents suggested that the
energy absorbed by pigments othel' than chlorophyll can béutiliaed,
for photoeynthe$i91. In 1950 Arnold and Oppenheimer suggested that
transfer of energy from phycobilins to chlorophyll (dissimilar
'molecules) could explain the high yield of photosynthesis in '
Ohroococcus turgidus in the spectral region where most Of. the energy
is absorbed by phycocyanin. This concept of energy transfer in
photosynthesis was not new although previous application by Gaffron
and Wohn (1936) had cOnsidered it bet'boen chlorophyll molocul,es
(similar) only. Dutton at al (1943) showed that enegy absorbed by
one pigment could be transferred to another in vivo, This work with
other results on sensitized fluOi'esoence '(Thittn and Panning, 1941) ,
supported the Engelxnann. theory that the acoessory pigments acted only.'
as light 'absorb's and not as photocatalysto.
Haxo and B1jtks (1950) determined action speàtra of photosynthesis in
several 'algae and compared them with the absorption spectra of intact';
thalli in each case. They conoluded that in Red 'algae chlorophyll a
.37Q 097,a tC in , P2~0 topyntmolo I1.th tho hJOthUD
ba tho poo of ctc iio Tho7. LQ otc O17
x bjy1 atrtr in th9 DZmQ-*,fv,-m aleaoica nag
(1io (24 cortrcot to tho oaito o'çto Lwo on
op (o toi n2ca .mQd
joo (32) obtaic cflc co clQmVh ho eodo
iU 0il1 t bo o ir O3t' O2O 3tO b7
~:Osraft 0 ô (i4). Ogial V10=0h o9 .ii (2) cmi to
ict Ot71A D12?S Dbb2 that ano j by otwj17
ya vmatleo cLo cb4c c tioüo oz
ot1 O rpacm
• 7U't Q3t3 OO 1003? o .t'o b1zo
vcalotaom &o to 001io abrouptlon mi,2 cozi o bo bi
t OQC t.t i&rj
• ta jthatho cnomt of t2 c
lnorc=coo ob=bo ác'a . oci thompwb 0
bc1 cO7 to 1ooyU c • io1io co c&o
offot to c ojac ofot tho 1t atcb to
cCt*3 o oLct u oaton of Io 12J
OnaTcy b ao1la lWol?p Am.Ally tho boio
to eg
3° 3Oi3O' QflO yhbtOO to . oti i
latar-U&l. Rod alrgao ta,o fo1 to ftmctj4Da no o 0000a
b,"alaot lnMblUn,,N'oon 1c1t 1VOM tho of• tho ozn (oo cn
Coio
Ua .
oro boo bom dono ' 04 to to b000l
MO L
mainly on action spectra of biliprotoin formation. It would appear
that their biosynthetic pathway dilTers fror that of the chlorophyllc.
For phycocyanin the anomalous alga Cyanidinium caldarium, uhoh
contains C-phyqooyanin and .aflo-phycocyanin but no chlorophyll, is
ideal for studying - Nichós and Bogorad (1962) workod on this and.
showed that a haem compound acts as a photoreceptor and possibly
also as a precursor of -phycocyanin,
Fujita and Hattori, (1960, 1962) worked 9nBlue-gx'eon
Toiypothrix tenuis whIch contain8 C-phycoorythrin, C-phyeocyanin and
allo-p}iycocyanin and reported that the bile pigment precursor of
C-phrooerythrin and C-phycocyanin was formed photo chomically and in
the presence of nitrogen sources' these pigento were converted in the
dark and via intermediateS into biliproteins, Bogorad (1963)
postulated two possible pathways for biliprotein biosynthesis, the
second of which is best eubstantiáted by the above work of Fujita
and Rattori. Guerin-Dwnartralt (1960) has reviewed the biosynthesis
of Rhodophyta and Cyanophyta biliproteins; Trozier and.Lester (1967)
have established that A-aminôlovullnic acid is a direot precursor
of phycocyanobilin whilst porphobilinogen and copz'oporphyrinogon iii
are direct intermediates in its biosynthesis.
• Ajisof the ReBgaroh
Xnitially.it was necessary to culture the Iei alga Porphyridium,
cruontum and then to extraCt and purify as hih1y aq posob1e
B.-ph'ooCr7thxin from it. Experimentally the N-'termi1 amino-acid(s)
of the biliprotein was investigated and then &t5sociation offSets
were closely studied. First of all natural dissociation. in aqueous
solution was toated for and then Chemical breakdown attempted.
1ereuria1 compounds were employed for thip and then a cobination of
41.
donaturinij solution uith a disulphilé bond breaker and suiphydryl
blocker tried. Throughout it uas attempted to isolate any subuñjt
produced and to estimate their molecular tioights, u3.th special
rofereaco to tho minimU molecular weight. Visiblo obeortion was
used to follow reactions and to characteriso any subunits whilst
separation and molecular weight estimations were attempted using
rnolecular siove chromatography; anano-acid anolysia was used to
check that samples of subunits were genuine biliprotein and not
artifacts 0 Finally thet technique of frontal analysis was used to
try and obtain further evidence for the presence of an equilibrium
system in aqueous solution. A small amount of work was dono on
R-phycoerytbrin to compare the propertios to those of B-phyooerythrin,
42
EXPERI11flTAL SEC PIO1
(i) UTTERAL , TZONUMBIS' .
Before deacrihing full practical details of 'all 'the various
experiments carried, out it 'Will be. convenient to describe for.
reference purposes some of the general techniques eployed.
Tutrasonioatton ..
Tiie is treatment of a substance using a oo1roe of ultrasonic
vibration, i.e.,, very high frequency sound waves which,, when used on
plant material 'such as.alCaep rupture the cell valls causing 'release,
• again in the case of the algae, of biliproteiñs into the aqueous
solutiOn. Tho inatrwent usedfor this purpose vasa Dawes
soniprobe (type 1130A) vanufactured by Dawes instruments Ltd., London,
Fng1and. 'As heat was generated by the excess onery available during
this treatment saple3. were always : eurrounde4 by an ice-bath as heat
• can caiise,denatmlation of proteins (see Psaeo 22-24 , ). One
treatment was usually sufficient but the residue could be subjected
to a secOnd if this seemed necessary. Ultrasonication was usually
øarried out in conjunction' With freeziüg and thawing, another.
effective technique for such cell breakdown,
Centrifucation S • .
Saaplee were centrifgod in as.s.E. (London, England)
'refrigerated centrifuge generally used at 1000g to 2000g with
25Ocrm ox' 50cn3 containers during the extraction and purification
procedures. Where this was not sufficient -(o.g,, to renove fine
precipitate after dialysis) a high speed head could be fitted
allowing up to 10 000g with use of 25cz 3 stainless stoel tube.
Very high speed eontrifugatión. (up to 160 0006, as used in
43
xtractionof B-phycoerythrin tiith n-butano],) roquirod an
tltracentrifu.e. Contrifugation v:as altirays carried out at 0-50C
to mini!iue riOk of protein denaturation.
(c) Dia1ysis
Dialysis is. basically a molecular ciove technique ofton used
to reovo omall molecules such as salts from solutions (usually
aqueous) of high molecular tieight o1ute anch as proteins. Under
vaouu it can also be used fçr concentrating solutions of
macromolecules. In this laboratory dialysis tubing (Cóllulose or
Viôking). was washed thoroughly tdth diotillod rntcr and tied at
both onds with the sonpie dissOlved iü a binimum of tmtor or the
•
buffer solution to be used for dialysis. About an equal volume of
space was always loft inside the tube to allou for expansion due to
• osmosis. Dialysis ties usually against running tap water followed
by several large volume changes of distilled water or buffer solution.
They tiers done in a cold room (50c) if poaiblo and in the dark to
avoid donaturation. About 36 hours was sufficient for most
solutions to be completely dia]ysed free of salts; much nore than
this could also causo donaturation. If the solute being dialysed
was relatively smaU (molecular weight likely to be less than 20000)
the dialysis tubing was first boiled for about fifteen thinutea in
distilled wator to docroase its porosity and thus avoid possible loon
of solute molecules.
(a) ectrphotómotr
Visible and uItra-violet.aboorption spectra wore recordod on a
Penn Elmer 137 U.V. automatic cpectrophotooter or on a Pyre
Uriioam S.P. 800 'spoctrophotometer uhon the latter bocmê available;
- _44
1Cm silica glass cells were used. If absorption at a fixed
• wave1onth was required for a large number of samples a Pys
iJnicam 8,?. 500 spoctrophotomotor was used with a Universal Cell
Vounting to alloti use of 2cm. path length micro cells for greater
sensitivity. Iater or an pprópriato buffor solution was used in
the roforeneecell. '
• ' Infra rca' spectre were recorded on a Pye Unlcam 3.?. 200
inStrument.
(e) Chromatograh, •
Chromatography was defined by f7artin (1950) as "the. uniform
percolation of a fluid through a column of more or loss finely
divided substance which selectively rotarde, by whatovor aoszis,
oertin comonte of the fIui". Provided finoly didod
oubotonc&' ° can be considered to include aper this is a convenient,
general explanation of the process. The physico-chonjoal -
phenomena involved in chromatography include abeorption, ion
exchange end partition :botteeñ phaeo although distinctions botweon
those can be quite arbitrary as often it cannot be stated with
certainty which of thorn is in operation at any given instant. The
definition includes paper chromatography (one and two dimensional),
thin layer chromatogriphy and column chromatograhr. Of these
paper and column chrornatographies wore used in this work,
particularly the latter, and aro described in more detuil:-
(i) Pager chromatora,hy 0
This was used ii section (4) to identify derivativo9 of amino
acids. The rothod involved use of Uhatmm chromatography paper
(number 1 or number 4), 20" by 6" 9 impregnated. with diluted buffer
45
solution and dr.od prior to being apotted uith the saplo(o). -
A capillary was used to spot the paper and a variety of solvent'
cystoas could be used. On óonipletion of a run the papor tias dried
in an afr oven at 90000 Spote were detected by colour, widOr UV.
light or by devolopment with ninhydrin reagent. Standard copoUndo
and stxturo3 were also ran, either coparatoly oi" concurrently.. A
full description of the 'techniques of paper chrosiatogi'aphy as well
as oanploa of rnny systoss can bo found in "A ianua1 of paper
ohroatograpby and paper eloctrophorocis", Ch. 4, soc. 2 by . Block,
Durrum and Zeig, or in "Paper chrosatography", paee 511 and 856..
by Rais and Pacek.
(ii) -GoIRM Cwonatorarhy
rndor this heading conos absorption on columns of trioalcium
phophato and moleotlar sieve chroinatoraphy. ..
A fufl description of.the oporisental details of a trioalciu
phosphate column is given later (section (3) ) and th'uoof
triccicium phosphate in the purification of proteins is fully
discussed by Swingle and Tisolius. (1951), Tiselius (1954) and
.O'Carra (1962). In the procipitotod state tricalcium. phosphate is
crystalline in otructure (proved by X-ray photographs) but it is
0 alao.a gel of high tiater content. Its ioot stable fora is the
• •• hydroxyl apatite of formula Ca 5 (PO4)3 OH which can be propared by
thecothod of Siogolman et al (1965). One precaution that must be
- taken when using the coripoun4 in absorption chromatography is to make
• the buffer solution 'oncentrations fa±' enough apart to provide clear
p1tion of the 'conponente of the iiture. This is necessary to
counteract tailing trhioh occur's unlosu tha oluting solution has a
oompooition ouch.that the R1 value of a givon so1ute.approaoheo
46
unity 1 This type of absorptó ch'omatoraphy was uoed to
purify the B-pycoorythrin from other biliproteins after the
stpges of ultrasonication and centrifigation.
The other type of column chromatography ttaed was itolecular
sieve chromatography. (gel filtration) and as the most extensivel
used technique in this projecVit will be t'i.uiy rsviá,i.
This technique was orignaily based on the use of starch
(Llndquiet and Storgrds 1955) and later extended to oross-1inked
dextraris (Porath and flodin, 1959), now called Sephadexgele, which
were found superior to all other rrateria1a tested. Gradation of
gels is broight about by varying the degree of cross-linking.
These particles are now referred to as xOrogels, organic polymers
which swell in suitable solvents to give particles containing a
three dimensional network Of polymer chains. When packed in a
chromatographic column these particles can then separate ro1eculea
according to their molecular size - the mode of separation is
illustrated in figure 7. Large molecules are oluted first (fastest)
and the smallest last (slowest). The theory behind this is quite
complex and is based either on separation according to molecular
size and shape, depending on the difference in the fraction of the
gel phase available for distribution of the ,aolute, or on the
importance of the djffusion rate. These theoretical approaches
are fully discussed by Anderson a.Stoddart (1967).
The deaáriptive explanation is quite traightforward, however,
(rale, 1967) - the swollen gel particles have a porous structure,
the pores being of Such a dimension asto exclude molecules above a
certain size (known as the exclusion limit). rolecules larger than
this cannot therefore enter the.gel particles and instead pass
IGVR.E1. PQINcIPLE or MoLEcoL. SteVE C $r06RIPm
••'O• fl . .
I' II II (a) A solution of high and low olecular weik
II 0 0 0 molecules loaded onto the surface of a ge:
•110011 •
00 . 0
column..
ILOO.11 .. .' •••
000 (b) The small molecules can enter the gel
I 00300 J . particles and are therefore retarded
0 0 0 • compared to the large molecules which havE
0 0
only the exterior liquid medium available
to travel through.
0 0 0 • ( c) Complete separation with the small
II. ?''ø:IJ S molecules still held within the column0.
110011 •
IIQ*OoOII • .
tto0000JJ S . •• . S • •
\\O 0 03
0 GEL PARTICLES 0 = LARGE NOLECULES ' = SNALL MOLECULES
47
through the bed in the iiq4d opacee surrounding the Particles
(iaotin as the void volune of the column, Vo). Smaller particles
can enter the gol partiolos and. therefore flotr freely through the
liquid inside and outside the particles, i.e,, they can travel
thrøugh the total volume of the liquid in the column, Vo + Vi (the
voluno of liquid contanGd by the gel partio1o)6 Obviously the
availability of thismuch.grester volunotothe small particles
coOnS that they will he retarded, i.e. 0 tillitravol much' more
slot7iy .thr0uh the column uhic'i ep1aina the separation of large and
email volocule0 a S
It is also apparent that no moloculee can bo oluteol before a
volumo of liuid equal to the void volume has passed through the
coLmn and sinilarly all will be oltod by the time the total column
voluco, Ve + Vi, has passed through the column (soo also figure 7).
Columns can be large scale (preparative, e.g., the recycling
column) or on a email scale (alytical), 'Various mathematical
relationships can be derived for analytical columns (e.g.,• u1ndoraon
and Stoddart, 19:7) but the one ueed mnst g&era1ly in this vorLt
was the oitcoilont linear correlation hetvoen log 10 rolocular veiht
and the ratio Vo : Vs (void volume to the elation volumo of a
fraction from a column). 1:oze exactly this is oxproseed as
iogt 4.01 2.105 vhoro 11. is the distribution cooffioiont of
the coluto and is found from X (Vo - Vo) / ( vi - ito) (mivisoón,
196e). ApplicatiOo of both of these relationships as uoll as
protical details of the analytical columns used are given in the
appropriato sections.
Columns have tuo main .ueou Thich are desalting, i.e., soparating
moIoclos of greatly differing siss, and frcoticnation, i.e.,
48
seprating molecules not differing, so greatly in 81z0. This latter
• approach was used extensively Both of thoso are represented
• echoiatically in figure T. The caterials used in the work included
various types of Sephadoz and Bio-Gel gels, Sehadex (Pha'niacia
'iue Chemicals, Uppeala, Sweden) is the trade name for,croos-linked
deztran gels (Porath and Flodin, 1959; J?lodin, 1962). This white,
inert, spherical powdered material swells in aqueous solution to give
gels .whidh have been widely and successfully used in molecular sieve
chromatography. The following table lists the various types of
• Sephadex available and gives the approziwete fractionation range (by
molecular weight) for each: ..
____ • . AUroximatd Fractionation Rajto. (r.txtd
Sophadex 0-10 . up to 700 (i)
" 0-15 •. •• ' " 1500 (i)
" 0-25 • 100' to 5 000. (1)
0-50 . 500 to 10 000. (1)
0-75 •, 1 000 to. 0 000 (i)
3 000 to 70 000 (2)
0-100. ' 1000 to 100 000(i)
000 to 150 000(2)
0-150 • • 1000 to 150 oóo(i) • '.
5 000 to 400 000(2)
0-200 ' • 1 000 to 200 000(1)
5 000 to 800 000(2)
(1) detorrined using polysaceharides (2) detoroined using globulsr
• proteins. . , . • •:
The change from 0-10 to 0-200 is that of, a decreasing amount of,
• . cross-linking and hence porosity and fractionation range, i.e,, 0-10
is the nost highly cross-linkedand the least porous vhilst 0-20O,is
49
the least crosslinked and the most porous. A neu SphO LH-20,
has recently been introducod wbicb has the advantage of being ub10
in polar organic L3olventE as well as aqueous solution. Also
avajb0 are the diethylaninoothyl (-anion) and Carboyrthy1
(Ct-cation) ronge of Sepho4ex saterials tihich 11oti jon exchange at
the oars time as fractionation.
• The other tridoly used tailgo,of nateriala are the Bio'-Gol P gels
•(i3ioRad laboratoriesp Ric1mond, Ca1forziia TLS.A). Phase are
grentdatod polyacrylanide gels of varying composition leading again.
to differing amounts of orois-linhing and hence similar properties
to.thé Sophadex gels. The fölloting table lists these gels:
Approtjmato iraotionation Rane (r.ut.)
• Bio-Oel P-2 ., 200 to 2000
500 to 4 000
P-6 . I 000 to 5 .000 • . .
- P40 5 000 to.17 000 •
" P-30. 20 000 to 50 00
P-60 30000to70000
P400 40 000 to 100000
P-150 50 000 to 150 000
" P-200 60 000 to 300 000
" P-300 100 000 to 400 000 •
Both companies. have -noi produced another range of materials by
sdding.a: cortnin.amount of agreso tq the gels. Xntead of chemical
oross-linking hydrogen bonds hold the po1yer chains together. By
adding differont concentrations of agarose gels of varying
fractionation range can be produced the lotror the concentration of
agaroso the higher the resultant fractionation range. The
50 ..
following tables hot these ratèia1o:
(i/w)grose in p!ox. Fract. Ran (i.t1itd
Sopharoco 0 approx. 4 300 000 to 3.106
2D. 2 2.106. to 25.196
Segarose 10 10.. • 10 000 to 250 OOO
0 8 25 000 to 700.000
6 6 50 000 to 2.106
4 . . 4 200 000 to 15.106
2 '. . . 500 000 tâ 10.106
Bio-0al -0.5t 10 . 10 000 to 500 000
8 10 000 to 1 500 000
• • 10.000 to 5.106
.4 40 000 to 15 .io6 .
• A-50za . 100 000 to 5040
A-150t 1 10106 to 10.106
For all the Cola there are various grades which allow further
• subdiviàibn on the basis of flow rate9 eg,p otandard and fine
• SephadeE gels and 'ary1ng teah sizes of the Bio-Gel A series. For
• optimurn resolution the finr grades should be used but when the
components .qf the aixture to be separated aro rolatio1y far apart
in riolecular weight a fee ter flow rate io zore important and the
ntandard. go1sro theroforo better. :It can thus be soon that a vory
• large range of gels is available to choose from according to the
fractionation range and degree of resolution desired. The •
fractionation range figures listed can only beapproxiraté, of
course 9 since the ohape and cheelcal structure of nolooulos affect
fractionation as iohl as aolooular size.
51
It is also possible to use molecular sieve chromatography
for molecular weight estimation of separated fractions which is
done by calibrating a column ueirg a very large mocroxnolooule,
uiti11y blue dextran, to determine the ,id volume and calibrating
n'aoromolecules in the fractionation range bf the gel which have
their molecular weights well established. This technique was also
extensively used and a typical calibration plot is shown in
Figure (io). Interacting systems, i.e., the equilibrium between
solutea in a system can also be studied by this technique, and an
application of such a use is described in section (s), These
techniques are 'nown as zonl and frontal analyses. There are also
many other applications which are ever increasing in number. A.
'full review of the development, theory, practical considerations and.:
various appliátions of I liar sieve chromatography is to be
found in Laboratory l'ractice, (1967); also Tisolius, (1960).
Finally, colunne were. Qlways packed and run according to
instructions in the literature supplied by the manufacturers. f'
("sephadex" and "BioRad");, buffer systems used and further details
are specified in the text. .' .
Lsurejnoxt
All pR measurements wore made using a Radiometer (Copenhagen,
Denmark) type TTT lop.L meter with a scale expander. An Activion
glass electrode (Fife, Scotland) was used with a Calouel reference
electrodei Readings were accurate to two decimal places whilst
the third oouldbe readily ested. from the scale expander.
Freeg'
Freeze drying is a rethodforcôncentratjng solutions of
tuj
52
• i-acroio1ocuiee in t7bjoh the oolution is fron and expoced to a high
•vac-,nam in the presonco of a substance thich•bind.a txater 9 Ice
oubliio3 frori the surface of thô solution and the solute is left as
an air-dry poxIor.
• Solutiono of samples to be froosodried were extensively dialysod
guinet rurning tap water and then again&t several changes of distilled
•
water to remove all buffer eolutiOn ealta as 'tho prosonco of Salto.
prvonte frçozo drying from beingeuccesoful. Tho solution uae then
frozen iaoing liquid uitrogon in on oven lyor round the sides of
• round bottomed flask by rotating the flosk, this providing a 1age
• oufco area.. Tho freeze drying was then carried out on aflanffold
foozo dryer, typo 31R, at a temperature of 65 9C dnd a pressure
of 0.05 athoophoroc. About 12 hours rras required for an averao sample
to bo cmplotoly froeze. dried. . •
(h) JkydrolZ3os.
?wo methods, of hydrolysis of oamplea,uero employed:-
• (i) The sample was heated in a scaled pyren gicac tube with
6cn3 of 611 conetont boiling' (redletillod)' hydrochloric acid for 24
.hours on a boiling wator.bath.. The hydrolysato was evaporated down
to drynees,'diocolvod in.a U.tle distilled water and ovanoratod to
• dryness again. This tiaO repeated three titoa to rmoo all the
hydrochloric acid. . The residue was finally dried and storod in a
• vacuum dossicator over sodium .hydrgxdo pellets until roqiirod.
•
(ii) The samolo wós dissolved in hydrochloric acid erntctly as'
above but in a pyrex hydrolysis tube. The contents were froson using
liquid nitrjon and the tubO then ovact',atcd (water aspirator) for
• &boit ten.minutos prior to sealing. ihe. tube uau than allowed to
.thw o't before being placed in a thermostatically controlled oven
• .4 •, ,.
53
at 10500 for 24 hours. After this the tube and contents were
cooled, again frozen with liquid nitrogen and the tube opened.
The procedure for removing hydrOchloric acid as described above
was then followed except, that doioziiced tratorwao used instead of
distilled. water as thie method of hydrolysis was. uced on samples
being prepared for amino acid analysis which requires the use of
deioniood water throug)out. Three wa3hinga were uuolly
sufficient and final drying was again in a vaouun dessloator.
(1) .an.orations ..
Evaporations were carried out under reduced pressure using a
]3uchi "flotavapor rotary 'vacuum evaporator in conjunction with a.
vater bath and water espirator. As a tomporature of greater than
about 4000 could cause denaturation, samples which would not
eaporate to ryriess below this temperature Or were in a solvent
difficult to remove tiërö evaporated ons Jones and Stevens type
VU vacuum evaporator used in conjunctiàn with an oil pump producing
a greater vacurn than the water apirator.
Arino Acid Analyis
Saml5les to ho analysed for amino acid content were first
weighed accurately using an electrobalanco (Cahn Instrument Co..
'ararnount, California,U,$.t,), About two ailligraris were
sufficient for a complete analysis and this amount was first
hydrolysed as deacribedabove. Amino acid analysis of tha
bydrolysed samle was then carried out using a Techaicon Automatic
Amino Acid Analyser. The method of analysis used was that of
3paciman et al (1953) as modified by Benson and Patterson (1965).
The columns used woro of Arborlito C. G. 120 (type 3) fractionated
5 4
by the nethod of Hamilton (1958). Laro columns (60cm. x 0.636cm.)
were used for fractionation. of the acidic and neitral amino acids.
and short columns (16cm. x 0.45cm.) for the fractionation of basic
amino acids. Sattples, after hydrolysis. in a sealed pyrex glass
tube as described (pages 52-53)wée dried overnight in a vactum
dessicator and then dis3olved in the appropriate amount of . pH 2,2
buffer solution (0.0667M with respect to sodium citrate) containing
the internal standorde norleucino N0L) and L-.t-amino--guariido_
• propionic acid .brdroch1oride (AGPA), both at a concentration of
O.l)di per 0.5cm3 of buffer solution. 0.2c63 samples hero then
loaded on to each column in turn. The peek area for each amino acid
was calculated by hare neasurement from the visible absorption
trace an the recorder chart, sing the standard equivalent values
• for each acid and the accurate starting teiht of sample the amount
of each amino aôid in the sample could then be calculated. 4
55
(2) Culture. of PorhyTidium.Cruefltuin
Porphyridium crcentwu, a unicel1lar red alga, is a primitive
momberof the. Rhodophyta, order Thxgiales (see Table 1) and was
first describod by Naegeli in 1849. It crows on soil and daitp
walls and has been reported from the sea. Attempts to culture it
on solid media met with no success but in 1949 Pringsbelm and
Pringsheim obtained relatively good growth on solid or in liquid
mEdia iade with natural sea-water which suggested that the alga.
might have a rnarine origin. This"was later confirmed when it was
Isolated from marine envirozuneñte (e.g., 'Starr, 1960; Allen, 1960;
Reth, 1961).
The growth, media moôt1y used included natural sea-water and
eoii extracts (Pringahoim' and Pringheiri) until Brqdy and Emerson
(1959) grew the alga in an artificial inorgthio medium. This did
not allow large scale culture but 4evelOprnent of an artificial
sea-water medium (A.s.w.) by. Jone3 Speer an&iury (1963) overcame thie diffinulty. Thisi 'mOdium o with alight modifications, was used
to grow Porphyridium cruentum in this laboratory. The oot
'composition t the medium 'was as follows:-
27 gms litre-I IaCl
6.6 " '
5.6 1 flgCl2.6H20
1u5 . CaCl2 .6E20 ,
1.0 " i0
0.07
0o04 ' !SRCO3
The solution was buffered with 20cm 3 per litre of 1I ris
i.e., ti13 c(cn2 oB)3)-}cl
Om-
• bi.ffor colution, pH 7.6 and also added tias lcm per litre of a
• chelated io,i solution (0o24ma PeC13 14120 por 100c6 of '0.05M
ethylene ditaininototraacetic acid, E.D.T.A., pH 7.6) and lcth3 per
litre of a trace metal solution ctaining the followina elements:-
.0,04 &po litre-i ,ZnCi 2 0.04 ms litre-.l 0412 .4H20
060 H3B0 3 0.40 " rnCl2.4H20
0001 " I' CoC1 2 ,6E20 . 0.37 " (04 ) 6 o7024 .41120
The trio bufffr and chelated iron oolutiori were aiwayc made up fresh
'tihilet the trace metal aolution was icopt in a rofriorator.
Analar 3rade chemicals tero used througIout. Finally, a onall
amount of Vitamin -.12.aoiuticn (O,Oio per litre) tras added to
the medium as come earlier work in this laboratory had shown that the
addition of vitamin colutiono In trace amounts could 'increaso the
initial grotith rate and yield (Paterson, 1967).
Stock cultures of the aIa were iept in 11yrezscrew.topped
• teet-tubos contàinitg the above medium plus 1 to 1.5 Difco Bactogar
• or in etoppored flasko containjng medium alone if a new oulture. waS
to be started within .abut six woeko. Culture was carried out in
Pyrex conical filter flasks ('litre) or in conical stopperod f1aok
(1 litre). although the latter had the d±sadvantago that It was not
posøiblo to agitate the xiedium by circulating air through it.,
Large scale culture could be achieved uSing 15 lit'e aspirators.
An improvement in growth rate wac obtained by bubbling through the
medium an atmosphere of air containing 5, 1) carbon dioxide instead of
air alone. ,
Onco, made up the culture xñediuc uan ctet'iliood by autoolavirg at
a steam preósure of 151bs/aqp inch for about fitteen inuto3.
Innooulation wab then, carried out under aeceptic cOnditionb oithor
57
by removing material from the agar slides with a tzire loop, flamed
to sterilioe it (as were the mouths of the culture vessels), o.
from a stock vessel using i.eteri1ioéd pipette. The innoculated
• culture vesselo wore placed on a white surface and continuously
• illuminated from above using a Philips "Cool—white" fltorescont
lamp (11.C.FoE. COw/33). The temperature of the culture room was
mointainod at 220C by moans of a theriostatically controlled heater.
The current of air and carbon dioxideS , provided aufficiont agitation
• for the larger vessels whilst the Smaller, otopperod vessels were
frequently shaken by hand to proiote air circulation. 4fter about
four to cix weeks gtoVth was complete and the alga ready for
harvesting. 1f left much beyond this time coil lyoié tended to
occur with consequent leathing out of the bliproteins into the
surrounding medium, • • • • •
(3) rvetiw of the Algae; tractjon and Purification of the
Biliprotoin
The dense red mass of cultured alCae tao oc±aped OfT the aides
of the oulturo vessels which were thon wached out with a solution
containing 276 glue, per litre of sodium chloride. The mass plua
washings ups, then centrifuged at 1000, for thirty ,inutes at 0.50C.
The supornatant was discarded and the centrifugato, suspended in a
iiinimum of oupornatant, was cubjectod to ultrasonic disintegration
for about fifteen ainutea q being kept cool by a surrouziding ice-salt
xizturo. The treated suspension could then be seen to, bo
ulüorosoent which indicatod that the desired cell rupture had oOcurrod
with roloaso Qf biliproteins into the solution. This Ouspension van
then frozen solid, left overnight and then thawed out at room
temperature to pror.otp further release of biliprotoin from the
ruptured cells.
The crude biliprotOin solution UaQ then centrifuged at 2 000g for
ttienty ninutes at 0-50C. If the residue centrifuged down was still
red it was subjected to further sonication end the procose ropeatod
until only green cellular mator (algal reraine, chlorophyll, etc.)
remained and this was then discarded. The fluorescent zupornetent
was then filtered undr pressure through a pad of wauhèd co].ito to
remove most of tho remaining cellular fraonts. Spoctrophotozotric
• anal3reiO at this stage showed that some impurities were still prosont,
notably chlorohyll (oe figure a).
The not stago was to precipitate the bilipro ltein, out of solution.
'2o do this solid emwonithn cuiphato (finely ground) ties olowly added
to the stirred and cooled golutjon, alao covered to ezelude oceee
light, until 30, weight by volume (w/v) had been added. The
59
solution was then loft in a refrigerator overnight and if
p$cipitation had not occurred a further 5 of amnonium. sulphate
was added, The resultant suspension of precipitate was then
oentrthged at 1600g for twenty minutes and the supornatant discarded
unless it wae strongly coloured in which case further ammonium
sulphate was added to complete precipitation. A sample of the
precipitate when dissolved in dIstilled water now showed little
absorption due to impurities such a chlorophyll but still had
absorption due to other biliproteins (soe figure 8). The
opeot,ropbotometrio criterion, for purity (ratio of the peQk at 565nm
to that at 230nm 'for B-pbycoeythrin) at 'this 'stage averaged only
about 1.5 to 2 which meant that further purification was necessary,
a ratio of 4 or more indicating good: 'biliprotoin homogeneity.
ricalcium Phosphate Absorption Chrothatoraphy
Pricalcium pho6phate obluma chromatography provided a noane for
further purification, The 'method developed by S4nglo and Tis3liva
• (1951) and applied by liaxo et al:(1955)' was used. The t4calciuá
pliosphtato aboorba protein and celite is added as a support and to
improve the flow rate, the ratio of tricalcium phosphate to celito
being 1 5.
A slurry of the rixture in vzator was poured into a water filled
column and a1ioed to settle under gravity to give a column length
of 20cm. (by, 6cm.). This was suspended on 'top of a pad of glass'
• Wool covered by 4-5on. of washed celite and with about the same
depth of celite on top. This, latter celite protected the surface
of the column and formed a much firmer surface for loading as uoll
as extracting any residual colloidal matter such as chiorophylland
denatured protein, it also had the advantage that it could be stirred
60
or replaced tzithotat affecting the separation wh?re otherwise, the
top of the column proper would have been clogged end the flow rtho
accordingly deorenoed, The column tac then eqilibratod tith
040025 r coium phosphate buffer solution, pH 6 6., made 12 with
respect to sodium chloride (v/v) 1 about 2 litres of solution being
required.
The protein solution, on average about 250om3 in volume at this
stage, was centrifuged at iGOOg for twenty minutes and tio
• precipitate then dissolved 'in the minimum of water, This was then
dialysod against running tap water for about tive hours followed
by several lArge. volume chonges of dietilledwater and finally by
• changes of the buffer solution. It wis thin applied to the column
• and' the protein eluted with increasingly concentrated solutions of
• buffer as .tabulàted.below. Tho colour of the elant at bach buffer
concentration is also noted'-
200 - 250cm3 of 0.011 buffer solution Cólpurlesa
320-400cm3 Of 0.02513'. ' Colourless to palo pink
• 340 - 400cm3 of 0io5p Palo pink to strong pink
350 - 400om of 0.075 Strong pink
400cn3 of 0.101) " Strong pink to pale pink
• 8000m3 of 0.'20I Pale pink; colourlo8s; blue
All buffer solutions were made 1, (w/v) •w.r.t.. sodium chloride, since
thin salt promotes absorption of the phycoerythrin to the. column.
• The column could bo regenerated, by washing residual protein off with
0,2513 buffer solution followed by several column volume8 of 0.0025n
buffer solution. ,
The pink fraction collooted now had a optical density ratiO:.
of around 4 on average. Its visible and U.V. absorption spectra were
61
consistent uith B-phycoerythrin havins 2 maxima and siou1dorin
the visible roion and three mia in the U.V. region. 4 sa1l
ziitnum at 60n 1as docreased co!npered to its intonsity before
application to the column but still .indioated that some colourless
a1lo-hycocyanin use prosont. The bluo fraction collected had
absorption spectra consiotent with C-phycocyaniii.. Those spectra
arc ahoim in fiaure 8.
The purfiod B.phycoerythrin thuo obtained was then prooiptated
t3ith amoniun sulphate (3o tilv) and atorod in a iofrigorator. t'Then
required for Ozporiontation a casplo tyaq contrifued, the precipitate
dissolved in water or the appropriate buffer solution and dialysod
aainot several changes of the oae until frao.from sulphate.
Occasionally aumoniuvi eulphatO precipitato could be used directly,
howoVer,
Extraction and purification as dosoribed based on tricalciurn
phosphate colurn chroinatoraphy was carried out for all the alao
crown OxCfpt on one occasion shoi tuo óthorentraOtior procedures
were used in addition to check if different ciothoda of extraction
had any afoct on the properties of the protein (see section (5) ).
traotion uoiM n-butanol (Pujimox'i and Pocci 167b)
The mass of algae .us collected., centrifuged, sOnicated, frozen
and thwod oao.tly as before. The euspension after thdAna tas
centrifuged at 300 000g for forty..five minutes to rciove the bulk of
the insoluble material instead of filtorinj. The supornatant was
then centrifuged at 100 0000 for one hour at 05 0 C tâ, romovo the
rcti. The final oupernatant was intimately sited with an equal
volume of n-butcrnol and tho resultant omulsion centrifuged at 10 000g
for thirty minutes. This resulted in two l±quid phases boing
62
• produced - a yellow upper layer and a lover red layer with an
insoluble gelatinous blue layer of p}ycocyanin at the interface
between thoso. The her red lcyer u'as carefully reaoved, made
01 saturated with eoñiui. sulphate (w/v) and then 'oentriuged at
30.000 for thirty ninutea, .. This resulted in the formation of a
copaot lyor of crude phycoerythrin on top of the tubes which was
tr3nsferred to the tniniriurn 'aitount of 0,11 sodtur phosphate buffor
solution, pH 740, required to dissolve it and dielysed against
coveral large voluo ohange3 of the se buffer solution, The
colution was then oöntrifued at 10 000g for fifteen minutes to
rexove any insoluble material. Phe aupernatant was made 35 H
satiated with amonlut sulphate, this time-using a saturated
eolutjon'of.tlie latter instead of solid, and after loming in a
refrigerator oveht, centrifuged at 30 000g for thirty minutes.
The red precipitate of B-phyooerythrin was th6ri stored ready for
use Mer saturated aemoniim sulphAte solutjoz and could be dialyeed
as required.
ztrotipn ustn SteWise Pirecipitation with Ammonium $ulphatp (Swingle
and Ticelius, 1951). .. • . .' •
The Standard extraction procedure deacrbed in. the first
subsection was followed up to and includixg filtration through the
óohito pad. Thoi, instead of applring the protein solution to an
absorption chromatography Co1mn, it was preciptatod with 303
amonium aulphate.using a saturato4 Solution of the latter, After
overnight refrigeration the preàip1tato wü. centrifuged, at 10 000g
for twenty minutes and then cUssolved in the rinimua of 0.11,1 sodium
phosphate buffor solution, pR 7.0, and dialysOd against oevoral
large vo1uno changes of the same for twenty-four hoursi The
WIII
suspension of precipitate was thon centrifuged. (10 00 .0a.for fiftea
minutes) end any ro±aining blue precipitate discarded. The
oupornatant us roprocipitated tith 205 waonium sulphate,
refrigeratod overnight and centrifuged as before, any proáipitate
disoei'dod and tho oupornatent brought to 30 asconium suiphato and
loft. This was thou contrifuge4 again and any blue precipitate
discarded, The remainiai.soiution was further dialysed, centrifuged,
blue precipitate discarded and the supérnatont brought to 15
enconium sulphate and loft overnight. One more eotrifugation
o11owcd with any blue. precipitate being discardod and the final
oupornatant was brought to 20 ainmoniurn sulphate. The prOtein
was at this stagO fairly h.tghly purified and availablo for
precipitation, atoring and use in the uaual canner.
The -sphycoorythrin produced by theso latter two eztractign
procedures was found to be virtiially spectrally identical to that
produced by purification using the tricalciun phosphate column
chromatography, although not quite so püro (both had a slightly
louer optical density ratio),. Theso could Oasily be irnproved by
further precipitation and dialysis, hotiover,
(4) I4enjfcation of , tho Ntorminaijgno Acid of BphycoOrthrin
To try and Idontify the I-tornina1 amino acid(s) of the
biliprotoin it use necessary topreparca deriativo of the sao,
hydrolyco this froo from the remainder of the protein chain and
identify it by comparison with otndard derivatives. For this
purpose the dinitrophonyl dorivativo was selected because it is
relaivo1y easy to identify by paper chrocatography. The method
°: preparation uas beod on that of Sangor (1945) with modifications
as introduced by O'Carra (1965) and the ozact experimental details
folloi:-
• (a) Preparation of the dinitrophenyl derivative
Tho reagent used was dinitrofluorobensene, the course of
• reaction being as follows (tising alanyl glycine' as an ezainpie):
WO M3CCI4CO$NCII3CODH
f .p. IC CM CO t*%CI4aCOOI4 t03 ) NM 411g 014
INCt1RO
• •,.wc.6. A(, 1
'0a. 4 Q-7 t414C14(OO.44 N4 C142 (0014 Nos ø(siiç ,
Some B-phycoerythrin was centrifuged and the ,ammonium sulphate
precipitate dissolved in the minimum amount of distilld water and
then dialysed., first against running tap water for twOlvo hours and
then against several 1arge volume changes of' distilled water.. To
• om3 of the final prOtein solution were uded 60 tnillirams of eolid
sodium bicarbonate followed by solid sodium carbonate until the pH
was 7.8 810 (fo11oied by use of a pri neter). Then 100 milligrams
• of dinitrofluorobénsone in 6c 3m of ethanol were added, the mixture
• stoppered and sbakefl, for three. hours at room temperature. After
this the mixture was acidified with 611 hydrochloric acid until, the
•
pH fell below 3.0, (shown using universal indicator paper). The
suspension was centrifuged and the yellow precipitate washo4 three
• timee with acetone (about 10cm3 pOrtions)'and .thenthree times with
• , ether .(aiso 10cm3 portiOns) until the washings were cOlourless in
order to revove any excess dnitrofluoróbenzene. The aqueous layer
was then, hydrolysed with 6ci 3 of 61) conStant boiling (redistifled)
hydrochloric acid ma sealed pyrex tube for twelve hours on a boiling
• water bath.' After cooling the tube was carefully opened,' the
65
hydrolyoate.dilited with 5 volumes of distilled water and then
eztracted fo.r titios with other (15J portions) shaken uith a fresh.
solution of ferrous sulphate to romovO any .peroxidos 'hioh could
cause oxidation. The coibinod other layers tiers thon evaporated dom
to .bóut lCn" in volume.. ,
(b) De.ein of Lh.0 4e.vativoj4
Paper chroato'raph3r tias uod to dentfy the derivative -
Uhatan nucbr. 1 paper (20" x 6") and the solvent syaorn t-criy1
alcohol saturated uith phthalate buffer solution (22.45 gns
otaastua hydrogen phthalato + 4 gins. solid aod.iumhydrozide dissolved
in distilled water and .ade up to 220c 3 with water) chosen. The
paper was inipregnated with buffer by drawing it through some of the
stock ooution diluted with water 1:9; the paper was thom allowed
to dry before being opottod, 4 little of the other extract was then
opottOd on the paper t2sing a capillary tbo and the c1roatogram run
• with the o1vent for ten to eighteen hours as necessary. After
dring in on air oven two yellow spots were seen one due to. the
D..P * onino acid and the other presumed due to. the suiphone of the
s&io produced by sOmb oxidation during hy4ro1yei (o tCarra and
O'hEocha, 1963). The spots showed up oven bettor under U.V. light.
PreVious workers had always identified aothiónine as the only
Io.torminal arnino acid oftho phycoerythrin2 (see pages 28-29).
Accordingly, the D$.Po dorivativo of tothioniao was prepared as
follows; to compare it with the unknoWn derivative.roduced. Sanger's
othod. (1945) was agaiii used wtth modifications due to Schroeder and
LoCetto (1953: 14.9 mil)igroms'(1O0pmolos) of Mothioniiie were 7 , -
discolved in 1.100en of distilled tiater.'. To 1cm of this solution
66
wore added 20zgms. solid sodiuni bicarbonate and then 0402cm 3 of
dinitrofiuobonzene dissOlved in 2cm 3 of ethanol. The mixture was
stoppered and eheken for threo hours at room tenporat,ure and then
diluted with 10m3 of distilled water. This was then extracted
with nevoral volumes of. etkier (four of 25cm3 and five of 10cm3 ). and
the cbbined ether extracts washed with water (three lots of
aci1fied with 2 drops.of 61hy4rOchloric acid and evaporeteddown
to 1-2cn.. A littlO of this solution was used to spot a oromatogran
as before. Rthr a lot of etreaIdn was observed on the paper and
to try and eliminate this Uhatman numbor 4 paper c used instead of
number I. this resulted in soe I improv6nent but streaking ww still
considerable and persisted even when much lose of the extract was
spotted on to the paper. It,seoed that thiomight be due to some
dinitrophenol produced and to try and overcoi2o this the preparation
was repeated except that "this tiie .thyl acetate (sane quantities) was
used for extrnction of the produOt instead of ether. This was
successful, almost all of the àtreaidng being elirninated although as
an extraction 'procedure ethyl acetate was not quite as efficient as
Othor, quite a lot of product remaining in the aqueous layer, For
mothionino some' suiphone was detected in addition as happened with
the unknown.
Samples of'the biliprotein derivative extract and the staMárd
extract were then spotted on to a chroatogram exactly in line with
each othor Ond the chroátograa developed. The resultant 'spots were
found to have travelled exactly the samo distance down the paper4
Also a mixture of the two extracts was loaded and run and this
appeared as one spot, .o., there was no tendency for it to'oplit
into two spots as would have been the case with differing dorivativOs.
67
any other solvent oy8toI1s could have boon trie& : to prqvie
booluto1y conolucivo proof (OCarra 1965; O'Carra and O'hEooha,
1965) but the ovidonce obtained., wbich ,us in cop1eto ogreemont
with that found by other torkoro, indicated vory otrongly that
otbionino was the 1—tormina1 amino aoi (and. the only one) of
-'phyOorythrin oztroctod. from Porphyridium cruont and
eoneidorod oufficiont proof. Accordingly the sootion was torinatod.
68 :.
(5) Djsoociatioi of Bpcoorythriu in Agaeous Solutipn
• Thrther purification of the bilirotein from other biliprotoin3
could bo achieved by coléculàr oiove chroaatorapby using a large
scalO cólwnn an 1,K.B. (Stockholm, Svteden) "Recychroiu' recycling
colwn, 60orn4cmo, was used for this purposo although the
recycling was not found to be necessary. Tho column uaa. packed
with Sephadox G.lOO, Which had boOn swollen in watOz' with constant
• stirrin; for twenty-four hours o doasrated by water pump auOtion and
then equilibrated with several column volumes of O.Olfl sodium
phoepliato buffer solution, (p1! 60 - 6.5 also d eratOd) with the
perintaltic pwp adjusted to give, a floU rate of atound 30om 3 per
Some protein was.centrifugo& and the ammoniuin suiphato
precipitate disolved in a miniaum of tho phosphate buffer solution
and dialysed against several large vlumo changes of the came. 1ny
remaining sulphate was removed by the desalting effect of the gOle
About 12cm 3 of this protein solution were thon loaded on to tho
coluz3n with the pump and olutod upwards with buffer solution. The
column was cooled by a water jacket and screened from the light to.
• yiininioo risk of donatiration. The reSult observed was a
separation with a concentrated pink band being elutocl first followed
by a blue bond with come pink closely elliod to it and finally by
another, more diluto, pin!. band. The two distinct pink bonds were
collected separately; web not found poosible to separate the
middle pink band from the cOntaminating blue fraction.
• These two banda were then, compared spootropliotornotrically with
each other and with a coriplo of the ntive phycoerythrin (iso,
pre-colunin). The firat (or larger sized) fraCtion had a visible
spectrum identical to that of native phycoerythrin with two peaks
69
and a èhoulder. with the peak at 620-625nxn due to contaminating
allo-phycocyanin diinihed in intensity as epeotedftethis
• purification process. The socond (or lièiiter) fraction,. however,
• had only One mtximun ii tho visible region, at 5454m, withy the peak
at 5654m and the shoulder at 500im:botb absent. Thoso are shoWn
in figure 9. I
Further investigation of this system was then carried out in
two way5
(a) by precipitation and (b) by use of an analytical nolec1ar
sieve oromatography coluimi. •• •
The light fraction was precipitated with W45 ome'onium
- sulphate (v/v) and then left at' to for several days. After this it was centrifuged, the' precipitate Iiseolved in. a minimum of
distillód Water and 4ialyed against Several changes of the same.
The visible spectrum was then scanned end it was found that there
was at least partial restoration of the 565n long wavelength peak
although there was no reappearance of the ehou)der at 500nin,
characteristic of the native biliprotein. The' indications Were,
however that some reagregation of this fraoton, had taken place
in the precipitated state,
An analyttoal rioleculur sieve ohromatogrphy column was
then used to try and establish the approximate molecular weights of
the species produced and also to check on this observod dissociation-
association.
A column, ?Ocmz'i,.25cm., of $ephadet G-lOO (doaeroted as before)
was packed and equilibrated with O.fl sodium phosphate buffer
• solution, pR 7,0. and the packing of the column checked by pasing
through a sample of blue dextran. The protein samples were
(b) Second (or 'light') fraction
isolated from the Sephadex G-100
column.
(c) Visible absorption of the
'light', fraction after treatment
to test for reassociation.
FIGURE 9. DISSOCIATION OF B-PHYCOERYTHRIN IN AQUEOUS SOLUTION
VISIBLE ABSORPTION SPECTRA OF THE SUBUNITS SEPARATED.
(a) First,(or 'heavy') fration,
isolated from the Sephadex G-lOO
co]umn.
.1.50 •'. UIiive.t. (J
fse
70
cioao1vd in this buffer solution and about 0.2cra3 lo$ed on o the
top of the, oolun by layering uith an Ala syringe. A little
cucroso ties added to each saiple, to inoroaso is density for easior
loading in this tiay. The eaploo tiers then eluted dotjntiarda using
tho smrro buffer solution at a floi 'rate of around 12 cm per hOur s
controlled by a periataltc ptnp. The oluant uno continuously
couitorod.at 253nn using a,'lox p1otoaotor and 0.5cii3 fractions
colloctOd using an optical drop counter and an L,K,B. fraction
colloctor, The visible abaoption of each eolicctod awplo could
then be chocked if decized using any of the otqndrnd viaiblo
aboorption aDogtrophotovotere t4th 2on path length micro cello for
ate aensitivity, All of this 'vorliz ias crriod out at room
tórnporaturo t4th the column shIelded from the light.
For quantitative tiork the colutn tree calibrated using the
o*reaoion To : To against lo fl .1t • To tx'as fowc'. using blue
dQtran, large enough to be totally oitcluded from the gel, and tho
protqino bovine sOrun albuminp ovelburiin and .trypsin inhibitor, all
of troll cubotntiAtcd colecuier usight, uGed for calibration.
PhyQooàihrin oanplootroro then applied to tho colurn and molecular
ueibto could be otimatod from tho calibration plot (oeo figure 10).
In practice, instead of the actual olution volumes being calculated
the ratio ta1en tree that 'of tubs nubero at the ilna as ehotm on
the 1,1.V., trace - the Uvioor4 coaitor tias connected to a Servoscribe
recorder tthiçh also had zarked on it each change of tube on the
fraction ,coljector trhich meant that W maiumn on the U,V. absorption
traco corresponded to' a certain tube nuabor for the banplo eluont.
Per this to,bO accurate all loadQd oanploo had to be approaiznately
the same volume and the recorder otarted at 'oactiy the sane point
• F%G'ucw too R%SOCITIW
Ce'co PLOI çot S%2tX G- Oo CO
• • • • •5o - -• •
Lcs Lcutv we
• 1 S
for each sample, iThiôh was irriciately on com1etioii of 1ac1iig,,
As well as a sample of native phyooerythrin sapplea of the two
fractions colleotea from the floeyohrom colunn were apnlied to the
analytical Oolunn and their nolocular weihto checked. Theso
roeulto and the calibration figures are given in the follotiing tablo
as u1l. sa being diagramctica1ly 1.1luotrated in figure 100
arp1 Q, O3Vo iprox.I.Ut. .6 i1ue dextran Vo 10
oino sorun albumin Vo (calibrating) 60 000
Ovalbuinin 11o. 13 . 45 600.
Trypsin Inhibitor. .• Ve 24 000
tatiVoDu.pbycoerythrin . 1.01 . 65 000
.1,66 36.000
1099. . 23 00.
Larger eearatod fraction aD native as native
Smaller " .. " 109 23 000
As nontioried the first and last of thO three fraction .
seen on the Recychroo colum oculd be isolated but not the middle
one. This fraction also showed' iap on the analytical column but
in too small an anount to be iaolatablô, To try end isolate ft
therefore, another, in botweon-aized coluDn 'tree tried. . 20cm x 2.5ori
of Scphadot G-100 packed as boforo. Sevoral attempts to isolate
all threo fraetjp, from this column were rado including running
Qa1n1o3 under gracrity instead of by pumping (i.e., at varying flow
rates) and alQo at various concentrationo (the Post conCentrated S
eamplo being obtained, by use of. .a pressure dialysis membrane filter -
8artor1u embranfiltar, V.A. Howe A Cc., London) but all wero
unsuccessful. It appeared that the dimOr, as' it seemed to be, was . S
72
probably in equilibrium with the ionoier with tho equilibrium
favouring the latter which meant that the diser could not be
isolated in the sso way as the othor two subunits,
All this uorc indicated the preseco of an asoociatin-dicsooiatin
ayoes and this was pursued further at a lator date. (see soction (s) ).
The rocults at this otago were uritten up in the form of a short
oonunication with additional similar results for R-pbycoorythrin
from Coramium rubrum (Brodie, 1966; Thxtehins, 1967; .Ja1l, 1965-67)
incorporatod and a copy of this is apponded
One other point arose from this work uhich was that splos
of I3-pbycoorythiin purified by the n-butanol and fractional
precipitation cothods (coo sections (3)(b) and (3)(c) ) viorooxsminod
as well as protein purified by the tricalciura phophaté Ooltann
chromatorapbj cothod ancI.11 aowod identical behaviour in aquoous
solution with roget'd to spectral differences and the some subunits
being separable on analytical colimno of Sepbado 0-100, This
neemed to rule out thO theoi"y that difforont mothods of purification
could result in changes in the propertioo of the biliprotein,
tricalcium phoophato column chromatography in particular having boon
ouspocted of this. (Scott and Berns, 1965).
73
(6) Dippociation Sica tI513g 1ez'curia1 Coipnde
The use of mercurial oompouzc1s as cu1b!iydry1 blookirij
roajento to bring about the dieociation of c3ome of tho biliprotoinE,
has already, been santionod (ace pw3eQ 18-19) and iork along thoe
lineQ hae been reported by jopos and Pujiiori (1961), Fujinori and Qulnion,
(1963), FlAjilnoro (164) 9 Fujimori a* n.4 Pocci (1966) using the cerourisi
oorinyunds pa a-chloromorcuribonsoato and paranieurip1ienylsu1phonic
acid on C-phycocyanin and. R-phycoerythria. . It ias decidod to, try
the offect of thoo compoid.a on B-pby000rythrin from Porphyridium
eruentum and the rosulte aro deacribedg-.
(a) ichioromorcuriboncot().
ias obsiried in cryotallino form from B.D.R. (Poole,
England).
B-phycoerytbrin samples uoro prepared by. centrifuging samo
protein at 1500g for twenty minutos and then discolving the
precipitatoirict minimum of O.]r oodiun pioophate buffoie solution,
pL7.10, to give as concentrated a ao1utionof protein as possible.
This solution tao than dialysed gctinst several changos of the same
bui'for solution uith stirring at OC and shielded from the light,
until froo from oulphato.
Lts a trial ezporimOnt en arbitrary snail amount of F,C.Fj.2.
was Qd4o& to about 3cm 3 of ouch dialysed protoin solution which was
stored in a otoppered silica glass 1cii, coil and tho vioiblo spectrum
of tho solution scanned at reg1ar intervals for cevoral days to see
if any changoc tooln place. Buffer solutiop containing F.0 0 11,13, at
the smo concentration was used as tho reference solution in the
back cOil. ThO results obeorved wero a gradual d.iOappoaranco of
fluorescence with a corresponding doceaao in the overall intensity
74
of 'rnib1e abeorption and eliiination of the lonavoléngth
(565nr) peak (coe figure ii). This indicated that dissociction
of oomo ttith tias taking place and that it was a fairly slOe process.
The axperiment was then repeated in a slightly more quentitativo
fanhion i.e. using a more accur4te concOntration of P.CflaB,
Pujimori and Quinlan had fOtnici a final P e C,P1.B4 conoontratioa of
13610 6411 to be quite catiofactory and this was tho conceutrttion
thereoro chosen. The required eount of ?.c.N.B. (0.485s,4) to
give a oOnoontratiom of 1.36 .10731.1 was dissoltrod in freshly ;ado up
sodiun hydrocide solution (o.in). lOJ of this solution could then
be diluted to 100cm? with 0.12 codiun phosphate buffer solution,
pU 7.0, against which the protein oanplo had already been dielysed.
It use found that protein solutions troato4 with P,C.Ff.B. in this
way docolourisod very quickly and precipitation soon took place which
indicated that either tho , concentration of P.C.Pt,B * was too high
(unhjIco17) or that the p11 of the final solution was too near the
isooloctric point Or too alkaline (see page 81, thus e&uoing
precipitation - dissolved minly in sodiun hydroidO solution
only slightly diluted with phosphate buffer solution would obviously
differ in p11 from the protein solution. To txy and overcono this
the oolution was sado up in a slightly difforont way - a 0.010
solution of ?.c.tI.B. was pado uo in fresh 0.l sodiun hy&rotide
colution and this was then diluted with phoQphato buffer solution to
finnI P.C.M.B. ooncsntratiori of 136.10' 3110.10a3 of this
solution was then added to iocm of protein solution dielysod against
the buffer solution and this gave a final P,C.I1JB, concentration of
1.36.10 as dooiroa. Oaking up the protein solution + P q CsFI4B1
in this way tiasuccCssful in stopping the precipitation. The
FiU( fl u . oc 1cutu.
MCP(UC IOf OB 0-
tw fOT(G' 6CUZ)
I
I, ••
I' • •:
•• ji I'
: 4• ! , I I
I
do ••
soo Sm • ()
• •• • .• ••
.•
•
(b) @-
P. tvewlitzy 704am (
• •• - 19
75
treated phycoerythrin sample was then loft in a dQrk room at room
temperature for some days at the end of tihch a constant visible
opeotrum with the 565nm peak aboent showed that the de3ired
dissoCiation had taken place!
To try an4 separate any subunits próucod by this tietxort with
P.c.r.D# molecular stove chromatography wse agAirl, used. an
enal,ytipal oolumn.(2Ocn. x.l.25om) of desorated SephadexG-100 was V
made up and euilib,rated with O,1P sodium phoqpbAtq, buffer solution
• p11 7*0, containing PX.MB O , pt the estie conoe4trt1on as in the treated
eavple, i.e., 1.610t A O2or?, samplo of the treated. -pbycoerythrin
was than loaded on the surfco, of the gel column by layering with
V Oyrizx3q.,qs before, aucroae sgain V being added for easier loading, The
&mpie was eluted downwards with the bui,fer solution at a flow rate
of about 12cm3 per. hour, controlled by a peristaltic pump, and the V
V
eluant monitored by use of the Uvicord at 253w!I. 0 drop fractions of
eluent Vwere collected (0,9cm3).uoirg the fractionóolleotor and photocell
• drop counter as described in the met section. A good separation was
obtained after initial, difficulties and two aubunit8 collected intee& of V
this way1 V The first (or 1rger) of these was purple in colour
in fact turned out to be a suspension of proteinprecipitatein
solution whilst the aecond was a red coloured solution which had only V
one absorption mi in the visiblo region, at 545nrn.e The column
• was calibrated as described in the previous seotion using the same
standard proteins., aiid from the calibration plot the zolecular weights
of the two aubtnits were estimated as being 35000 and 36 300
V respectively.
V
V No attempt iao made to bring about roassociation Of thosb • V
subunits and in fact before any ftrthor work along these lines could
76
be carried out Fujimori.nd Pecci (196Th) publiohod ft11 reu1te
and observationa for phycoythrin from Porphyridium cruentun
unin p.e.n.w, The roulto obteinod i9i this 1aboratorr shotiod
ostiefaoto'y aroeient with thoao publiehod the formation of tto
separable btbunto, one of tihich uas purple end insoluble and thd
0 thor ahotiing opcctrl difforenes fron native pby000rthrin. In
addition there was the estiation of the nolcouler tjoihto of these
Oubun1t, however g tihich they had not celoulatod. Pi*ther uork
aimed at deciding tihothor or not one of these otbunito represented
the smallest possible subunit of the biliprotoin is described lutor.
(b) j ohlorophen1sulhenic aoid
Prior to becoming atiaro of the uork of, Fujimori ond PecciAt
was decided to try P.taCJ.5.A. to ooe'if it had MW difforing
-effect on the phycooxytbrin9 Pujimori and Pooci (1966) having used it
on phycocysnin from Anacystie niduleno with resultant separation of
trio subunits by to1ecu1ar sieve chromctography. -
As the compound is rather ozponivo. it tias cythesisod from
p7Aonykorourichlorido4 This involved suiphonation, vhich proved
relatively difficult to control. Oloup tiao tried first using im
oquivalont amount of the ocie at 600C for one hour; oceaa acid tiao
noutralisod by oddition of solid sodiiaincarbonato and then sin
volumes of ice cold 6aturatbd sodium chloride oolution tloro added
to precipItate out the sodium salt of the suiphonic acid. In fact,
although some of the latter may have boon present and non-dotoqtable,
the built of the product uas mercuric sulphate,, i.e., dbmercurisation
had t&ton place. Oloum .tiao therefore rather too vigorouo and the
proccos tias repeated using concentrated sulphuric acid in enceso at
77
100C for three to four houre. The eime rocult teas .bbtainad.
indicating the treatment atill to be too, vioroiw. The next atteit
iao to iwo lose conoontratod sulphuio acid at under 100 °C for one
hour s notralieo the product pith calcium carbonate instead' of sodium
carbonate and r6citth1liGo it from uater. The precipitate
diceolved in vater ancl tz'atod tith colid sodium oulpate end then
filtered to ioinovo insolublo Calcium cuiphato.. The residue tree
vapoatod doun until the sodium salt of the suiphonic acid
• depocitod. Thio highly doluble product iia then. carefully
recryetólliead t7ico from wititor, An infrared Opootruu indicated that
thio troationt trith tho .modifiod otaction and purification
proceihtre had boon successful trith p0duCtion of the sodium salt of
the oul,honio acid as desired.
A omiiplo of phycoorythrin tree propore& in tho usual tey by
dialysis of some arnonium suiphato precipitate and P.H.C.P.S,Ai addod
to the solution to give it a concentration of 10T. The rc&ult uao
for almoCt innediate prooipItaion to tCo place as happoned uhon
• too mueb PXJLD, tree used, a deep puplo precipitate boing forced.
This roont that no spectra could be taken, the precipitate being
insoluble in tiater. The concentration of P.fl.0,P,5.A. 17eo gradually,
rôducod until precipitation no 'longoa occurred and spectra o0ul bo
run, The observed cheea trero identical to those brought about by
troatrmont with P.C.Pis.B1 as farea spectral chthgoe troro concerned
but before any attccpt at soperatiQa of the oubunito by molecular
sieve obromatógraphy could be started the results obtained by
'Iujicori and Poeci trere received and thore ties therefore no point in
continuing thooe:oiperiments tritb the mercurial coupounde.
78
(c,) vercuric Ion
By this time it was clear that dissociation in aqueous solution
took place depending on the pH of the solution and also that some
dissociation could be brought about by treatment' Of the biliproteth
with the riercurial compounds mentIoned. ' It was thought that the
dissociation might be due to some interaction between the chromophore
group of the biliprotein and a matal ion, possibly the mercuric ion,:
and it was therefore decided to study the effects of merCuric ion
direct on the biliprotein In aqueous so].ution. 'For this purpose the
soluble mercuric salt tnrcurio acetate was chosen.
A series of experiments was carried out uettig different
concentrattous of mercuric ion, The 'samples of pb-coerythrin used
were native (undiaeoci4ted) and the first (or "heavy") fraction
obtained from the Reoyorom coiuin to try and 'déternine whether any
such interaction was talçing place. Fercuric acotate solution was
added to dia1yed samples until preàent at the dsered concentration
and the resultant solutions Iept in atOppered Silica glass ic. cells
at 0°C with the visible and fl.V. 'spectra being regularly scanned to.
see if any change was taking place. The saluó oonoentraton of
rni'cric acetate in water as in the protein solutiOn was used ,,
the reference solution in the back coil. The observed results
are suamarised In the following tables: ':
79 •
Unciesociated phycoeEXthrin Observed at
Concentration of Time after g&di Onn 545nm 500nm
2 hours peak peak shoulder
24 hours shoulder peck shoulder
48 hours shoulder peak shoulder
5.lOr 4 hours shoulder peak shoulder
94 hours shouer peak. - shoulder
• 2fr hours shoulder peak shoulder
24houra precipitation
Henvv fractio
• 1 hour pesk peak shoulder
24 hours no pock peak shoulder
2+ hours shoulder peak shoulder
24 hours precipitation ....,......
5 minutes peak peak shoulder
18 hours no peak peak shoulder
24 hour3 . preOip1tati6i............
In all oases (with both samples) the intensity of visible
absorption overall decreased with time (ooe figure ii) and the intensity
of absorption in the U.V. regibn correspondingly increased. All
solutions lost the characteristic fluorescence and became purple in
colour purple was also the colour of any precipitate formed. A
concentration of 5,10 9 Hg was about the most satisfactory and
succsaful as it brought about disooiation fairly quickly and did not
load to precipitation.
Attempts to separate the subunits using molecular sieve -
chromatography as before were made using an identical set—un to that
described in tho.provious section.. The oluting buffor solution
30
contained mercuric ion at the eamo concentration as in the treated.
cainpie. The onplos in each cace after troatmet with meronric
ion at 5.10tl both split into ttiroubunit5 hieh sboted up on
the Uvicord trace. Calibration of the colurin as boforo ahotiod
theco fractionc to Itave molecular teighto very close to th000 of the
fractions ceparated after treatment with Genorally it was
obQorved that treatment with mercuric ion produced reeultB V0Z7
• cimlar to those. brought about by the mercurial compound,o although
to a o1iht1y1oeor extent,.e.g., thoohouldor at 500nm.in the
viciblo region alye remainod. Incroacing the concentration of
morcurlo tori. cimply led to precipitation toking plco tiich meant
that thero was no advantao to be gained in ucing it over the
mercurial conpounde.
81
(7) Dissociation Studies U jg Guanidine Buffer Solution
As tentioned in the introduction (see pages 18-22) possible
methods of bopd breaking include the use of very.conoentrated tonic
buffer solutions, in particular urea and guanidine solutIOns.
Accordingly a series of experiments was embarked upon using six molar
guanidine solution in which samplee of Bpbycoerythrin were dissolved
and left; diSsociation was then tested for by spectroseopy
(changes in the visible and UV. abeoptton Bpèctra) and molecular
sieve chromatography (to try and separate, as well as estimate the
utolecular Weights of, any subunits formed by this treatment). The
main aim of this treatment Was to dissociate the pbycoerythrin as
much as possible., i.e., into the smallest possible subunit, the
molecular weight of which would be the inimal molecular weight of
the biliprotein. . Initially the. strong guanidino buffer solution
alone was tried and then bond breaking reagents employed in
conjunction with this solution (sections (b) and (C) .).
(a) Guan&djneBuffex, Solution plone
Some work had already been done in this laboratory (Brodie, 166;
Rutchin, 1967) using 613 guanidine and 3fl urea solutions on B... and
R-phycoe!7thrina. UnSatisfactory results had been obtained, however,
with only one sub nit usuallybetng obtained and that having a
relatively, high moleOular weight in. e&ch case, . well above the
minimal possible.inolecuiar weight expected (from aiiino acid analysis
figures). . However, a reason for this was sug€stod when Tanford
(1967) poiutod.out that the use of such strong buffer solutions as
media for bringing about the dissociation of proteins depended to
some extent on the pH of the solution uied. Be oxplained that
generally disuiphide and sulpiydry1 bonds are fairly reactive and
32
re1atie1y eaeily broken but in. aadition at alkaline. pH valuo3
there rerö' the po3Qibilitio$ of polymerloation (i.e. rocombination)
of eubunits formed and / or precipitation (seepage 74) both of
ubich tioré much boa in acid conditionce ror a rnaiuxn poraeneflt
oplitting effect, thereforoo an coid rcdium ohould a1isyo be employed
(althouh 0ei4ng above the isooboctrIc point 9 i.e., botuóen the
iosbectric point ed neutrality ohoiald be ideai)s The uork
referred to cbovo had all been oerricd out at alkalino pH values
end for this roacon it tas docidod to repeat some of it but uoin. an
acid mediui iutoac of im aika1inomodiun, •.
Thzouhout this t7ork UBiflJ gonidino solution a molecular sieve
• ohroatogrAphy analytical Column 20cm. n 1.56m9 uca.usbd of either.
Sophado C100 or 075. Samples were aluayo 0.5cm3 in volume tiith
•
euOrose added for eaoiór loading by 1arorin3 uith A syringoon
top of the gel; olution was dbi1ardt3 using a porioteltie pump
and the clwrnt tias monitored with the Uvicord at 253nm being collected
on the fraction collector uith the photocell drop counter in 20 drop
(0.550m3) fractions. Coiwrn.calibration was tiith blue 4extran
top the void voluno and the pro tois bovino sbrum albumin 9 ovalbumin
• end tryp3in inhibitor as standards, all dissolved in o.iri soium
phosphate buffer co1uton (smgme per. cm 3 ), pR 640 and oluted with
the eage., • . .
Per the first oxperiiient buffor solution use riado up by
dicolving solid guanid1num bydrochborido (supplied by
Poolo, Eng1am) in 0.Olfloodlun phosphate buffer solution to. give it
a concentration uith respect to guonidino of sin molar (354.24 gnio i-i)
and the final pB adjuetcdto 60,. Some Baphycoorythrin uae
centrifued and the anironium oulphate precipitate die1vd in
83
little of this bufor Oiution to give as concentrated a.
• solution es oseib1o; the sample was then left for twelve hours
in the dark and at 0 °C. Soiie of this was then loaded on and run
through the Sophaden 0-100 eolwnn as outlined abOve, although
elutod tiith phosphate buffer solution alone. Only one fraction
was observed in the elwmt and this had a molecular weight of
around 86 0000
• . The O4erimnnt was than rpoatod but this time the eluting
buffer collition contained guanidine instead of being phoophato
buffer solution .alOnO. An inmiodiate problorn then arose which was
• that the. vey concentratod guanidine buffer soltion tended to cause
excess swelling of the gol particles which rosultod in the column
becoming slowly blocked with a consequent diminishing of the flow
rate until it was doun to about lam, per hour which tras far too low
to be prqtico1. This was slightly improved by changing the
poriotciltic pump head Speed, lowering it, this reducing the back
pressure, but the flow rote was atill abnormally slow. 8ephade 0-75
was then tried instead, of 0-100 end this also helped to some extent
but still not enough. Tho diffioulty was finally resolved by
doasrating both the gel prior to packing the column nd all the
buffer solution to be passed through the column. after this a otea&y
flow rate, of about 12cm3 per hOur was obtained for the co]umn of
Sophadex 0-75. Sorno of the treated phycoerythrmn eamplO was then
tried using this ayst6m and it foufld that the esmo bain fraction
appeared at a volume corresponding to a inoloqular teight of about'
06 000 but this time was foUoied by a Very osall amount of substace
.haing soleoulor weight around' 21 000 i.e., as obered before in
aqueous solution (seoton (4) ),. but not tàling place to the sac
Ul QW
• extent. This was not therefore the desired dissociation into
minimuni molecular weight subunits.
The next stage was to repeat the experiment but this time at
a lower p1!.. accordingly the guanidinG buffer solution was made up
using 0 0 11 sodium ácotato. buffer solution instead of the phâsphate
buffer solution and a final: pR of 40 obtainOd. Tide Value was too
near the ilooleotric point of the pro tfin, however, and caused
precipitation of the phyooerythri.n,
A pH of 4.9 was then tried, again using the acetate buffer.
solution, Title tine a sample of pyooerythrin in the final' solution
was left for seVeral days to try and ensure that any dissociation.
would becomplote. ReaulteWers quito incoholusive g however, with
the main predominant peak again corresponding to a molecular weight
of around 86 000 0 although very email, broad peaks at lowor molecular
wóigbt èlution volumes were seen. 0
From all of this it was clear that varying the pH of the
guanidine solution was not going to bring about the desired total
dissociation although indications Were that it might be taking place
to a very small extent, It was therefore concluded that more than
buffer solution alone was rciuired to bring about complete dissociation
and the obvious step was to add a disulphide boztd breaking reagent'
(see paces 18-22). A reductive cleaving reagent was chosen and this
is described in the next. section. In addition, the sensitivity 'of
the U.V. monitoring system was iMP roved at this time by introducing
a logarithmic amplifier into the circuit to booat the signal reaching
the potentiometric recorder. This resulted in a considerable
improvement in the senthitivity of the recrder and it became. possible
to detect much smaller amounts of protein or any U.V. absorbing
05
atcrial in the eluant from the column than was the case before.
Finally it is of interoetto note that since this work doubts
have been cast by Bezkorovainy at al (1968) on the efficiency or H
effeót1veneas of strong gusnidino solutions a, providing a inediux
for oucoesaful dissociation of the type aimed for here - the
foregoing results tend to indicate the name.
(b) oianicuneSolution with J3-meroartoethan! The thiol -mercaptoethanol (or 'ethane thio1) 9 03-CH2-SII, was
chosen as, a suitable reductive cleaving reagent (see pages 18-22),
its mode of action on a dioulphide bond being as. follows:-
R3-$R + R'Sfl - ) fish + R'S-SR
- It'S-SR + R'SI , ) RAR + R'S-fl'
i.e. RS-SR + 2R'Sfl. 2RSR + RS-3R' óveral].
The equilibrium constant for this type of reaction is near to unity
and therefore an' excess of thiol is required 'to drive the reaction
towards completion unle8a ,a blooing roaent is used in conjunction
with the thiol, in which - case an eq4Eo1ar quantity of thiol- will be
sufficient,
Since )3-meroaptoethnnol -has a highly noxious aroma as such as'
possible of the work involving use of this compound was carried out
in a 'fume cupboard or in sealed, containers When reactions were to be
left for some time to try and ensure that they would be complete.
Buffer solution uas made lip as in the last subsection using
guanidino hydroohloride.dissolved in acetate buffer solution to give
a final Fi=idine concentration of six molar and a' final pH of around
500. Solutions were deaerated by vacuum pump for about half an'hour
por litre' and then completely deoxygonated (aiiy oxygen present could
load to reversal of the reduction) by PasainT, oxygen-free nitrogen
86
through the solution for about two hours for each litre0
J3-n6rcaptoothanol was then added to the buffor , solution at a
concentration of a& and the SephadeE 6-75 analtical eolusn
eqiilibrated with this final buffer solution. Some fi-phyooervthrin
was contrifuged and the amoaiuni sulphate. precipitate dioolved in
a littto of the buffer solution to give as conoontratod a oe.mpie as,
pseiblo; thio was than loft in the 'dark at 090for twenty foux
hours for reaction to take place. A oaplo was then applied to
the gel ààlunn under onac'ly the Osmo conditions and in an identical
fahion to that described befré and eluted downwrds.tith' the
guanidino-marcaptoethanol buffer solution s collection of fractions
and ronitoring again as previously described. Pros this onporimont
only one fraction was oboered 'in 'thö oluant o coming juet after the
void voluijie of the column 9 i.e. g as observed when guanidiné buffo
solution alone was used.
The oporisent' as repeated but thin' timo the caziiplo was loft
to react for foxty eight hours. This made. a difference and the
oluant now ohowod two peks on the recorder trace compared :to ono
boforo. The first of theso (ropresonthi the leger molecular
weight fraction) was in the sass positiofl just after the voId volume
as before but the socond pk appeared óome tio ofter this.
Calibration of the column o exaCtly as previously decribéd indicated'
this fraction to have a molecular weight of around 23 000 (tho
larger fraction being about 86 000 as usual).
Those reoulte wore slightly surprising to the otent that the
larger fraction Ghould not' have boon precnt if complete dissociation
had taken place as hoped for and also the second fraction was colourless?
The first fraction was rod and had a visible 'absorption maximum at
87.
545nm whih indicated it to be a phycOerythrin-t3rpe but the second.
• fraction, although having a molecular weight in the expecte4 region,
was colourlesa which was moat unexpected as it indicated that the
•
obromophore groups must have been split off which the treatment
ahould not have been drastic enough to bring about. The
alternative to this was that this fr&otion was not a phycoerythrin-type
but some interfering protein not renved during purification or some
artifact formed during reaction. It was decided to study this
fraction further to try and resolve the question and it was therefore
collected by combining all the tubes whithi.the U.V. trace showed it to
be present in. The resultant solution was scanned in the visible
and U.V. regions- there was virtually no visible abeorption' but a
broad peak with maxiniul at 25Onm in the tT,V. region. This lack of
visiblO absOrption allied to 'presenôo of U.V. absorption suggested
that the fraction might be an artifact such as nucleic acid instead
of a biliprotein subunit. Further identification could best be
provided by total amino aóid analysis, comparing the results with the
figures for native 13-phycoerytht'in.
To preparo the sample for amino acid analysis buffer salts were
• first removed by dialysis against running water for twenty four hours
followed by several changes of distilled water. The residue was
evaporated to near drynese and then hyclro].ysed with 5cm3 of 6N constant
boiling (rediti11ed) hydrochloric acid. Immediately' prior to this
a U.V. scan was taken which showed the sample' to have very little
• absorption which tended to confirm fears of extensive bond breaking
in the protein or the presence of some artifact rather than the
desired dissociation having taken place. After hydrolysis at 1050C
for tweity four house the sample wai again evaporated but it was not
88 .
found pocsiblo to evaporate to coniplote drynoss, px'esumcbly duo to
the presence of glycerol from the dialysis meDbrene,.. The solution
was thorefóre mixed uith dolonised water and then alcohol 1n an
attempt to removo this interfering glycerol but' with no cuocesoe
ron exchange was then tried - a column (150n. z 1.50m.) of
Zeólcarb 225 oatton exchange resin wae packed and equilibrated by
pasing through hOt 0.21 Sodium hydroxide solution foilow&1 by hot
O.2 hydrochloric acid. The sample was then applied and first.
olutod with water to bring off the unaffected .glycorol and then with
21 hyth'OohloricaOid to o].uto the samplo. 1vporation to completO
dryness was now found to, be possible ubich indicated that this process
had been successful in removing the glycorol. The residue after
evaporation was dissolved in a little dOionisod water, evaporated
and this washing and oapo ration repeated tbreó times.' The final
residue was dissolved ma little doionised wator and before being
applied, to the amino acid analyser wao checked for the presence of
free amino acids by a spot tect with niithydrin reagent. The result
was that no purple opOt vad obtaincd, i.e., no free amino coins wore
proSent. It was possIble that the ion ezohane column had retarded
at least some of the amino .aoids bit there waS obr1.ously no point in'
continuing with the 'analysis. It waa thoroforo 'deidod to repeat
the initial separation but this time to remove any buffer salts from
the collected smaller traction by passing thO solutiOn .thvough a
dosaiting column of Sophádez C-25 instead of using dialyaie'
There was also the question of why the higher molooular weight
fraction was still present, however, and therefore instead of
repeating the above procedure It was decided. to repeat the whole
experiment with addition of the introduction of a blocking reagent
89 U
to see if its added presence could eliminate the higher molecular
weight fraction.. !lkylat,ion by iodoaoetic acid was choSen for
this purDoeo (see pages 21-22)..
The experiment was 'first tried tiith urea colution instead of
gunidine solution to cioo.if this Made any difforenoe. :l uro
was made up. in distilled tator and then pasas& through two deionioing.
001U5n3 a cation oxchanger prepared by washing tiith hydrochloric
acid and then distilled water followed. y an anion exchanger prepared
by washing with oIium'hydroxide and then dietifled vater. This
process was to remove any ions in ,tho urea solution which sight
intorfore with the roaction, To 12cm3 of the urea solution. collocted
after this treatment were 6dded.5o? of 06DI sodium phosphate buffer'
'solution, pH 6.0, and 0.5cm3 of 0,05nE.D.T,A solution (as a metal
ion cholater) . Oxygen-free nitrogen was then bubbled through the
solution for several hours to remoe all oxygen.
sample Of D.phycoerythrin was made ip in a little of this
solution exactly as previously described and then 0,5cm 3 of
-iorcaptQothanoi added followed by bubbling through of more
oxygen-free nitrogen for tho and a half hura • For alylation a
solution of l.5guis of iodoacetic acid. in 1cm 3 of fresh 11 sodium
hydroxide solution was made up and tho pH brought to .6.0 by addition
Of a . littlo solid sodium bydrozide the ohango in p being followed.
on a PH retor t A little )3morcaptosthanol was then added to use up.
ny oncoe iodoacetic acid as this could produCo froe iodLto which
could cause oxidation; for the same reason tbb iodoacetio acid was
twice rocryataflisod from distilled water prior to use and the
alkylation was carried out .i the dark. The result after treatment
of the 13-phycoorythrin in this way was for 'precipitation to t±o plaoe
so
cxnd the preciitato ou1d not redisaolv0 9 even at a1kaIno pL
vcluce. Accordingly the trhole oxpQriment was repeated but this time
roturning to the uco of guanidino solution inated of the urea.
Precipitation vas egain found, houoior q. The other poesiblo i"oason
for tio eoecod to be the mozcaptoothenol and oroof it uas
thoroforo rodistilled prior to use on another camjle of' p1ycoerthrin.
Thi time there crno no procpithtion, i.e.., rodiôtilling the
corcaptoethno1 had the &èirod effect and this was aubsoquoxitly
alte carried out tihonovor mercaptoethanol tc to be used for:.
aEporirnontation.
Boforo applying this eamplo to the gel column the. column tins
repacked (after deacration of the G-.75) and equilibrated with the
buffer solution prepared by caking up 6fl guanidino in 0,011-3 codiun
phosphato buffer eolution&nd adding 0.68cm 3 )3..morcaptoothcinol per
of buffer t3o1ution. giving a morcaptootbno1 'concentration
of O.111uhich vac that recommended by Ulnann ot a). (1968), The
buffer oolution trno filtered to remove any imoolubie impurities trhich
night clog up the gel column end then deaoratod as before; finally
oxygon.4roo nitrogen tiai passed through the solution for several
houre to remove any remaining. oxygen, The column wAs then equilibrated
with this buffer bblution.anda ôainplo of the treated phycoerythrin
applied to it. Application of the. sample and collection and
mouitoring of the oluant. uee oact1y ap proviouoly described.
TuO peaks on the recorder t'aCo ¶7er'e again oborved - the high
molecular toight poak uhich was present in a small amount only this
time and a omallor molecular weight peah which appeared to be quite
concntratod. This 1attr fraction seemed to have a molecular
oight of around 10 000, hotever, which una very unlikely for a
91
oubunit. It was collected by cornbining all the tuboo as before
and the vioiblo rnd U.V. opoctra thkón - again thero trac virtuefly
no vioiblo. abzorption but relativo].y strong TLV., absorption tith a
moxiriuzi at 243-250ris. 0 i.e., very nuchno obtinod previously.
Again it rrae dooldOd to try and ideiritifr this lower moleouler
troight fraction further by oubjeoting it to total arino acid. enalyoic.
Thic time, inoted of removing the buf'for aelto by dialyio or column
desalting, precipitation tro uoc1, amonium sulphate being added uMil
the point of saturation uaa'reached. After being left at 0°C for a
lou hourO a fine whitioh precipitate could be soon and this was
contrifugoct dovu (2000 for twonty minutes), di000lved in a littlo
distilled wator. and dialysOci, first againot running tar, uator for
tt101vQ hours and then against several large volume changes of dictiflod
tater, The resultant solution tiae evaporated to drynoso (no troublo
uith glycerol on this occasion), dissOlved in distilled t'ator and
ovporated again, this process being repeated three tiniop. After
the final evaporation the Xooiduo was dissolved 'in 6cm3 of 6I conotait
boiling hd.rochlorio acid and hdrolysed at 1050C for twenty four
hours. The hydrolycato was evaporatod to drmose and dissolved in
doion±aod water and the prO 0050 zepcatod three timos.' Pinal drying
of the residue uas. achieved by leaving it overnight in a vacuum
dicator over 8odiuri hyd.roiid.o pellets,
ror amino acid anc3lypic the residue wee then diocolvod In 5cm3
of a Oolutjou containing the atmdarde IOL and AGPA (aso pageo53-54)
and 05ori oaiples of 'this oolution applied to the baoio amino acid
and acidic/neutral amino acid doternining ion: onchango columns in
turn on the autoratic aalycor. The resultant enelysic charts
and amino acid content figuros (see table 3) tero.comparod. to thO
rReL 3. Cok 1p i'qgfiTive A rt wa MCiV
OF 1IV 3riiu. Sucuvrr
t 6 a C. A Pto lVNOL TrL1V?
N iwo A ID .L 3- PINC68al(r"kilo Svus it
736
w it r.ttwe o•qi
AspArn
At cp 24.14
cy.sTEIc. Rt! I Oli 23
Gvric Ac3 6•q14
33
066
.Lsi• 3•Sl
5eiwE
I 10 I
14t3 LeSt,
T$oNIVV, I•21
VLIw kq6 I II
c&& (t4vt Wei3hi c' OF
92
oae for natjve phyôorythrin obtained by rtnq lyoin d acip1e vzhioh.
had been frôse..dried. hycL'olysed rind thor treated as aboe Iact
quantitative cornpa'1aon tyab not possible a6 an accurate teicht of the
untioun fraction ras•not'uso4 but the ualitatvè pictu e uas
sufficient to Oho merited differences betn the two aaalysod.. In
'.-partiôular the ceparatod fraction bad ouch greater aiotuito (relatively).
of thO. amino ,fjj :03T5jO (oid and glycino than native phycoerythrin
and loso,ok thOat of ihe Othors, especially mathionine , (tibich was
hardly døtootod at ali), tyrosine and arginino. This clearly inferred
that the fraction uas not a coer thrin.-typo but some artifact or
iinpurity
'The lileliest oEpicntion for this fraction coomed to be that
it usa on intorfering protein not rornovd during purification of tho
B.'phyooarythrin from the algai There tias also the prOblea of the
•
absence of any plycoo'ythrin-otype subunit of the also eapoctod and a
posiblo op1anation for this tias that long time standing in the
buf for eolutlon had brought about diseociation to ouch an otent that
the rosultant oubunit moleoulos had boon sncU enough to pass through
the dialytic tbrano into the surrounding solution, Such
diooiation bad already boon observed in t4S laboratory (Brodio,
166 aid Hutcl4no, 1967).
• To. resolve those problems it uae decided that the ezporinont
should be repeated using B.phycoerythrin purified as highly as
poopiblo i,o., crystalline, ubich should thorefore be tr6o of any
intorforinproteino. Both fractions produced should be collected
and procipfntod, 4ydrolypod and amino acid analysed, This uould
• confift uhether the larger fraction ras phycoerytbrin-type as colour
and visi10 absorption iMicated. Tha cocond fraction should be
93 .
split into two,, one half to be precipitated and di&lyed as before
and the other to be desalted by passing it through a column Of
Sephadex 0-15 or 0-25 an, then treated. in the samo way'as the first
half. This would show whether or not any low molecular weight
material was being, lout during dalyis as the samO could nat oCcur .
with the half desalted by column cbroatography.
The experiment was therefore repeated on highly purified .
B-phycoerythrin and the resultant two fractàns, which appeared exactly
as before, collected., Before they could be treated as outlined, above,
however, it was deCided to change from use of -mercaptoetharioi to
another thiól,. dthiothreitol, thiqh had been reported as being much
zore ettoceseful es' a disuiphido bond breaking reagent. The
•mercaptoethanol, een when used in conjunction with a blocking reagent,
was still not bining about the desired dis9ociation as higher
riolecillar weight subunfts were always being isolated, albeit in smaller
amounts than with the previous treatments,, This could always be
returned to if dithiàthreitol, as described in the next subsection,
was no more successful, . . .
(c) Cuan&din lutigfl with Dtti4othrei.tol. . .
It has already been mentioned that thiols' like -mercaptoethanol
suffer from the considerable disadvantaes of being relatively
inefficient (exceSs usually *equired) and having a nozious 8roma.
Cleland (1964) used these compounds as disuiphide bond breaking
reagents and found such drawbacks to their use. He then studied the
bond breaking reaction pathway and came to the conclusion that if an
intramolecular reaction could occur at'soe stage during the course
of reaction which would lad to the formation of a btericAlly
favourable product the whole process would be muoh more efficient,
* 94. .,
flo qorked out 'thitT a 10-dithiolbutono ntorrneditd co dicad to
such a oterically favourable product on roarrangoent and experimented
41ong theso.1inds to try and 'find a Oultablo, praótical compound,
To result was preparation of the tuo ioonora of 2,3-dihydroxy-1 0
44d.thiOlbutanO named dithiotbreitol and diorythrothreitol,
• abreiated to D.T.T. and D.TiP S, iespeottvoly. The ,ne of reaction
of these copcunds in 4ioulphido bond breaking is as fØllOs, D.T+P!
boiric taken as an exoinle:- .
-sa + 2scn2(cuon)023u - ns + i-scR2 (cHoO2cR2sR
S / .
0'
2 '
/,
. . 9E2 V ESH + CUORH CH SH ' s,2. j * CttQE CE2 . .
• '
'For the first otago of this reaction the bquilibrium conetent is again
found to be near to unity, i.ô,, it is no sore' favourablo than the
firet stage of tho etanda±'d tbol reaction. Eotiover, the socond otopp
which involves an intrexnolooular rearrangomoflt to fore the six-moribored
Anq q is highly favoured atoric4ly and aO a rosult this reaction stage
quickly goes to' completion and has a high eguilibriun.con5tcnt, O.C. R
for D.',T. with cystirió thO equilibrium constant for, the first stage
of the roaótiou is near unity but for the cocond, rearrangement stage
is about 1.104 which is hihly favourable to completion of the
• , 'eaotioz. It can also be seen from these equations that the use of
ono solo of D,.,T.rasults in the formation oftwo soles of protootdd
aulphydryi groupings *uhoreao peroaptootaflol and other thiolo reacted
•
on 'a one to ono,baeic, joe,, D. is twice as efficient as these
thiola.
95
Olelond had marked success in maintaining thiolo in the reduced
state with D.T.T. (there being no danger of rovoreal of reaction as
with the óthôr compounds and héncC no blocking agent required) end'
aloe in obtaining quantitative. reduction of disuiphide !o • He
marketed D.T.T. (it being the easier of the two isomers to $ynthestse.
although they are identical in properties for this purpose) as
Cleland'o reagent and it has since become widely used for such
raacti=4 it has other aventacoa in addition to' iti erficioncy.
of reactiQn at it is a solid soluble in both wátor and alcohols due
to its two hyth'oyi groups; it has only a vary oliht thiól -odour
which ceano that it does not require to be kept in a fume cupboard;
in. solution it is resistant to air oxidation and generally is vory
etiablo.
Up until this time atteipts to d1sociate -phycoerythrin into
its Trinimub rolocular weight. form had not been 'conspicuously successful.
riowover, Castelljno and Brker. (1968) managed to dissociate sever1
multichain proteins into their monomoric forms using' giaanidine solution
with 3mercaptoothano1 at concentrations of 0.1 moles 1-1 and
0.05 moles11, They did not find it neCessary 'to aZkylato to protect
the sulphydryl group3 formed in any instanCe, There seemed to be no
obvious reason therefore why Bphyooerythrin should not dissociate in
a ailar =nner when treated with guanidine solution and
• )3-niercaptoethanol or oven more so if wore. used, with no
alkylation by iodoacetic acid being necessary in either case.
£ccordiagiy it was decided to try guanidine solution in conjunction.'
with D.T.P. in view of its superiority over J3a.rnerOaptooth6nol as
• mentioned. '
• The heifer medium to be used was made up :by dissolving guaniditie
hydrochloride in 0.01t sodium phosphate buffer solution to give a
final gunidine concenttatiOn of six molor and adding DTT r (supplied
b Calbiochem., ftichmond, California) to this to give it a concentration
of 0.005)! (half the concentration of mercaptoethanl. used in view of
the fact tht D.T.T. is twice as effeCtive). The final p11 of the
solution was 5.O5 and it was deasratect by bubbling through oxygen-freo
nitrogen for seversi hours per litre
A sample of phycoerytbrin was preparfd. by dissolving some ammonium
aulpbae precipitate In a' minimm volume of 'this solution and dialysing
against. several chagee of the same for i.ip to three days. No
precipitation occurred, which was an immediate improvement. Some
spectral óhangos were observed: the peaks in the viible region all
disappeared to be replaced by a general, fairly high intensity ,
absorption over the 0016.visible reion with no distinct maximum and
the U,V, absorption increased slightly in overall intensity compared
to that for native B-phycoerythrin.
A sample of the treated phycoerythrin was then applied to. the
analytical $ephadex G'75 column but the results from this were rather
inconclusive .. there seemed to be dissociation tO 'some extent but not
into minimum molecular weight subunits as desIred. This was also
indicated by the speOtral evidence 'but it did not eeem possible to
achieve a good' separation of whatever subunito were being produced.
A possible reasOn and solution for this were suggested when
Daviason (1968) published his observations on tho.behaviour of a number,
of proteins in 4eriaturixig solvents wuOh as guanidine solution when
applied, to gel columns. Ho pointed out that these concentrated
denaturing reagents expand the el network in such a way as to
considerably reduce the pore sizes. i.n the gel network and as a result
of this the denatured voleouleo are exclded from gel ined.a which
tould norrally admit the native molecules, In particular ho noted
that Sephadex G-100 could only be , used sueceosfufly for denatured
roleoulea of up to 20 000 in tolecular weight. Pore porous gels
are requixed if a lar3er exclusion limit than this is desIrable and
Davisson found io4ol.5m (containing agaröse; ese agee 49-50)
to be the best gel of theSe he tried over the most useful range of
molecular weights, its upper exclusion limit being about 100.000
molecular woight, Froin all this it appeared that the cause of
non-isolatjox. of the deairod minimum molecular ieiht subunit of
D-phycoorythrin was duo to it being ezøluded from the gel and not due
to incomplete breakdown as thought previously, e.g., when
cercaptoethanol was used. In fact this subunit probably was
produced by the latter bofore D.T.P. was tried but in all cases the
eubunit was being excluded by the gel and a false estimate of its
moleciisr weight given for this reason. It was therefore decided to
replace Sephndex in analytical columns with Dio-Gel A-5m to see if
better results could be obtained,
A Colunn of the Bin-Gel A.-5m (supplied preewollen in 0.0011,
tria-E.D.T.A, buffer solution by Calbiochezn, Richrond, California) was
rade up, 60cm, x 069cri. 9 and. equilibrated with 0.Olfl sodium phcsphte
buffer solution, pfl 6,0, The packing of the column was checked and
the void volume found by passing through a sample of blue dextron as
usual and calibration was with bovine serum albumin, ovalbumin and
trypain inhibitor as before. Davidson also found it necessary to
detCrmine the internal volume of the column for better calibration
the expression E = (Vs * Vo) / (vi - Vo) being used for this instead
of Ve / Vo directly as before (see section (i) (e) (ii)). In fact
98
Lacas.o at a]. (1968) have since ohoth that logK against log U,Ut.
ohould be used to obtain a propor linear calibration . for these
aarose gale, pleri of K against log being' slightly non linear.
In the abovø ozpresaion, Ve jreprosonts the e1uti4 vGlwiIo of any
saplepVothoe1ution volume of blue doxtran, i,o, the voidvolumo
and Vi the elution volume of. the iner.1 $tandard, i.e., thö internal
volumO of tho coiwri, Ninitrophony]Ainino uao used as this internal
tn.ai'd and was prepared from puro aisnino by the aethod of Songor
(1945) as follous. (Soc zedtion (4) ).
C 3CRCOOfl 3 + 021T"9-.F .- or liCCRCII3
slaninO dinitrofluorObensene W 20
- . S :0 fl NffCIWOOR
N- dini trophonylAlenine
To 100 of an aqueous colutton of aThnine'(0.089 gsa in ioøcm 3 of
distilled uater, i.e.', o.oii), were adcd 0.2gius of ,olid analar so4ium
bicarbonate folloued by 0.2cm3 (excess) of dinitrofluorobensene in
of ethanol and the mixture shaken for three hours • The product
tao extratéd into ethyl cotato" (uflucd dinitx'ofluorobenzeno remainina.
in the c.qudou. layer) 'five' tunes (tto volumes of 25dm and three of
ior?) and'tho cosbinód ôstor layers iashcd vith a little uator The
• ethyl aôotato tac then evaporated off leaving the roeldual solid
• N-dinitophonyRlez4no. A little. bfthis solid Qould then 'be dissolved
• • in a smail amount of the approriate btiffer solution being uood. for '
olution of the calibrating protein sc'lnplos and some of the pole y-ollou
solution loaded on to the column as usual to find the internal volume.
Quite good calibration for the Bio 'Gel 1-5m colunn could be uchievod
99
in thie way (see fitro 12).
Davisson in hie studie3 used a buffer solution eontainirg 61
uanidine in uater ttLich to ã10 0.0517 with respect of lithiui
chloride (p'eont as an e1ctro1yto), Olti tithrospoct to
• rnQrcaptoQthano1 end 0.01 tzith rospoct to (present as a
netal cholator). The pl of tho final solution waa altiayo adjustod to
bottieen 64 oAd 840. Por the work on B-phycoorythrin a relatively
sii1ar colution uas made up - 6M guanidino in 0,Olt: sodiun phoophté
buffoz oolution (tib.toh also acted as the olectrolyto), O,O1
and 0 0 00514 D,T.T o instead of mroaptoothnol. The final pH of thic
solution ub, adjusted to 6.5 by dddition of coljd sodium hydroxide,
being foilowe& by a 1A soter. The solution use deeratcd by bubbling.
through oxygon*froo nitrogen as usual and in addition was filtered
bofóre use# Thecolumn was equilibrated trith this solution thilet a
esmplo of B-pliy000rythrin was allowed to roaôt in it for some dsyc
(aiwonium cuiphito preoipithte dIssolved in Ond dialycod against
solution so usuai).
Considerable flow rato diffculties arose and were only Ovorcoiie
whom both the gel and tim equi1ibiating biaffor solution wore doaerated
with a vou11n pump iwtoad of the oxyen-freo nitrogen. 1 samplo of
the treated phycOerythrin tras thou app1o4 to the equilibrated column
and elutoa with the semo buffer solution, Sample treatmont and
collection of Oluant were exactly as previously described; about
twontr four houra was required for a cosploto analysis • Tue peaks
were oboorvod on the Uvicord, monitor tracop the first fraction being
red/purple in colour and the cecona colourless. Visible spectra
comfirscd. thia with the first fraction havIng low intensity visiblo
absorption over thà whole region with a broad. eanivium . around 545nm
T. I
CLI6TI PI.Dc '!
(3-G. Cotu"ws
(s Pi -ioo) 0
•
: • •
Laq I0 tcu(g w&3L
• • 100 ••
(low intensity •aainly due to dilution during development from the
chromaographic column) whilat the socond fraction'hai.. no ''isible
absorption whatsoever. This indicated the first fraction to be a
phcoerythrixt...typo subunit but there were doubts as to the nature
of the second fraction, as Was the case aftor treatment with
J-meroaptoethano1. . . . . .
The whole experiment was.therefore repeated to see if those
results were reproducible. This turned out to be the case and the,
Calibration plot showing the poattions of the two fractions is . shown
in figure 12a. The molecular weights of theao fractions appeared to
be about 32 000 and 14 000 respectively (soépae ioi).
To Check further the experiment was yet again rôpeated with a
newly treated sample of phyooerythrin and the column recalibrated
with fresh protein solutions. The eluant was'oolleàtod in smaller,
amounts tO allow more accurate calibration and molecular weight
estimation • Very eimiiar results were again obtained with the
B-phycoerythrin sample displaying the same two peaks on the monitor
reCorder chart, the first of those being coloured and the second.
colourlesS. This time their molecular weights were estimated as .
being 36 006 and 10 600 respectively. This calibration plot is shown
in figure 12b. .• • • •. . •
The data obtained from the two experiments is summarised in the
following tables:- • . • . • . . • •
Experiment 1 •,. • - ' .. . -' • •
.Elution Vol.. (as tube no.) .. io].. Wt,
r3lue dextran 10.6 (Vo) • • 10
Dj.P,-alavj.ne 32 (vi) <10
Bojo serum albumin 13 . . 0.114 60 000
Ovalbumin 18 0046 45 000
10] S
Eiujonvol.'(as tubo no.) . ol.Ut.
• Tr'ypsin inhibitor 25 •' •. 0.678 24 000
y000rthrin 22 (first fraction) 0.533 32.00O
32 (second frGotion) 1.00 13 650
•
1ution vo1uiies are GiveA in terms of tubo number (ceo page70)froxn •0
the titio of loadi$ of the atLmple. (constont for all oai1es); 'ttiont
• drops per tube (0.550m3) were colloctede
0 '
Blue deztr3n I (VO) , . 106 0
P.,P..a1az4ne 59.3 (vi) .
• ' •Eovine aerui Albumin . ' ' '• 0.203 60 000
- 0vibumin • 0 34.1. 0 • 0089" 45 000.
Trypein inhibitor 51.8 01318 24 000
D..phcoerythx'in 42 0.581 35 080
• 0 •• . . 0.976 lB 60
This time ton drops (0.28cm3) per tube were collocted,
Other features of the reaction yore also obOorved the'coiour of
the Bphycoerythrin coluton, dtIring treatcnt eanged gradu1iy (over
a period of days) from orange-rod and fl'orosceflt to a deep red with 0
0
no fluorescence. 'The simp1é prior to loading had a general visible 0
absorptjon vith no maxim diatriguiohablo. and 1o, intensity T1.V4
• absorption. The firt collected frotion had similar absorption 0
•cbaractcrietics but the second had no visiblo tbsorption and quito 0
hQ'h U.V. absorption. :' •'
•0 ••
0
It was decided that the only ay to resolve those roculte was
O :0 total amino acid analysiD of the two fradtiono isolated ac ',carried out
provtuply after treatment tiith J3-corcptoethono1 had led.to similar • 0
• resuite. Too lit't1 of each fraction vas obtained, cfter coparat±o 0
102
on the analytical oolunn and it was thei'efoae necessciy to ropeat
the ttholo oerixont on a vich 1ro' scale to obtinnoih of
each fraction for accurate amino acid ana1sie.
•Sóao procipitctcd B-phycorythrin vac centrifuged, tho precipitate
diesoiUOd in diotillod iator and thefl dialysod oitoboivo1y cgaivat
aovoral laro voluio chanoo of the Gaae until free from sulphate,
Thic, aolutton tac then tranofered to a 'large round-bottojed flask
and fror&on round the sides of thio tiith liquid nitrogen and thou
freoao-driod. 50igzno of this frQeze.-&ied protein were thou weighed
out accurately (using an o1oCtroba1anoo)and disoolvod in 5om of the
6:1 Cuanidiao ooluton, 0.0111 ith rospeot to E.D0T.. and 0.051- with
respect to D.T.T,, final pR 6.5 The damplo was left to react ft11y,
progress of reaction boin' chôekod regalarly by scning of the visible
and U.V. regions.
In the zoautino a Pharsaäia (p rracia, ipp1a, Sweden) coluin
(20on. z 2.5cm.) wac thdo up tifth Bio4e1 A.5m,' doaorated with a
vouUti pump. Tho gel vae equilibrated with tho sane buffoi co1uton
as bqvo:ozoopt that it did not ôontain any D.T.T. Jpwara elution
through the column tiao achieved using, a periotaltic pump giving a flow
ato of about 10J or hour. The packing of the column was choc1od
by running through a eaznple of blue dotan diosolved In the buffer.
cöltition which pasood up in an even band s indicating the packing to bo
tiofotory.
The pjcoerythrmn had chargod In colour from red-orange and
fluorescent to a doep rod with no fluorescence eactiy as observed before.
ljioibjo absorption was dininiohed in intensity and was genezal over the
whole region whilst U.V. absorption was slightly inóroaeod. AwOek
wag. required for those absorption: characteristics to become constant
103
thus indicating comp1otio7A of reaction.
The sanpie was then loaded øn to the column usng tho pump and
eluted upwards with the buffer so1ution ttienty drop fractions
(0.55cm3 ) being collected and the eluant monitored as usual. The
some two bends as observed from the sm4lor analytical ôolumn wore.
again oluted, the first beng reddiah in colour wid the second virtually
colourless. Both of those fraction tore colloced by combining tubes.
The first had visible and tI.V, absorption very sinlilar to that of the
loaded aaxp1e although &irinihod in intensity, presumably due to
dilution, whilst the second had no visible absorption and U.V.
absorption similar to the first fraction and original sample,
Both of. chese fractions wore then precipitated with anuionim
euiphate, the first requiring addition of amnior4urn sulphate until the
solution was 30i (u/v) with $spct to the satie whilst the second
fraction required it to be present, to to point of saturation before
any preoipttation toofr place. The solutions were centrifuged and the
procipitutee (red nd whitish respectively) were then each dissolved
oprtoly in mininurn volumes of distilled tatet and dalysed eztensively,
• first against running tap water and then againot soveral large volume
OhtxigeS of distifled wAtor. For the sècnd fraotion the dia1yis
tubing was first boiled in distilled ustor for about fifteen ninutes
prior to use in case any very small subunits were present which rnight
otherwise be lost through the porous membrane. This extensive
dialysis was necessary to remove the very high concentration of buffer
olut1on salts, as freeze-drying will not be successful if appreciable
amounts of salis remain in the solution. Sóe considerable time
elapsed before the samples uero sufficiently thoroughly dialysed for
the freeze-drying to be successfully carried Out,
104.
Tuo milliaraze of each of the froee-dried ssflples were then
weighed out aecuratel (uoing .on e1ectroba1ano) eM. hroIsod as
before (6cn3 of 6N constant boilnjirOchloric acid in a sealed tubo
for twenty four hours at 1050). The hydrolysates were ovapotted
down to dryness and washed several times with deinioed wator, The
final residues were dried in a vacuum &essicator overnt and then each
dissolved in 5cm3 of standard solution for amino acid analysis. 069a3
oeziplee tiore ued for. analysis and the results for each freotion
ôorn,ared titb the other and with a sample of native B-phycoer'ythrin
froeze..clriod and analyeéd in an identical monnez'a The eflalysos figures
tea' all three samples (given in terms of residues per 100mgus of protein)
ore given In table 4. in table 5 these are adjusted to have all the
values for valino the sime (valine being chosen as an amino acid present
in en aproxinatoly average mo*t) and si4larly in table 6 they are
liotod with their histidine values all equal (histidine being prosent
only in 0 very small amount).. These adjusted figures give e cloeror
• comparison (and contrast) of the results;... they are alsO ropreseritod
disgrarnatioally in figure 13. • Prop the tables and figures it seems clear that the, first (or
larger) isolated fraction obtained from this etporiuont was a
B.phcoerytht'in-typoj as also indicated by its colour and visible
abaorption; but the second fraction was very different in amino acid
analysis figures end was also colourless. It would therefore appear to
be some interforin protein or some artifact formed during the coux'so
of reaction. This will be more fully considered in the diOcussion.
Pevertholese, the ioo of DT.T. did seem to brine about the desired
TABLE lo.. COM PRA'rIvt! AII1WO ACID Co,,o$rrIows
OF
NATlva-
AFrCft D.T T TE,iTv1WT
NAriuf FiRST 150LT $t:o.siii!o
ActZ 4 g...pHegYT*rni
A%GIWiW S 6I2. •i•2.I
A s maricActv
Cstic Ac') lOu. S 043
Gwir oftle ACID S
066 o.6i 0•31
tSo.-L'uC.IHE 348 3.7, S
LGuc'viE S g.qg 620 s•a
3-61 3.98 2.17
NI.APJ1WE
119 2•2.6 O1L.
PRói.uiE ).O 0- "77
SERP '11# S
S pJIw€ 3•31 3.i5' 110
_______ 5•o9 3.9w 013
VALIiis 5.11 1'Ia
F5ures Eac'reucd as i?u4ite Wchb }gv t0Oi O £ 4(C
TAI3LE 5. As F0(I T A OLLS , u. V$LUES
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'105
(a) Fi,onta1 and Zonal Aa1's
?rontal and zonal. analyses are o1ocu1ar ciové chroiiatography
teohiquoo for studyina intOracting protein oyetore and trero first
introduced by Lathe and Ruthven (1956) and Porath and Flodin (1959).
Proviouoly the aoving bOundary techniques of electrophoresie and
oediraontation using an ultracentrifugo bed been uso 'for stu4ying
ouch systems but rzolpoular ceVo chromatoaraphy rae fotthd to hao
• beora1 qdvantnaes over these zothods in qualitatively detocting the
proeenco of interaction and also qantitativo1y, in dotrminir
oi1tbzitHn constants. Tho tochi4ques ae nou used mainly for two
typos of syotOD. A + -D-K ubere A and B am diffo±ont n2aCroIóiocuioo,
and on po1yzerioing eye toins involving one type of macrDnioleoule.
in frontal analysis (Tiaeliias,' 1943; Ujnor and Shoraga, 1963,
1964) Quff4ent protoin Oolution is ap1iod tO agol co1un for a
plateen rogion to be. prOduced in tho oluant at tyhioh tho concentration
of olutod prptelacquals that of the cp1c originally loaded,' Zonal
anezlyoto (Audreu, 1964, 1965) to similar in tochnique but norim11y
reserved for, popai'ativo and ana1ytica1 UOZ'k. It 'is niost useful tthOn
applied to OystOic tihere the rate Of equilibrium is slou compared to
tho rate of oopaation of cOmponents by. differential tieport, i.e..,
for detection of interaction,in a syotom vhorO the molcoulee bobive as
if indopendeM of each other and in 'such cases the technique pro'ideo
a, relatively otp10 method of 'dotormining the oquilibriun conatant fo
intraotions, For cyoteme other than this typo application of the
'tIniqio bçcomoc rathor complicated and frontal analysis to to be
preferred, in such stosqui1ibrium battOOn tho':subapeoies 'is"
restored rirtut4ly as soon as it is dictuithod vrhich moons that separate
boundaries cannot ho ascribed to any.parttcur ouspecioa so. can
106
happen with the other typo of cyetem.
Such rapidly polyrerioing oyetes uore considered thooroticl1y
by Gilbert (1955, 1959, 1963) who made oeveral predictions abott their
properties. The moot relevant of these predictions to thie work was
that if the equilibrium o yatem is between a nonomr and a diner only,
tho the first derivative plot of the trtling olution profile will
consist of a single aoinmetric peak (the leading ego of this being
charper than the trailing edae) wbeeae if higher polymeric species
are involved in the eilibriur partial reoolttion of this plot may
be obaered. The whole effoct depnd3 to a considerable extent on
the concentration of tlje loadd sample as well, there being a linear
corrolation between concentration and olution volume and this further
complicates the process, e.g., the partial resolution referred. to will
not be observed at extremes of conoontraflon for como maaz'Ocoleoulea.
Uere no1ecilr sieve cbromatoraphy has an advantage over sedimentation
as it cat be used at much loror conoentrattons (as low as 0605mgmo per
compared to a minimum of about 3.5mrs or. cm 3 for sedimentation
expericonto) with the aid'of spectroecopy and in fact this allotred a
series of exporiznente to be carried out which showed that partial
resolution was not Observed at very low concentrationa, a prediction
of the theory. This feature of molecular sieve chromatography is
also very uoful as itallows much easier extrapolation to sero than
can be obtained by the other methods and this is much used to try and
detez'mine moflomeric violooular weights, as will be mentioned later.
Other advantages of the teohniqQe include the fact that a second colvent,
which may be detrimental, to a pro tom, is never required and also
• oltition rato is dependent on molecular weight which is a necoesary
criterion for the Gilbert effocto of rapidly associating systeis to be
oboOrvd.
'07:
• it very good demonstration of various aspects of the Gilbert
theory is represented in figure 14 for studies using frontal analysis
oii -chyotryssin and dilcopropyiphosphoryl (DIP)-ohymotrypain
(ujnzor and 5hera, 1963), The plots shown are of absorption at
280nm against elution volume for both proteins (14 a t b) and these
show the oharacterietic greater sharpness of the leading edge over the
trailing edge as well as showing the plateau region. In addition the
trailing edgo of.-chyrotrypsin 8hows points of inflexion which was a
prediction byBothune and Iege1es. (1961) for a rapIdly polymorising
system involving aggregates greater than diner. Thie. is not 'the case
for DIP..chymotrypsin, )ioweve, indicating that it dIffers in this
reapot Also shown are the first derivative plots of these absorption
curvea'(14 o, d), which are obtained by plotting the oalâulatod change
in absorption (sodulua value)JAAJ/4v, for an inorenont in elution
volume, AV, against the mean value of the eluant volune in the
interval AV, V. It can be , seen that this plot forcC-obyiotrypsin has
three poaks and thie iseactly as predicted by Gilbert for a systorn
involving rapid, reversible equilibrium •betuoen species involving at
least one pOlymer larger than diiner. For D17?-chymotrypain there is
only the :one peak on the derivative curve and this is consistent with
the prediotOn for a system involvingequilibrium between monomer and
ditner only. Also seen is the predicted greater sharpness of the
leading edge of these derivative peaks compared to the trailing edge.
In figure .15a a comparable plot is shown for ovalbumin which. is
known to be a non-associating protein and here it can: be seen that
there is no observable difference between the derivative plots of the
leading and tailing edges of the oluant profile, both being symmetric.
Finally, the offset of eoncentration.dependence as reflected in the
Fi&UI E M. Of Fot4FL ALY$IS h%.Ui( PDLtS FLSc TIERiV .ATIVG Gins
çc 5Hci, t&3)
0
I $
V ()
(h) Tn4 !Ir ___ Vm
1oiYatiVe rnttorn to i1luotr6ted for tho trailing ojc of
. ccotryain (i'igro 150. ¶ito rate of clutton also deonde on
ctcentration, of COuroo. it to ona4nt1n1 to note that the peIta g
oboerved on the dcriv4tive - plo.ta are tiev6r , fully reoolved and for tbiø
'eaeoflo peals cC ever be olesaified Ao correapoMing to an
parttouItr speoiea present in the system,, ronoeric or po1y3erio. tloo
the tact that the peakS are Vot observed at certain eoicentratioie
(ftiro ].b) does not rien that the polyorio oecieo oonernod to not
it can be seen, there?ore, that fDontol snal.ysto to a sip3.o but
• very eff7ective tochaiqe for qualttntiveli doteotiva the Preaence of a
x'a$d17. easocistin diesociating etlib*iur eye tern. It is more
4ifioult to extend thin sztttatvely as rolocultr sie in not the
only factor whiah afteotp the, rate of iirstiOn, althou it can be
extended to ci'o a weight average eetitation of the eta, of the
oo,erto epeciea (7tnzor and i3heraga, i964) • !i'kts requtreo
orr1tion of the rOlocular sievø: chx'oratography data with tiolecular
*teiht detereinatione by otler direct nethoda, auch as iiltracentrifugotion.
tor !3.phcorytbrin the dissociation effeCts noticed earlier (ens
soction () ) had indi&ted that tbez'c' was pousibly. an equilibrium
oydtei of. the type vonomner.dim1er4.hexarer4.do8eoatner being present
in an aqueous solution of the protein vhiih should be tell suited to
otudy by the frontal .umaiycia technique, with a view to deterinin
qualitatively' hother such on equilibrium eysten was pzeooat, initial
oxporirente along these lines were carried out istng 3eDbadez ()'lOO;
a 30ct4 X l,7cr, oolum of the deterateQ gel was made up and equilibrated
uitb seVra1 colwn volee of 0.O1 sodiutt pboophate buffer solt&tion,
a px'esirnre head was wijuetod to ct1re a otea2y flow rate of
• 109
about 6om3 per hour0 A caple o B-pbycoorytbinuao'preparod in
the etandard ay by diesolving amtoniun giAphate prcipitate in and
•
dialyeing agoinot DoUeral large volume chengoc of the oato buffer
aolution. I Yith eucroso added for eaeier loading l0om of this coiplo
ycoerythrin solution were 1oadd on to the top of tho oolthmi by 1ayring
pith a cyringo. I
Tuonty drop ractidno (05500 ) t7er6 collected ixeing
tho photocell drop counter and frac .tioa collector and the oluent
monitored x7ith the Uvicord at 23nn to indlOate the divisions betron
loading odge e plateau region and treiling edge although the traOo tao
not good onough to be used to road absorption values dirctly. In
fact only a very 0a11 p1o.eau region tme obaorvodo indicating that
inoufficiont protein aemplo had boon used 9 but the loading and trailing
boundaries could still be ditinguishoda The plateau region cad
tr&iling boundary fractions iere then Pall individually onitood at
280nm (using the Pye Uicam S.Po 800 pectróphotometertith 2átas
dcro'collo for geator aonsitiiiity). All eamplos tero diluted.rith
0.15cn3 of buffer aolution to bring the volume up to that required
to fili a eioro-coll buffer solution cas taeod in the roforonco coil,
T collted fractions all then had a volume of o.54-0.56G ho oc checked
by toasuring a large ccmnlo of. them using a grduntcd syringe 9 end this
tie averaged out as 0.55cm3 and tao taken aa the atoedy volume
incDomont bottoon each collectod fraction uhich made calculation of
the derivative ratio owiero Abeorbeoo uasAhon plotted against
olution voluo over this trh1e region of eluant and thent4( plotted -. • • AV
against V 9 the values of each being calculated by uoe of a oouputar0
The scattor of pointc on bOth of those pioto tnas very conoidorablo.
but if all points havingl4I g6 001 on the derivative plot were • AV
óooidored to equal an arbitrary eoro 9 peake could b dietinguishod in
110
addition to a peck for the plateau region. Coroøpondingly. thrce
points of infleion could be obeorvod on the trailing boundary on
thö olution profile plot.. .Eouovor 9 thaoe rou1to looked zore tha
a little unrelab1e due to the oonsidorablo tcatter' of the points and
accordingly the eporiiient tTas ropoatod.
nothOx'.co1uxan of Sophado C.-100 ties propad 9 29cr. x i.7ca., as
before tiith a foen rubbor pad placed on top of the gel to try end
improve tho evenness of the loadina a6any atroalting completely nullified
the oporisent. Only devon 4rops per tube (0.30cm) were collected
for greater accuracy; the flow rate ties 12ci 3 'per hour, The. oóUecte
fractios wore egain individw11y monitorod but this time it 550mm
end 20a (1ng the Pye Unlcam S.F. COQ) and also at 280mm (twing the
• S.P. 500 tiitb Tynivorcal Cell ounting for greater oensitivity). Onco
• again very compliôatod plots for both the '<1utton profile plots and' the
first dörlvativo plots wore obtained for all tho 'series of readings.
L sicilar goneral picture to that outlinod above could again be
diabornod but those roCults wore still not satisfactory, and in fact
were oven leso ôo• than thoo from the first ewporitaent,
change of technique tias thon tried with 13io-0o1 A-5m bo±ng used
intsad of Sephndox 0-100 and the Pharmaoa column with pi'ugor fittings
allovW tox'e oven loading also used (20cm. it 26cm,)4 The seine
buffer tolutioft ties used, again pH 6.5. and the 3-phycoerythrin ocinpie
prepared oa be'ore. 15cm3 of thie colution . troro loaded and elution
tiari dotyntrarda controlled at a flow rate of 120m3 per hour with a
periotaltic'puirzp. Printy drop fractions (O.55cm) uore collected
and again individually monitored at 550mm and 280mm (using the S.?. 800)4
The roe4ings at 550mm yroidod' the better plots which were in fact
oupetior to those previously obtained - the ocattor of points was much
.1.0
AV
.FIGuRE 16. ANL'iSI5 0F IYCORVT14tN
1•0
0I I
V4Lqmw 1)
(ft) Ei.urtow Pl%ocILE
% I
s 0
• . ••, •
(A) Ft.si ew,,'tve CwvE 07 Tg*iuwc, E'DGE
111
less, although still quite considerabloand enough to raise dOubts
as to the validity of a third peak on the derivative plot as it was
represented by onoroading Only. However, the other two peaks and
points of inflexion as seen on the elution profile plot were far
clearer and this therefore.repreaented a considerable improvement on
the previous experiments using Sephadex 0-100. :
Individual monitoring of tubes uas obviously not being particularly
successful, cOnsiderable scattering of points always, being observed.
The automatic U.V. recorder traOe was not accurate enough for direct
roading of absorption to be taken from it although this method of
recording was obviously preferable to the collection and individual
monitoing of tubes. It was therefore decided to try using one of the
automatic amino aoid analyser colourinieters at 550nm to record the.
eluant absorption automatically to see if this wOuld eaccurate enough.
The io-e1 A-5m column was again used and the result was very
favourable, a clear and. even trace being obtained which was quite good
enough for accurate readings to be takei at fixed intervals. By
careful measurement ofe1ution volume at regular intervals the recorder
chart could be calibrated.
The absorption trace obtained is shown in figure l6a and clearly
has one point of inflexion with a second almost certainly present. The
derivative plot (figure 16b) has two asymmetric peaks with two others
in between which may represeüt individual peaks or one other peak -
failure for complete resolution to take place (as is always the case)
makes it difficult to decide which is the case. However, the overall
picture is clear, more so than the previous attempts but all are similar
and indicate that the type of equilibrium erstom postulated is Indeed
present.
• < vQ ©3 • to QIto
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fl) a &
rt' CtC fl 3UO13 c1P thcco o:
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cctma 61alimc2,
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IB vol or, elsutulca t78tO. ¶t
PI& J(E 11. 13$oPrIOW 'EC1UP O PHYCOYTHIW
V 1•O
V V
VV
V. ...
113.
was centrifuged to reovo any insoluble particles and was then appliod
to a tricalciumphosphtte - colito column eiactly as vida described for
a-phoerythrin. The bilip;ot0ih was olutod at a sodiwn phosphate
buffer' solution concontratlon of O.251;1 coniprod to the 0.051 to O.751
buffer oolution t'eouircd to élute B-phycoerythrin1 tloro conc'ontz'ated
buffer solution then eluted' a blue band of phycocyanin.
The visible absorption spectrun of the phycOerythin solution
collected from the column showed three peaks at 56nm (which was the
Most intense), 545nrien4 500nm (see figure 17). The optical density
ratio was 6pproxiiftatoly 310. 'This visible abo'ption spectrum
indicated the' biliprotein to, be R-.phycoerythrin. Further purification
was obtained by reprecipitatton and dialyeis and the biliprotein was
finally stored at 0°C in 'the precipitated state as for B..phycoerythrin..
The first experirent 'carried out on . sample of this R-phycoorythrin
Was treatment with J3-mOrcaptoethanol and alkyiatioi with iodoacetio acid
in the presence of 6fl guonidino o1utton, the details being exactly
• as doscribOd for B-phycoerytbrin troatod at the same time (see pages
89-9Q). Precipitation was always: fond to take place, however,, and the
• experinient was not repeated 'uàing distilled ))-morcaptoethanol as for
B-phycoerytht'in. A. amp1e was then treated with D,T.T., again
àactly as descrbod for B-phycoorythi'in (èeo pages 99-100). No
picpitation took place and results were similar to those for
13-phycoorythrin - quenching of fluorescence Tiaa ,obperved, the ao1ition
becoming a deep red colour, and the visible absorption was diminished
in intennity with the peaks a t 565nri and 500mm disappearing. The U.V.
• absorptior increased slightly in intensity. The iidications were that
effects similar to thoso described for B-p}iycoorythrin were also taking
place for the R-phycoorytbri, ' As the former was the ,bilipretein
FIGUt%Ei& frioNTrn. Aiv,i.ygis or. f-PHY(OE1?YTH1tr1.
0
C, • V01u4.'t '"!!
(p_) ELUIION •Ptocii.E
(.b) F •IRST ')el%tVA't%v6 CuavE osr ThA%LING'E3r.2. • • •
114
priasrily being studied a sample of it was applied to the molecular
sieve chromatography column but Rphyoeythrinmas not eKamined
further in this way. It seenis likely that a similar dissociation
into riirimuin m3leculd. , weight subunits had taken place, however,
An âquooüssolution of R-pliyooerythrin was also atwlied by the
frontal analysis technique using the Iiio-Gól A-5m column with
automatic moiitoring at 550nr. as described for the B.-phycoerythrin,
An Oxcellent olution profile was obtained (see figure iSa) showing
• one' point of inflexion very c1ealy, and the derivative plot
:(figuro 18b) shows two asytnmétrio .paics very ölearly. The indications
from this are that on equilibrimui systen is also present in aqueous
solutions of this biliprotein as previously indioted.
Time precluded any fui thor uoi L. on the R-phycoerythrin being
carried out.
115
(io) B±ø41ase S
&tother material uhiàh can be Used for coluan c oaraphy (ao
t:oll aa thin 2.ay0r ga-eo1id and gae-liid chromatograhioo) is
poroue g1ac, a caries of matorialo being available uith differing 0
c10001ycoiitro11ó. pore eizes (20O1 . 2 500). Thoec materiale are
rii non-compreeoiblo high oilicate g1ac 4th a not7ork of
interconnected poroc. As a column pocking thøy have somo unique
properties for Oxamplo t the rigid otricture allotio coluna. to be
uood in any posit.on and high t'Ot rates can be obtained. /iqucous and
not.equeous niodici can be ucied; coluranc can be sterilicod by heating
and organic contaianto can be ronovod by passing through hot nitrio
acid. S S
It tras dooidd to try a poroum glase coluan for asparation of tho
phy000rythrin eubunIts in equeous Golution to see if it trcc aW more
offootivo than aephddoz or J3io-Cc1. Some Aquapek (mesh e±zo 37-70;
supplied by 7aters Aoao. 0 Inâ, 0 Praniaghnm,1asa., u.s,..) wasuiod
In a alurry in 0,0111 codiuc phosphate buffer oolution end a coluiun
(10nie x 04cm.) made u. A óapo of phycoerythr:Ln eolution trac
iayorod onto the top of this column but uaa t'ound to bo irovoroibly
absorbed, not oven 611 gtianidino or 1211 urea olution' trould. oluto the
sample0 nor uould proaouro aplisCt by ucing a porifr1tio pvp.
This abeorption in duo to poz'ouc g1oa being a xYezli cation exchanger
with iydroon lone no the ativs groups.. To reduce this offct
DioRad. (fliebmond, California 0 U.S.A.) developed a vacuum oi1aniati6n S
tocuiniquo which involveo coating the glass partièleo with . S
S hoxamothy1dici1eane, this, compound. noting as stx obcorption-rsduoing 5
coating. It was docidod to try this and the glass wa p put in a
horiaontai glass co1unn (25cm. x 15mm internal diameter) mirroundod by
116'
a hoating tape. The tbo was ovacuated at 13000 for one hour and
then hexzimethyldioilazane vapour allowed to porimeato throught for
thirty Dinutos, the co1vztn uao thon ro-ovacuated and t'io procen
repeated, to. ty and onoura coating vould be Complete. inally the
column uas evacuated etjain. 0
The coated Class waa tried in a chromatography column as before
but exaCtly the sane roenite were observed, i.e# irreversible
abso'ption In caoe the coating proc003 had not worked properly it
was repeated, this time with tho glase in the coils of a condenser
and heated by rofluxing xyloue (boiling point 13600,. Botever, the
sace reoulto were again ob3erved and the conclusion seets to be that
porous glass is not a slit table column paoidng medium for the
biliprôteinae ' '
117
)ISCUsIoN
Culture of Po rph ,yridium Cruentum
This Red alga was euccessfi1ly. grown on a relatively large ocale
(up to twenty litres of meduia) in an artificial beaa-water medium based
on that developed by JOnes et.al (1963). The thediwn wae always
cterilised before. jnnoóulatjon and trans for of st I oak, solution carried
out uder 'aeoeptie donditions .st ThitG light was used to illuminate the-
cultures and they were grown at a constant temperature. Iiddition of a
traäe amount of a vitamin waG found to help growth and the' rate of
growth could be increased by igitatng the solutions 11th aix' containiflg
• 5.41 carbon 'dioxide jnstead of air alone, The cultures were harvested at
about siX weeks after which time cell lySiG tended to oCcur with release
of the biliproteiri into eolution. 0,
Etraction and Prifiation . .
The rnethod of extraction and, purification of the biliprotein generally
employed was that extensively developed in this laboratory by Paterson
(1967). 'Px'eshly harvested Porphyridiurn cruenturn was always used and
• cell rupture achieved by a comb±nation Of ultrasOnic disintegration with
freezing and thawing. The bulk of the cell reaidues were removed by
centrifugation and finer re'fliaitiing rosidue8 by filtration through celite.
• . Pwifioation of the Orude extract was basOd on precipItation (with
solid ammonium sulphate, 30-35 being required), dialysis and then
abóorption chromatography on 'columns of triosicium phosphate. . This
chromatography separated the 13-phycoerythrin from the phycocyanins and
any other impurities. It also led to' a dtatinCt improvement in the
'spectral pattern of the protein solution with the characteristic peakG
in the visible region (two maxima and a shoulder) and the UV. region
118
(three taxita) being clearly dstinguiohab1o. Inthe. visible region
the rain residual peck due to iipurity was that at 617-620ni, believed
due to allo-phycocyanin and this could he decreased further by
application of noleoular sieve chromatOraphy. The spectral purity
ratio (see pagoll) was generaUy in thc region of 3..35 after the stage
of absorption chromatography but .5 furthr stage Of precipitation, usually
raised this to around 4 which was indicative of high purity. Such
repeated fractional precipitation (with a decreasing percentage of
ainmonium sulphate being used each tiie) resulted eventually in the
formation of orystalline B-phycoerythrin, the crystals being either
needle or platelet shaped, depending on the pH of the solution # Purity
was then very high With the apetra1 ration being well over 4,
Extraction, by fractional precipitation alone is also possible and
was carried 'out on one occasion. . The methOd is, howev5r, aozewhat more
tedious and tinS consuming than absorption chromatography and has no
particular advantages. A13o tried on one •'ccaeion was extraction using
n-butanol as a solvent a This method was quite successful and reasonably
fast but required very high speed centrifugation which was not readily
available and this need therefore made use of the møthod 0me'what
impractical. Some of ,the biliprotein purified by each of these methods
was conpared but no spectral differences were observed. All three
samples were also seen to be identical in behaviour when dissociation
in uqueotia solution was tried, which eeexns to indicate that the method of
extrnotion does not affect the properties of :the biliprotoin absortion
chrocatography has been reported to. irreversibly change C-pbycoerythriu
(ScOtt and Berns, 1965).
119
UoInG, the iothod of Sanger (1045) as codifiod by 0 'Carra (1965) the
- torcinal cnino acid of the B-phycoerythvin, usa idontifiod by the
preparation of a dinitro3hony1. cle'ivative. This acrivotive uao
extracted uith an oranic o1vont after acid.hydrolyoi3 of the
Mr.—protolp and identified by paper qb±ónatoraphy by cosprioon with
inttrophon1 dorivctios of otendad anino acids. A nixturo Of the..
standard prepared fron cothionino and the un!aoun ohoued no tendency
to oplit ac tioll as travolling the soio distance iheñ run independently
;hich identified nothionino as the only I-tcraina1 anitno acid of the
D-phycoOrythrin confir-ing pro?iouo vork on this and the other,
phycooi7thrina extracted from Various eourco (oe paoc 28-29).
Only one Oolvont sytoa was sod vhere tony could havo been but this
ootpIoto agroaront uith proviOua rosuito taa considered sufficient
evidco. - .
Dieoociation of
(a) In. AnooQ. Solution
Diasociattoi of the biliprotein in aqueous solution tiao first
observed in this laboratory uhon a aetplo uae beina purified beyond the
absorption chrorator pby tao by colecular sievo cbroatQrsphy.
Soothina oiriior had previously been noticod for I-phycoorythrin both
in this laboratory and oleouhoro (3olan end O'hEocha, 1967) but no
detailed studios appeared to have boon carried out. Accodiwly it
tins Cocidod to investigato this oyoten further. -
It was found posoiblo. to ioolatá and colloct tzo of the threo
fractions forced. Those differed opoótrally, the laraer being virtually
identical in vioiblo.aboortion to nati'ephycoorythrinbu.t tho amller -
1mving only one caiiui in the viGiblo re.ion. This latter, after
120
precipitation and storing, had certain visible reior, spectral
characterstice restored uhich 'indicated that at loost partial
red zeation had taken place. By voo of an analytical' iio1ecular sieve
ohroratoarap!ly co1urn the tioleoular tzoihts of these cuunits were
eotiirate 6.35 000, 36 000 and 23 000 and the presence of a
dis000iatih,g-awociating equilibrium syoton of onome44imer#po1yiier
• uao postulated. It tzas not found pocoible tisolato the ridd10 one
of those three subunits despite repeated attopts - this was probably
duo to it being in oquilibriun uiththo monomeric speoieQ, the
oquilibriun favouring the latter.
The idea of such an equilibrium 5yetowao not new, the same having
prCviously put fcrtiard for core other btliprotoine 0 o,., C-phycocyanin,
for tihih the system onomer dimer or trinohczanordodecamer had
been poôtn1.ted (see paOo 14-17).
- Thte tioE was then tiritteliup in the form of a short communication,
a copy of tihioh is ppondo4. The equilibrium system was later studied'
further by application of the steady state approach (oo pao 124-125).
(b) rorcurjal Compounds
..roatent of D-~phvaoorythria with parachioromercuribensoato brought
about dissociation of the biliprotoin into tho fraôtions which could be
seDarated end charactericod with respect to nolecular weight by use of
an analytical molecular sieve' chromatography-column, Evidence for tho
dissociation came also frorn the colour of tho treated sample (fluorescence
-quenched) and the visible absorption spectrum (diminishod overall and
ootain spootral characteristics destroyed). The xolocular tyeiahto of
those wcroostiriiatod to be 385 000 and 36 000. Dofore any furthor
studioo could he carried out'l?ujinori and Pecoi (167b) publiohod their
øboorvations on the protein which worø ionticaI to thoso found here
121
excopt that they had not estimated the olecu1ar uôihtaóf the
subunits.
Also tried -was paramercurichlorophonylaulphonic acid, prepared by
suiphonatton of phenylmrcuribloride, and this compound was fotind to
bring about changes very sImilar to those resulting from treatment with
P.P1D. tercuric ion direct was also tried end again Vary similar
• results obtained, If anything the mercurial compounds were more
• '. effectIve than mercuric ion which seems to disprove any. idia that the
dis000iatae might be due to some interaction bottreon the chroinophore
group and a rotal ion such as mercuric ion, If this was the case rapid
and complete dissociation should have been observed when mercuric ion
zaa added to protoin solution and this uas not in fact ebsorved. It
would therefore nppav that mercuric ion and the rercurial compounds
behave in an analogouc fashion to bring about the oberved partial
dissociation.
(o) Cuaidtno Solution .
Cànoentrated guanidin hydrochloride solution: at various acidic pE
values (Tanord (1967) having pointed out the dangers of reaggregation
and/or preOjpitatibn at alkaline p}I values) was tried as a dissociating
edium. The aim was to. dtsociete the B-phycoerrthrin into its smallest
possible subunit and to eGtimato the mo1ecu1ir weight of the. same, I.e.,
the minimal moleclar weight of the biliprotein. For this purpose
roleou3.ar sieve chromatography using analytical columns was again
omployed. Initially guanidine solution alone wa. used at various acidic
pfl values but with no success, only one fraction over being isolated from
the cOlumn and that having an apparent molecular weight of . 5 000.
Do -tot. , Concentrated solutions of guanidino hydrochloride and urea are
• Imown to bring -about dissoCiation by breaking down the secondary tertiary
122
OtrUcture$ of proteins (eee page19-2O), also Dozkoeoviiny and
6rbh1jch 1967), 1.e. 9 tho non-covalent bonds Rowevér # tho above
eritionod uor: as usil as other pork (e.cc0, Davisson,
13e&corovainy ot al, 1969) indicatea that further di000ciation, uhich
roq iroc breaking of covalent bonds, cannot be browht about by such
stroe ionic solutiono nloiao. 'or tius reaeo' it is necessary to
introduce a covalent bond-breaking reagent in conjunction irit1i the strong
ionic nedium to comploto the desired dissociation. A certain de,ree of
rogrogation of the subunits produced can take place but this effect
to ninirised in such concentrated media (Bron ot el, 1968; Bazkorovainy,
et al, 1968).
P r_orcaptoothanol uan introduced as a disc lphido bond-brcacing reagent
in conjunction with the strong guanidine hydrochloride solution and this
combination uao rather more cuccesfu1 thaa gizanidino colution alone.
3poctril cbanoa indicated that some reaction uae taking place and to
fractiois uero separated on an analytical r:olccular isieve chronatography,
coluan. The larger of those otiU had an apparent iolocu1ar toiht of
around 5 000, hotrever, which sooied to indicate that the dissociation
was still inoot]pleto. The erialler faotion isolated tras found to be
colur1oes and had no chaczctoristic phycoorythrin absorption. This
raised doubts ac to whether or not it was a phycoeryhrin-typo subunit,
the other possibilities being aoo inipurity or some artifiot formed
during reaction
Id àaeethero had boon come revorenlof dissociation thiol alkylation
with iodoacetic acid tree introduced to protect the -SU groups formed when
the disuiphido bondo wore broken but sinilar results to the above wore
obtained after thie ovet when crystalline (higaiy purified) phycoerythrin
tao used. To resolve doubts no to the nature of this smaller nolocular
123
weight fraction it was analyoed for atino acid content and the
figures compared to those for native B-phycoorythrin. The results
hoted clearly that the fraction differed marlthdly in rany respects
in amiro acid comteut from natjvo l3-phycoorythrin and it would not
therefore appear to be a phy000rythrin-t,po subunit.
13-iercaptoethano1. was replaôod as the. d.tsulphide boud-brescing
reigent by the less uô;ioua and itore effioioñt Clelandto reagent,
dithiothroi.tol (D,r.Tj. This compound was also used in conjunction
with strong. guanidino hydroc4oride oolution1 Columns of Sephudex
gels were apparently coparating fractionofrou these proparations but
Daviesoi (l96) pointed out some disadvantages in the use of these gels
when such strong ionic odia were to be used for elution, the rost
important of which uao that false ootir.atee of moiecuir weight
froquontlyreoulted. He discovered that ouch difficulties could be
Overcome by use of large pore size Agr'oue gels and for this reason
Bio-GeL -50 was substituted for Sophadox. The results after O,T.P
treatnent were rather more favourable on Agarose gels and two fractions
were aopsrated for which molecular weights of 35 000 and 18 000 iere
o&1c11ated. The larger fraction was very similar to the treated sample
with reapOct to colour And abóorption opoctrum but the smaller fractioa
was colourless end had a Vsiy different absorption spectrum. The
: molecular weights iedict'ed that theos thigbt well represent monomer
and dimer but there wae again doubt as to whether or not the smaller
fraction was a phycoOrthrin-typo.. To resolve this eampleé of both
fractions were isolated in relafively large amounts from a large boale
repeat of thb sOparation and each was analysed for amino acid content.
The figures wore compared with. each other and with thoèe for a sample
of native D-pbycoerythrin. It was clear from the results that the
124-
ancuysio of the laror fraction was similar to that of phycoerythrin
but the analysis of the siaallorfractjonwacj not,- this low molecular,
weight. mateial would. appear to be some interfering. protoin not removed'
during purification' or some artifact formed during. roaction, '
• It was therefore concluded that disooeiatiointo what ceorned to be
the ninir.um molecular waiht subunit had been 'brought, about by treatment 21
with D ET,., and that this aoleoular weight was aroufld 35 000. It is
also quito possible that the same rosults vorie produced by treatment with
j34eroaptoethano1 but were not reconisod da'auch,due to the use of
Sophadez, the disadvsntao' of which were not reaiise,d at this time k
'ronta1,An,iyQia
This to1ocular sieve -chronatoaraphy tochn'iqua inVolves applying
sufficient protein solution to a' oolü5n for a plateau rogion o equal in • ' concentration to the. løaded eamp1, to be aeon in the aiiiant which allotqs
the loading and trailing edges of the citation profile to be studied
independently, It wa& used to study the equilibrium cy$ten thought to
ist in aqueous solutions' of' the Bpbycoorythrin, previoua work having
led to poe tulato of. a ononor dimorpolymer'. dissociating-associating
'system. ,' Initial results from this work were not very clear sinôe the
ocattoring of points on the '.elution profilO and the .firet derivative
eurvo of'hO trailing'edgo was considerable, but later szperiinento
conffrmôd the' early indicatiois o' the presence of at lenot wo points
of infloion on the trailing edge of the. olution. profile and two
• . ' aeymr2otric joavs on the derivative Curve of. the same. Two smaller.
poais were also observed an the lattor which were not vezy well resolved
but certainly represented one moro peak, if 'not two.
According to the theory of frontal analysis (coo pages 105-108), ,
this indicated that in aqueous solution Dphycoerythrin'formo an
125
equilibriun systen involving at. least one polyrjoric species (greater
than dinec) i.e., confirring the earlior. york. The technique does
not allou any particular point of iriflexion or poac to be assigned to
any particular species, boyover. This ctn be done for the ronoEoric
apecios by carrying out a cones of expsniiaents ovor a range of
conoontrationi and extrapolating to zero concentration but as this vould
have thvblved using freeze-dried protein (ihtoh vould therefore be
doatured, i.e., no longer in ite nativó state) this t'as not attempted.
It nit be posSible to accomplich thxo by using an aqueous solution of
procipitated protein and progreocivoly diluting the suns, changes in
coacenration being estimated by the abeorptioz at 550nn.
It is worth contioning that this closely related biliprotein seems
to ethtbit effeoto vorjeinilar to those described for B-phycoerythrin
throughout this project. t oro specifically, tratront with
-orcaptoothanol brought about spoctrol changes similar to those
observed for, B-phycoonythnixt although soperation of subunits was not
attenptod.. In aqucous solution the protein has been observed to
dicsocicto cc previoucly rontioned with the smallest subunit dotootod
having a molecular weight of around 35 000. R-phycoorythrin was also
shown to form an aesociating system similar to that of B-phy000rythrin
by tho frontal analysis tocbniquo and results indicated that polymonic
species were also involved in this oquilibr'iun,
Genoral Conclusions
This work has been principally concerned with the dissociation of
D-phycoeryth.nin. Changes in the absorption spectra flone usod to follow
or show up the Sane and rolecular sieve chromatography was used to
126
oeparate any subunits formed SB troll as to eatirnate their molecular
teights. 115 a rothod of, estimating , mplecular weights this is not
particularly acctrato but neithearo any other motiods of macromolcoule
1ecu?ar troight determination. Ilevertholoss, results obtaine(j voro
gonrfly uto•reproducib1o.
Tho d&sooiatjon,rssult f'allinto two catoCorioB - natural
dIssociation 1n aqueous solution and dissociation brought about by
6her2jcal Bothode of botd-brqakin~ g and those donot seem to be entirely
coxnplomentaz'y to erie onothôz'. Dissociation in aqueous solution was
proved by the isolation of two undoubtedly phycoorythrin-typo subunits
on a large scale and the partial reasQociation of the smaller of theSe
as woll as by the frontal analysis uork. Chemical breakdoirn reulted
intho separation at a cubuntt having tiiolöcular weight of 35 000 (from
D.T,Ti troatmont) to 36 000 (from mercurial coxpound treatment) which
• would appear to be tho minimal molecular weight of the biliprotein -
the smaller molocular weight entity ieolatod after both )3-meroaptoethanol
and D.T.T. treatment proved not to be a phyooerythrin-type in each case.
• This latter result led to Come confusion had this esallor fraction
b6cri a p eythzin-te its molecular weight of around 10 000 would
havo fitte4 in troll with thôiargar fraction andtho aonoierc.diiner
polymer potu1ate from tbe tioriç on dissociation in aqueous solution.
• IIowover, ao it would appear that 35 000 - 36 000 Ia ';iie miniimim mbleoular
uoighto this fitting in well with the pz'opàsod 'dimer', molecular weight
36 000, thero is tho question of what the fraction having noleculer
weight 23 000 roprcsonte, there being no doubt &out it being a
phycoorythrin-type. A posaiblo oaplanation is that tho kinetics of
tho d1ssocItion-o.s3ocjatjon process are not negligibly slow and for this
roacon the molecular weight uiguron obtinod from the relatively elow
127
na1ytica1 aolecular sieve cIuomatographr iothod tiere less accurate
than xpeotcd. Thero would he no suáh problern for the estimates
obtained after cheiical breakdown which therefore favours the
35 000 - 36 000 miniial molocul&r weight figure :as being the more
isliabie. It is possible that this night be resolved by roDe
quantit4tive frontal analysis work at a aeries of o nhden trati on's in
aqueous solution which should lead to a more iccurato estimate of the
ninimei moleculak weight.
overtheloso, the prinoial aim of, this projct, which the to
dissociate 13-phyco6rythrinas much,as possible into its mini imuin
molecular weight forrsi and to. estimate this inimum riolecu1r weight
would seem to have been achieved. In addtion, the presence of an
• disoociating-aocooiatiig system, obsorved oriina1ly by chance, has
• been dèmoustratOd both by the isolation andoharactèriaation of two
of the sbuni.te foz'mcd aa well as*by the frontal analysis technique.
= l3oth methods have indicatod that this eyilibriun involves at least
one polymoric species (groator than die). The aaio has also been
shown to be true for the closely related R-phycoorythrin.
•Retaining to be established, in addition to the monomeric molecular
woight froF aquooua aolution çôri, is what species are involved in the
equilibrium it night be that the onomer+dimor or trimerhexamer.
• dodecaor byston observed for C-phycocyanin may be present in solutions
of phycoorythrn as well. Frontal analysis work in both Cases would
appear to be the likeliest rnethod of resolvirg these questions.
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127
Sub-units of the Algal Biliprotcin Phycoerythrin
• By G. A. MIE1As and R. A. WALL Department of Chemistry, University of Edinburgh
(Received 14 December 1967)
l)issociatiOn of the red biliprotcin phycoerythrin fdablO from many species of red and blue—green jne) has been studied hy'Fujimori &.Pccei (1967).. The' invcstiated the action of various inercurials an the intact protein and demonstrated the existence of several sub-units from which the protein (mol. wt. øpprox. 200000 by sedimentation and gel filtration; d. Eriksson-Quensel, 1938; Nolan & O'hiiocha, l97) is presumably assembled. These workers did not describe any dissociation in untreated samples of the protein and showed gel-filtration elation d&agrams indicating only one component. How-
er, Nolan & Q'hEocha (1967) reported separation of it component of lower molecular weight (45 000) in It -phycoerythrin preparatioiis from two species cf algae on columns of Séphadex G-100.
During isolation and purification studies on two I hveocrytl inns, one from Porphyridiuim cruentunt nad the other from Rhodyinenia palniata, prelimin-
v to chemical investigations in this Laboratory, 'pavation of three red fractions was repeatedly
ebervcd on columns of Sephadex G-100. These obgervations were supplemented by further work tith analytical columns monitored by spectrophoto-m't rv, which showed that this separation was a intant phenomenon. Different methods of
rtr8ct,ion and preliminary purification gave pri)arat.ions that showed substantially the same nut ion patterns on Sephadex columns.
)xpernnentai. (a) Preparation of protein samples. '' phycocrythrin from P. crucntunr (Agardh)
na-geli, Cambridge Culture Collection of Algae and S'aPtozoa no. 138OA/]a (grown on the ASW medium ( J}iiCs, Specr & Kury, 1003), was extracted by
ttsfltineiut of an ice-cooled aqueous suspension :ornl.) of freshly centrifuged and washed cells
it l)awes Soniprobo for 15mm. The cell debris Pflrated from the bright-red aqueous solution
b centrifugation in the cold and the Protein was Vr~v il)itatccl from this solution by addition of •Aitrnte(I (N}1.4)90 4 solution in the cold. The
1 l)Itated protein was either stored under 50%-'urnted aqueous (NH4)2SO4 solution or dissolved
the minimum volume of 001 M-sodium phosphate 1)116-4, before application to a Ca3PO4-
4It column and 1)UrificatiolI by the method of flihoehs & Haxo (1900). A small amount of
luble material in this solution was removed by
filtration through a Celite pad before application to the column. The parts of the red phycoei-ythrin-containing eluato from the column that did not show admixture with allophycocyanin or phyco. eyanin by its visible ipectnini or by disc eleetro-phorosis was used for these experiments. Samples of this protein were also prepared by the butanol extraction techiiquo of ii'ujimori & Peeei (1967) and by repeated fractional precipitation with (NH4)2SO4.
(b) Gel filtration. The analytical columns were packed with deaerated suspensions of Sophadex 0-75 and C- 100 aecoi-ding to the clii-eetions of the manufacturers (I'harmacia, 1963) to give a bed 1-25cm. diam. x 25cm. tong. Samples (0-1 or 0-2inL) of pi-otcin solution, to which sucrose was added, were loaded on the columns by layering with an Agla syringe and the chromatograms were developed With 0-1 ru-sodium phosphate buffer, pH7, containing 0-1% NaN3 at 6-12m1./hr. The eluates were either continuously monitored at 253 rn/i with a flow photometer or collected as 0-5 ml. fractions on a fraction collector actuated by an optical drop counter and their extinction at 550 m1i measured with a spectrophotometer. Ultra-
• violet and visible spectra of isolated fractions were determined on a Perkin—Elmer model 137 or a Unicam S P. 800 spectrophotometer. The analytical chromnat;ograms were all developed at room temperature and were all protected from the light. Largei--seale prepni-atii'e separations were done in cooled (8-16°) columns, which were also protected from the light.
Results and discussion. The ratio of the void volume, V, to the elution volume (of a peale), I'. was plottcd against the logarithm of the molecular weight of several reference proteins (Andrcws, 1964) for each analytical column used and on the Sephadex 0-100 column the thu-ce peaks givcnby the phycoerythrin from 1'. cruentunm had ratios 1-01 (mol.wt. >85000), 166 (mol.wt. 30000) and 1-99 (mol. wt. 23000). The minimum molecular weight of a possible sub-unit of this protein was calculated from amino acid • analysis results (assuming 1 histidine residue/sub-unit) to be about 18000, which suggests that a monomer—climei--
• polymer system was what was observed. A. less detailed investigation of the phycoerythnin isolated
128 G. A. I'tLTERAS AND R. A. WALL loOs
by Ca3PO4 chromatography from aqueous extracts of Ceramium nthrum gave similar but not identical results. On Sephadex columns this biliprotein also showed three components, which had apparent molecular weights of :35000, 70000 and 85000, suggesting a possible dimer—tetramer--polyIfler system (minimum sub-unit mol. wt. by amino acid aialvsis 19000; O'hEocha, 1905). No traces were observed of a lower-molecular-weight component with the C. rubruin protein.
When solutions of the lowest-molecular-weight fractions of the phyeoerythrins of P. crue alum and C. rubruns were treated with sufficient solid (NH 4)2SO4 to precipitate all the rod solute, a solution of the precipitated material in 0-1M-sodium phosphate buffer, pH7, demonstrated only one component on Sephadex G-100 columns. The olution volume of this ¶rcaggregated' component was the same as that of the 'heavy' fractions in the original preparations.
A decrease in the intensity of the longest-wavelength absorption peak of the visible spectrum was observed with each isolated 'light' fi-action, just as reported by Nolan & O'liEoeha (1967). Visual observations suggested that the fluorescence was also decreased, although not completely quenched as is the case after thermal denaturation of biliproteins. The 'rcaggt'egatcd' protein pro-duced by (N}I4)2SOt precipitation of the 'light' fraction from P. cruentum showed at least partial restoration of the long-wavelength absorption towards that of the 'heavy' fraction, although the shoulder at 500mc present in the spectrum of the 'heavy' fi-aetion was not recovered.
These results indicate that phyeoerythrin, like phycocyaniti (Scott & Barns, 1965; Hattori, Crospi & Katz, 1965), forms an associating system in aqueous solution. The qttantitative aspects of
the association will need detailed study by ultra. centrifugal and diffusion techniques. The sub-unit peaks on the analytical columns were very diffuse, a condition that became very serious when th elution rate was decreased in an attempt to increase resolving power. It would appear that the kinetic of the association process are not negligibly slow, at least not for periods of several hours. This means that these values may not he aecurato, although fully reproducible, so that it may be that all bili-proteins follow the monomer—trimer—hexamer--dodecamer pattern of dissociation observed in the C.phycocyanin system (Seotv & Berns, 1965; Hattori et at. 1965). It is hoped that the Gilbet-t-Kellett (Kellett. 1967) steady-state approach to this type of gel filtration problem will yield more reliable answers.
We thank Professor C. O'hEocha and his stall at the Department of Biochemistry, University College, Cal,v, Irish Republic, for much assistance mcI good advice duri
ayng
a working visit (by H. A. W.). Ihis work was supported by the Browne Research Fund of the Royal Society and the Science Research Council.
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