I95I

Purification of Horse-Radish Peroxidase and Comparison ofits Properties with those of Catalase and Methaemoglobin

By D. KEILIN AND E. F. HARTREEMolteno In8titute, Univer8ity of Cambridge

(Received 13 September 1950)

Although the peroxidatic properties of animal andvegetable extracts were recognized already bySchonbein (1863) and ascribed by Bourquelot (1898)to special indirect oxidizing enzymes whichLinossier (1898) named peroxidases, the firstsuccessful isolation and purification of an enzyme inthis' category was achieved between 1917 and 1924by Willstatter and his co-workers. They describedthe main properties of horse-radish peroxidase andgreatly improvedthemethod ofestimation ofenzymeactivity, expressed in terms of 'Purpurogallinzahl',P.z. or purpurogallin number, P.N. (for a definitionsee p. 89). The unusually high values of P.N. recordedby Willstatter & Pollinger (1923) can only beascribed to some error in their calculations. Thus ifthe total iron (0-064 %) found in their preparationwith P.N. = 3070 belongs to the prosthetic group ofthe enzyme, then, in the light of our present know-ledge, it can be estimated that the purity of theirmost active preparation was probably 40-50% andthat the P.N. values given in their paper are too largeby a factor of about 5. However, to obtain at thattime a 40% pure enzyme was in itself a remarkableachievement. It not only paved the way for furtherstudy of peroxidase, but stimulated similar work on

other enzymes at a time when it was still believed(Warburg, 1928) that owing to their inherentfragility all attempts to isolate and purify intra-cellular enzymes would have no chance of success.

Willstatter's classical study of peroxidase was

followed by extensive work on the purification ofthisenzyme with a view to determining its chemnicalnature.The presence of haematin in horse-radish root

itself (Keilin, 1927) and in enzyme preparations ofwidely different purities (Kuhn, Hand & Florkin,1931; Elliott & Keilin, 1934; Sumner & Howell,1936) suggested the possibility that the enzyme is ahaematin compound. However, it was only afterfurther knowledge had been gained of the propertiesofmethaemoglobin and especially of the compoundsit forms with hydrogen sulphide (Keilin, 1933), withsodium azide (Keilin, 1936) and above all withperoxides (Keilin & Hartree, 1935) that the haematinnature of peroxidase was definitely established byKeilin & Mann (1937). This opened the way for a

much more precise study of the properties of theenzyme and of the kinetics of its reactions.

Peroxidase offers one of the most instructiveexamples for the study of the chemical structureof an enzyme. Thus the criteria upon which thehaematin-protein nature of peroxidase as well as itsclose relationship to catalase and to methaemo-globin were established (Keilin & Mann, 1937) can besummarized as follows: (1) Presence in all peroxidasepreparations, from horse-radish root itself to themost highly purified enzyme, of a pigment with anacid methaemoglobin-like absorption spectrum.(2) Proportionality between the intensities of theabsorption spectra of this methaemoglobin-likepigment or of its pyridine-haemochromogen de-rivative and the activities (P.N.) of different enzymepreparations. (3) Formation of spectroscopicallywell defined compounds between this pigment andinhibitors of peroxidatic activity such as potassiumcyanide, hydrogen sulphide, sodium fluoride andnitric oxide. (4) Formation of well defined com-pounds between this haematin pigment and hydro-gen peroxide which is the substrate of the enzyme.(5) Rapid decomposition of haematin pigment-peroxide complex and the liberation of the pigmenton addition of 'acceptors' like pyrogallol, quinol,ascorbic acid and others which can undergoperoxidatic oxidation. (6) Demonstration of theabove reactions between the enzyme and inhibitorsor substrate (i.e. hydrogen peroxide) not only withpurified enzyme but also with crude root extractsand even slices of horse-radish root.The peroxidase-peroxide compoun'd provided the

first example of direct visual demonstration ofunionbetween an enzyme and its substrate. According tothe amount ofhydrogen peroxide added two distinctred compounds are formed with somewhat differentabsorption spectra. On the other hand, there isno spectroscopically detectable reaction betweenperoxidase and substances such as pyrogallol,quinol, etc. Peroxidase also resembles methaemo-globin in changing into a redder alkaline form athigher pH (> 10) and in being reduced by sodiumdithionite (Na2S204) to a ferrous form which cancombine with carbon monoxide.The purest preparation of Keilin & Mann (1937)

had P.N. about 950 and contained 1-2% haemin. Itwas also shown that milk peroxidase is also a met-haemoglobin-like compoundwhich will combine withpotassium cyanide, sodium fluoride and hydrogen

88

PEROXIDASE, CATALASE AND METHAEMOGLOBINperoxide and, after reduction (Na2S204), with carbonmonoxide.A large-scale preparation of horse-radish peroxi-

dase described by Theorell (1942a) enabled him toisolate the enzyme as a pure crystalline materialcontaining 13-2 % nitrogen and 1-48 % haemin,estimated as pyridine haemochromogen. The mole-cular weight of the enzyme calculated from thesedimentation and diffusion constants was found tobe 44,100 (Theorell, 19421b) while the catalyticactivity or P.N., estimated in the usual way, was

about 930, the highest value reached being 1100.With this preparation Theorell corroborated themain results of the previous work (Keilin & Mann,1937), especially the haematin nature of peroxidaseand the compounds it forms both with its inhibitorsand with peroxide. However, in addition to the twored peroxidase-peroxide compounds previouslydescribed, Theorell discovered a third enzyme-substrate compound which is of greenish colour andshows an absorption band at about 652 m,u.(Theorell, 1941). Since this green compound is thefirst enzyme-substrate complex formed, the number-ing of these derivatives has been modified accord-ingly. Thus the first green, the first and second redcompounds are now referred to as peroxidase-peroxide compounds I, II and III respectively.Theorell (1940) was also able to split peroxidase intoits protein and haematin components and to recon-

struct active enzymes from the native protein anddifferent haematins (Theorell, Bergstr6m & Akeson,1943).In the present paper a description will be given of

a comparatively simple method of preparation ofpure horse-radish peroxidase. The spectrophoto-metric curves of peroxidase and its known reversiblederivatives will be given both for the visible and theSoret region, and the properties of peroxidase will becompared with those of catalase and methaemo-globin.

Nomenclature. The catalytic system in whichperoxidase is involved is composed of (1) enzyme:

peroxidase, (2) substrate: hydrogen peroxide, and(3) a substance undergoing peroxidatic oxidation,usually called an 'acceptor', but which will bereferred to in this paper as a hydrogen donor.

EXPERIMENTAL

MethodsActivity ofperoxidase preparations was determined through

the purpurogallin test as carried out by Elliott & Keilin(1934), Keilin & Mann (1937) and others, which is a slightmodification of the original method devised by Willstaitter &Stoll (1918). Suitably diluted enzyme (0-1-0-5 ml.) was

added to 500 ml. glass-distilled water at 200 containing1-25 g. resublimed pyrogallol. No commercially availablesamples of pyrogallol were satisfactory unless resublimedunder reduced pressure (approx. 40 mm. Hg). H202 (0-4 ml.

6% w/v) was addedwith stirring, and after exactly 5 min. thereaction was stopped by the addition of5 ml. 5N-H2SO4. Theyellow purpurogallin was extracted by shaking with threelots of ether (80, 30, 30 ml.) which had been freed fromperoxide by distillation over FeSO4 + CaO. The volume of theether solution was measured, the solution dried with an-hydrous Na2SO4 and the concentration of purpurogallinestimated in the Hilger Spekker absorptiometer using 1 cm.cells and Ilford 602 filters. The instrument was calibratedwith ethereal solutions of twice recrystallized purpurogallin.The quantity of enzyme taken for the test should be suchthat less than 10 mg. purpurogallin is produced.Enzyme activity is expressed in terms of purpurogallin

number, P.N. =W/vd, where w =mg. purpurogallin formedunder the above conditions from v ml. of enzyme solutionand d =the dry weight in mg. of 1 ml. of solution.The number of enzyme units in a peroxidase preparation,

E.U. = Vw/1000v, where V is the total volume of the per-oxidase solution.Dry weight. Solution (1-2 ml.) was evaporated to constant

weight at 105° in a weighing bottle. Prolonged heating wassometimes necessary and the dry residues were often hygro-scopic.

Nitrogen was estimated by the micro-Kjeldahl method, theNH3 being distilled in the Markham (1942) apparatus intoHCI which was back titrated with 0-01N-Ba(OH),.Haemin concentration was estimated as follows: A

suitable volume (1-2 ml.) of the preparation was pipettedinto a 10 ml. volumetric flask containing 0-4 ml. N-NaOH towhich 2 ml. pyridine was then added followed by about50 mg. Na2S204. The flask was made up to the mark withdistilled water, stoppered, well mixed and allowed to stand0-5 hr. The concentration of haemin, as pyridine haemo-chromogen, was measured in the Spekker absorptiometerusing 1 cm. cells and Ilford 605 filters. The blank cells con-tained all ingredients except peroxidase. For calibrating theintrument haemin was recrystallized from pyridine-CHCl3-acetic acid. A stock 0-1 % solution of this haemin was madeup in 0-1 N-NaOH and measured volumes of it were taken forpreparation of standard haemochromogens. The final con-centration of haemin must not exceed 4 mg./100 ml. Theestimation of haemochromogen by spectrophotometricmeasurement of the height of the sharp a-band at 556 m,.was considered to be insufficiently reliable since a slight shiftof the band can introduce a large error. An additional errormay arise from variation in the height of the a-bandaccording to whether protein is present or not. The measure-ment with a Spekker absorptiometer as described aboveobviates these difficulties.Throughout this paper results are expressed as

haemin', i.e. ferric protoporphyrin chloride, since thiscompound is weighed out to make the standards. In earlierpapers results have been given as '% haematin' (ferricporphyrin hydroxide, formed on dissolving haemin in alkali),although in fact the analyses were based on haemin. Since inperoxidase one ofthe two residual valencies of Fe is linked tothe protein and the other either to hydroxyl (Theorell &Paul, 1944) orto a molecule ofwater (Keilin & Hartree, 1949),the molecular weights of haemin and of the prosthetic groupof peroxidase are 652 and 635-636 respectively. Hencehaemin values will be approx. 3% higher than the truevalues for the accepted prosthetic group. However, for thesake of consistency we shall refer to 'haemin content'.

Spectroacopic observations were carried out with the Zeissmicrospectroscope and Hartridge reversion spectroscope.

VoI. 49 89

D. KEILIN AND E. F. HARTREE

Ab8orption curves of peroxidase and its derivatives weredetermined with the Beckman photoelectric spectrophoto-meter using 1 cm. cells. The following units are used inplotting the curves: Optical density D =logl0 (I0/I); ex-tinction coefficient K = D/l; molar extinction coefficiente= D/lc, where I= optical depth in cm. and c = molar concen-tration of haemin. In an earlier paper (Keilin & Hartree,1945 a) we plotted the absorption constant , =log,, (Io/I) /lc,where c is expressed as g.mol./ml. The absorption coefficientis therefore 2303 times greater in terms of fi than in termsof E.

Estimation of peroxidase in horse-radish roots and crude.enzyme extract8 was carried out by means of a microspectro-scope ocular and double wedge trough as described by Elliott& Keilin (1934) and illustrated by Keilin & Wang (1946). Theprinciple of the method consists in matching the absorptionbands of pyridine haemochromogen obtained from per-oxidase against pyridine haemochromogen prepared fromcrystalline haemin, or in matching the absorption bands ofone of the reversible derivatives (i.e. fluoride) ofperoxidase inslices or extracts with those of the corresponding derivativeobtained from pure peroxidase.

Calcium phosphate gel. To a 101. glass jar containing 1 1.tap water and 250 ml. 0 6M-CaCl2 was added, with stirring,250 ml. 0 4M-Na3PO4 followed by sufficient N-acetic acid tobring the pH to about 7-3. The jar was filled with water andthe precipitate washed six times by decantation. It was

finally collected in centrifuge tubes and suspended in tapwater to give about 400 ml. of a thick cream.

Catalase was prepared from horse liver as previouslydescribed (Keilin & Hartree, 1945a).

Notatin was the same material as described in a previouspublication (Keilin & Hartree, 1945b).

Alternative procedures. For the estimation of purpuro-gallin concentration the Spekker absorptiometer can bereplaced by the King photoelectric colorimeter (with bluefilter) or by any standard visual colorimeter, while the Kinginstrument with the green filter gives very reliable results inthe estimation of haematin by the above method. Since it isin the visible rather than in the Soret region of the speotrumthat the reactions of peroxidase can be followed, a visualspectrophotometer (e.g. Hilger-Nutting) is adequate forquantitative studies of peroxidase.

Isolation of peroxidase from horse-radish root

Preparation of crude extract. Roots of wild horse-radish(Cochlearia armoracia L.) were gathered in spring at thetime when the first shoots are appearing and the interferencebyroot polysaccharides duringfractionationis at aminimum.Well washed roots, after removal of damaged portions, were

cut into 2-4 cm. long sections and passed through a mech-anical mincer. The juice was extracted from 50 kg. mincewith a stainless steel hydraulic press and water, equal involume to the press-juice, was well mixed with the mincewhich was then pressed out again. The process was repeatedonce more and the three extracts were mixed. When dealingwith large quantities of root it is advisable to work in theopen air, or alternatively to use a respirator for protectionagainst the strongly lachrymatory mustard oil (allyl iso-thiocyanate) which, during the mincing, is rapidly liberatedfrom the glucoside sinigrin by a specific glucosidase.

Firstfractionation. About 69 1. of the above crude extractwas treated with 47 kg. (NH4)2SO4 and the mixture (0-92saturation) left standing overnight. Next day the precipitate

which had risen to the top was collected, placed on two27 cm. Buchner funnels containing a bed of kieselguhr andthe residual fluid was sucked off. The hard cakes were in-verted on to sheets of paper, the kieselguhr scraped off andthe precipitate suspended in 21. water. Since a suspensioncan only be obtained by kneading with the fingers it isadvisable, on account of the residual mustard oil, to wearrubber gloves and to work in a well ventilated fume cup-board. The suspension was placed in cellophan tubing anddialysed for 3 days against running tap water to remove(NH4)2SO4. The contents of the tubing were then filtered togive 4 1. of solution A. This solution was treated with 1248 g.(NH4)2SO4 (0 5 saturation) and the precipitate (b) which wasformed only after several hours was filtered off. The filtrate(4600 ml.) was brought to 0-85 saturation by addition of1150 g. (NH4)2SO4 when a sticky precipitate (c) appearedgradually and adhered to the glass container. The two pre-cipitates b and c were separately dissolved in water anddialysed until salt-free, giving respectively the solutions Band C. Solution B contained much coloured impurity, whichgave it a greyish-green colour, and had a P.N. of 24. The bulkof the enzyme was in the brown solution C which had a P.N.of 129 and contained 2040 E.U. At this stage the fractiona-tion was interrupted while a second batch of horse-radishroot (48 kg.) was worked up in the same way to give thecorresponding solutions B1 (P.N. =32) and C1 (P.N. = 112;2100 E.u.).

Further purification offractions B and B1. These fractions,which contained 15% of the total enzyme units, were com-bined to give 1025 ml. solution, treated with 100 ml. trical-cium phosphate gel and centrifuged after 5 min. The super-natant fluid was treated with a further 100 ml. calciumphosphate gel and the gel cakes were discarded. The solution(1050 ml.) was brought to 0 40 saturation by addition of264 g. (NH4)2SO4 after which the precipitate was centrifugedoff and discarded since it contained only 89 E.U. of P.N. = 27.Asecond precipitate obtained byaddition of232 g. (NH4)2S04(0.70 saturation) was collected on a filter, dissolved in theminimum amount of water and dialysed until completelyfree of salts. The solution B2 thus obtained had P.N. = 163 andE.U. = 568.

Isolation of the pure enzymeThe solutions C, C1 and B2 were now combined to give 2 1.

solution D (P.N. = 125; E.U. = 4630), the starting material forthe next stages of purification.At all subsequent stages, unless otherwise stated, dialysis

was carried out first against running tap water and finallyagainst distilled water. On all occasions where ethanol waspresent, the liquids and centrifuge tubes were cooled in ice,but the use of a refrigerated centrifuge was found to be un-necessary.

(a) Precipitation with ethanol. One-third of solution D(i.e. 667 ml.) was divided equally among four 1 1. centrifugetubes to each of which was added 1-5 vol. (250ml.) 90%ethanol. The tubes were centrifuged for 10 min., the stickysolid was discarded, while the supernatant from each tubewas poured into another similar centrifuge tube containing416 ml. 90% ethanol and 10 ml. 0 6m-CaCl2; the final con-centration of ethanol was thus about 72 %. The tubes wereimmediately centrifuged for 15 min. and the supernatantfluid discarded. The precipitates from the four tubes weresuspended in a little distilled water and kept at 0° while theremainder of solution D was treated in two lots in the sameway. The suspensions of precipitates were finally united and

90 I95I

PEROXIDASE, CATALASE AND METHAEMOGLOBINdialysed overnight against 5 1. distilled water at 5° to give567 ml. solution E (P.N. = 586; E.U. =4000).

(b) Fractionation with calcium phosphate and ethanol.Solution E was added with stirring to a mixture of 510 ml.water and 76-5 ml. calcium phosphate (37.4 mg./ml.). Thecalcium phosphate was centrifuged off, washed with 100 ml.water and the cake eventually discarded. (Elution of thecake with 2% potassium oxalate gave a dark greyishsolution containing only 56 E.U.) The supernatant solutionand washings (1147 ml.) were diluted with an equal volumeof 90% ethanol followed by 408 ml. ethanolic calcium phos-phate (a mixture of equal volumes of 90% ethanol and thecalcium phosphate suspension). The mixture was centri-fuged for 10 min. and the cake was immediately suspended inwater to a final volume of 200 ml. The supernatant fluid wasfurther treated with two successive lots of ethanolic calciumphosphate (200, 100 ml.) and the practically colourlesssupernatant was discarded.

(c) Fractionation with (NH4)2SO4. The combined browncalcium phosphate suspensions (500 ml.) were mixed with anequal volume of saturated (NH4)2S04, allowed to stand 1 hr.and centrifuged. The cakes were extracted twice more withsmall quantities of0 5 saturated (NH4)2S04 and the washingsadded to the dark-brown supernatant fluid. The total volumeofsolution at this stage was 1290 ml. By successive additionsof solid (NH4)2SO4 to 0-60 saturation (84 g.), 0-68 saturation(68 g.) and 0-85 saturation (171 g.) three precipitates wereobtained and filtered off. A little peroxidase was held insolution by the ethanol present even at 0-85 saturation with(NH4)2S04, but by keeping this solution in an open dish over-night a fourth precipitate, containing the remaining enzyme,was obtained.The first precipitate was rejected, the second was dissolved

in water and dialysed to give 222 ml. solution F (P.N. = 753;579 E.u.) while the third and fourth were combined, dis-solved and dialysed to give 232 ml. solution G (P.N. =831;3520 E.u.).

(d) Heat denaturation of impurities followed by (NH4)2SO4fractionation. Solution G was diluted with water to 1334 ml.and treated with 306 g. (NH4)2SO4 (0-38 saturation).Quantities of :* 200 ml. were heated rapidly to 620, held atthis temperature for 10 min. and rapidly cooled to roomtemperature. The combined heat-treated solutions werecentrifuged to give a grey cake insoluble in water, and aslightly cloudy fluid which was clarified by filtration throughWhatman no. 5 paper. (Although centrifuging did not yielda clear fluid, it accelerated the subsequent filtration.) The

clear fluid (1340 ml.) was fractionated by successive additionof (NH4)2S04: 168 g. (0-56 saturation), 75 g. (0-65 saturation)and 44g. (0-70 saturation). The three precipitates werefiltered off, dissolved separately in a little water and dialysedto give solutions containing 700, 2550 and 344 E.U. re-spectively. The second fraction, H (0-65-0-70 saturation),had a total volume of 104 ml. All attempts to increase theP.N. and haemin content of fraction H by further (NH4)2S04fractionation were unsuccessful. In fact, the variationsbetween different fractions were within experimental errorand the average analytical figures were: haemin 1-61%,nitrogen 13-2%, P.N. = 1220±50. The behaviour of fractionH in the ultracentrifuge showed that it was almost homo-geneous (see Addendum to this paper, p. 105).

Absorption curves of peroxidase and its derivatives

The positions of the absorption bands of per-oxidase and its derivatives have been recorded byKeilin & Mann (1937). The spectrophotometriccurves of free peroxidase and 'paraperoxidase' havebeen given by Theorell (1941, 1942a), whereascurves for the Soret bands of the three peroxidase-peroxide compounds and of the peroxidase-fluorideand peroxidase-cyanide compounds have beenrecorded by Chance (1949 b).For measurements of light absorption of per-

oxidase and its derivatives in the Beckman spectro-photometer, solutions were prepared as shown inTable 1. They were suitable for density determina-tions at the red end of the spectrum while at lowerwavelengths the peroxidase concentration had to bereduced five to twenty times. In making suitabledilutions the concentrations of the other ingredientswere kept constant. The absorption curves of per-oxidase and of its derivatives except those withH202 are grouped in two figures. Fig. 1 representsthe absorption curves of free peroxidase and of itscompounds with fluoride, cyanide and azide, whileFig. 2 shows the absorption ofalkaline peroxidase, ofperoxidase reduced with Na2S204 and of the latterafter treatment with CO.The comparison of these curves with those of the

corresponding derivatives of methaemoglobin and

Table 1. Preparation of solutions for spectrophotometric measurements on peroxidaseand various derivatives other than the H202 compounds

(A stock solution of peroxidase (0-386 mM, r.N. = 1220) was used to prepare the following solutions.)

Other ingredients8 ml. 0-25M-Phosphate buffer, pH 5-60-3 ml. 0-7m-KCN in m-KH2PO40-6 ml. 2-5Sm-NaF4-0 ml. 0-7m-NaN3 in 0-25M-acetatebuffer, pH 4-25

3-0 ml. 0-6m-K2CO3 (pH 11-4)4-0 ml. 0-25M-Phosphate buffer,pH 6-8, 20 mg. Na2S204*

Saturated with CO*

Final haeminconcn.(IM)77-470-364-577-4

64-542-9

42-9

Spectrum obtainedPeroxidaseCN--peroxidaseF--peroxidaseN--peroxidase

Alkaline peroxidaseReduced peroxidase

CO reduced peroxidase* Solutions covered with layer of paraffin during measurements.

Peroxidasea 2-0 ml. stockb 3-0ml., ac 3-0ml., ad 1-0 ml. stock

e 0-6 ml. stockf 0-5 ml. stock

g asf

VoI. 49 91

D. KEILIN AND E. F. HARTREE I95I

O O~~~~~~~~~~~~~~~~~~r

-~~ 0*4 CN ~~~~ \~~o~CN7004 660 62Peroxidase580Feroxidase 6)0

0-2 440

0s8Reducedtj/ j^<\>J( / | q~~~~~~~~~~~~~, /

02~ ~~~L> 20I

- --<-~~ ~ ~ ~ ~ ~ ~ -' ./' ~~~~Soret0 0700 660 620 580 540 500 460 420 380

Wavelength (m,u.)Fig. 1. Absolute absorption spectra of peroxidase and of its fluoride (F-), cyanide (ON-) and azide (N8- compounds. For

deta ilso fsolutionsused see Table 1. e = 1/cl logl0 I,I, where c = molar concentration of haematin and. optical depthin cm. Ordinate scale (e/104) reduced 20-fold for Soret bands..

Reduced CO

1.0 /

0-8 - \~// / ~~~~~Reduced CO

~14-0

I, " ~~~Alkaline12~~~~06 I / ~~~~~~~~~~~~~~Peroxidase

II / ~~~~~~~~~~~~~~~-10*0

0-4 eroxidase -lklie8-0

0 ~~ ~ ~ ~ iI I

7M 660 620 58 540 5/ 46 420 38Waveengh I \.

Fi. bslteabortinspcta f eoxdae,alanepeoxdae rduedpeoids an C -reue eoiae02 ea/ fsouinue eeTbe1 o dfnto funt e i.1

92

PEROXIDASE, CATALASE AND METHAEMOGLOBINcatalase will be discussed at a later stage. Here weshall note only a few characteristic features of theperoxidase curves.

(1) The height ofthe band in the red of free peroxi-dase is somewhat greater than that given by Theorell& Paul (1944). Their value for p640=0-585 x 107 isequivalent to Em0 = 0-254 x 104, whereas our value is0-284 x 104. A similar proportional increase in thevalue of e for the Soret band was found on comparingour results (see Fig. 3) with those of Chance (1949 b).

(2) The azide derivative of peroxidase is onlyformed below pH 4*5 (Theorell, 1942 c), while at pHlower than 4 the compound becomes unstable asindicated by a drift in optical density readings.

08r-

I.

.1I0110 6 _ t~~~(H202)

IH2020-6-

detection and estimation of peroxidase in roots andin crude extracts is more easily carried out after itstransformation to one of these derivatives.

(5) The Soret band of CO-ferroperoxidase is muchhigher and sharper than the corresponding band inother derivatives of peroxidase.

Peroxida8e-peroxide compournd8Peroxidase, as was mentioned previously, forms

three compounds with H202: the green compound Iof Theorell (1941) which is a precursor of the two redcompounds II and III of Keilin & Mann (1937).Compound II, which shows absorption bands at 555and 527 mix., is formed with as little as 1 mol. H202

120

Peroxidase - 10-0Peroxidase

880

dase~~~~~~~~~~

700 660 620 580 540 500 460 420 380Wavelength (m,u.)

Fig. 3. Absolute absorption spectra of peroxidase and peroxidase-H202 compounds I, II and III. The pecked linesrepresent the Soret bands of peroxidase and of its three peroxide complexes as obtained by Chance (1949b). Thefull lines are based on the results of the present investigation.

(3) On addition of fluoride to peroxidase the ab-sorption band in the red is replaced by a muchstronger and sharper band at 612 my. which is,however, not as strong as that of the cyanide com-pound or ofreducedperoxidase and itsCO derivative.Nevertheless, peroxidase in horse-radish roots ismore readily detected as the fluoride compound thanas any.other derivative since the main band is at awavelength where there is no interference fromother pigments and, at the same time, it is moreintense than the peroxidase band at 641 mp. TheSoret bands of the F- and CN- compounds have alsobeen plotted by Chance (1949 b, Fig. 6).

(4) Since all derivatives of peroxidase show muchsharper absorption bands than the free enzyme, the

per Fe atom and even in absence of acceptor itrapidly reverts to free peroxidase. On the otherhand, the addition of 15-25 molecules of H202 leadsto the formation of compound III which is also redand shows absorption bands at 583 and 546 mp.Furthermore, a peroxide concentration high enoughto yield 100% of complex III will bring about afairly rapid destruction of the enzyme with an inter-mediate formation of a compound showing a distinctband in the red at 670 mit. This compound alsoappears when any of the peroxidase-peroxidecomplexes (I, II or III) are repeatedly reformed bysuccessive additions of small amounts of H202 toperoxidase liberated from the complex. The com-pound with an absorption band at 670 mbt. resembles

93VoI. 49

D. KEILIN AND E. F. HARTREEthe complex IV described by Chance (1949b) asbeing formed in the reaction between peroxidase andmethyl hydrogen peroxide where, in absence of aderivative corresponding to peroxidase-H202 III, itfollows directly the formation of complexes I and II.It is, however, quite conceivable that the band at670 mit. belongs to a peroxidase with a modifiedprosthetic group (a verdo-type as was suggested byChance) not necessarily combined with peroxide.This complex therefore is not comparable with I, IIand III and the term complex IV is hardly appro-priate for it.

Spectrophotometric curves of the peroxidase-peroxide complexes (Fig. 3) were determined by usonly for complexes II and III in the visible region.Since the light absorption is constantly changingduring these determinations, it is necessary to obtainrecords of changes of density with time at each of anumber of wavelengths and from such records toconstruct the curves. Under the experimental con-ditions of these determinations no oxygen bubblesappeared. The Soret bands of all three complexes aretaken from a recent paper by Chance (1949b).

Peroxida8e-peroxide I is the first compound formedwhen a very small amount of H202 is added toperoxidase. Direct spectroscopic examination re-veals a band at 657 m,u. As the compound is veryunstable and rapidly passes into complex II itsspectrophotometric curve cannot be recorded by thepresent method. However, its Soret band, de-termined by Chance (1949a, b) using his rapid flowmethod, is given in Fig. 3.

Peroxida8e-peroxide II. The stock peroxidasesolution, 4-52 ml., described in Table 1, was dilutedwith 0 25M-phosphate buffer, pH 5f6, to 25 ml. anda solution of H202 (0.00446M) was made up in0 125M-phosphate buffer, pH 5-6. The formersolution (2-5 ml.) was mixed with 1 ml. of the latter,the mixture placed immediately in a Beckmanabsorption cell and readings of density (D) weretaken with the miimum delay at 595 and 586 mjt.Records were kept ofthe times at which each readingwas taken, and six further concurrent sets ofreadingswere then taken alternately at the same two wave-lengths. Thus during 15 min. seven values of D atdefinite times after mixing were obtained at each ofthese wavelengths. The above operations were re-peated at seven other series of two wavelengths sothat data became available for each of the followingsixteen wavelengths: 595, 586, 583, 580, 578, 573,568, 564, 558, 553, 545, 538, 530, 520, 500 and 470m,.In the region above 595 m,u. the changes ofD wereslower and it was possible to 'scan' the followingwavelengths using one peroxidase sample: 750, 700,680, 672, 660, 640 and 615 m,u. From the variousdensity-time curves, absorption curves at varioustimes were constructed and those for 1-5, 7, 10 and13-5 min. are given in Fig. 4. The 1-5 min. curve

represents a mixture of complexes II and III inwhich the latter predominates. Since the H202slowly decomposes the later curves show the a- and#-bands of II. The ratio of the band heights ap-proached a constant value at 13*5min. and thecorresponding curve may therefore be taken as thenearest approximation to that of peroxidase-peroxide II (see also Fig. 3).

K

0

Wavelength (mp,.)Wavelength (mp,)

Fig. 4. Changes of absorption spectrum of peroxidase withtime (min.) after addition of H202. Series II experiments.Peroxidase, 5{00 x 10-EM; H202, 128 X 10-8M; 0.2M-phos-phate buffer, pH 56. K = 1/i loglo IO/I, where I =opticaldepth (1 cm.).

Peroxida8e-peroxide III. For the study of thiscomplex the same solutions were used as for complexII except that the H202 concentration was five timesgreater. Since D now changes more slowly eachperoxidase sample could be used for the study of thedensity-time changes at four wavelengths instead oftwo. The region 595-750 m,u., as in the previousexperiment, could be covered with one sample ofenizyme. It was found that there was a drift inreadings during the first minute after which theabsorption remained constant until the end of thethird minute. Subsequently there was a progressivedecrease in D at all wavelengths indicating a de-struction ofperoxidase. The 3 min. absorption curve,which may be taken as a close approximation to thatof complex III, is shown in Fig. 3. Since a slightdecomposition ofthe pigment has occurred the bandsas represented are a little too low.Of the three complexes the peroxidase-peroxide

III shows the most distinct absorption spectrumwith two bands in the visible region resembling thoseof methaemoglobin-peroxide. The absorption spec-trum of complex II is of similar pattern, but the

94 I95I

PEROXIDASE, CATALASE AND METHAEMOGLOBINbands are less distinct and nearer to the blue. On theother hand, the Soret band of III is lower than thatof II. The absorption spectrum of I is of the samepattern as that of free peroxidase, but it has anexceptionally low Soret band (Fig. 3).A summary of the absorption maxima of different

derivatives, based upon the spectrophotometriccurves represented in Figs. 1-3, is given in Table 2.

cooling a solution of alkaline methaemoglobin andinterpreted it on the assumption that the sixthoctahedral position around the iron atom in acidmethaemoglobin and also in neutral peroxidase isoccupied by a molecule of water. The transition tothe alkaline form would then involve not the additionof a hydroxyl ion but rather the dissociation of aproton, as was previously suggested by Coryell,

Table 2. Wavelength8 (mp.) of ab8orption mzaxima and (in parenthese8) the corresponding values of 6 x 10-4for peroxida8e and derivativeB

Peroxidase (Fe+++) 641-5 (0 284) 497 (1-003) 403 ( 9-10)Alkaline peroxidase 574-5 (0.686) 545 (0 864) 416 ( 8-85)Reduced peroxidase (Fe++) 556 (1-122) 437 ( 7.73)Reduced peroxidase CO 572-5 (1-140) 542 (1.177) 423 (13-80)CN--peroxidase 538 (1;020) - 423 ( 9.39)F--peroxidase 612 (0693) 487-5 (0-755) 404 (13-00)N3-peroxidase 635 (0-170) 534 (0.820) 416 (11-40)

The wavelengths of the absorption bands of theunstable H202 complexes recorded in Figs. 3, 4 and 6differ slightly from the figures obtained on directvisual observation with either the microspectro-scope or reversion spectroscope (Table 3). This is duepartly to the differing experimental conditions andpartly to the fact that close agreement between thetwo methods can only be obtained when the ab-sorption bands are symmetrical.

Effect of low temperature on the ab8orption8pectrum of peroxidaae

On freezing and cooling to the temperature ofliquid air, the absorption bands ofperoxidase and itsderivatives are sharpened, greatly intensified andslightly shifted towards the blue end ofthe spectrum.In this respect peroxidase behaves like some otherderivatives of haematin and many other substancesshowing selective absorption (Keilin & Hartree,1949, 1950a). Freezing in liquid air also makespossible the direct observation in horse-radish rootslices of the absorption band in the red of freeperoxidase which, owing to its diffuse absorptionspectrum, is hardly perceptible at ordinary tem-peratures.As already mentioned, peroxidase, like methaemo-

globin, shows very marked changes in colour andabsorption spectrum with changes in pH. Thus atpH above 11 a reddish alkaline form of peroxidase isformed with bands at 574 and 545 m,u. (Fig. 2).WVhen a neutral solution of peroxidase is cooled itsabsorption band in the red is intensified and shifted7-9 m,u. nearer the blue end ofthe spectrum. On theother hand, when alkaline peroxidase is cooled, itscolour and absorption spectrum revert to those ofneutral peroxidase with the appearance of thecharacteristic band in the red. On warming thesolution to room temperature the colour andabsorption spectrum of alkaline peroxidase rapidlyreappear. We observed a precisely similar change on

Stitt& Pauling (1937) in the case ofmethaemoglobin.The shift in equilibrium observed on cooling alkalineperoxidase or alkaline methaemoglobin could thusbe ascribed to a suppression of ionization. This isstrongly supported by the fact that similar changesin colour can be observed on cooling solutions ofsomeindicators (Keilin & Hartree, 1949). Below a certaintemperature cooling affects the colour of indicatorsby suppressing their ionization, a phenomenonwhich is general for electrolytes (Harned & Embree,1934). The spectroscopic study of peroxidase at lowtemperatures does not support the view put forwardby Theorell & Paul (1944) that betweenpH 5 and 11the sixth co-ordination place is occupied by ahydroxyl group while at pH 11 two hydroxyl groupsare attached to the iron atom by covalent links.

On the 8o-called 'paraperoxidase'It was claimed at one time by Theorell (1940,

1942 a) that horse-radish roots contain two differentperoxidase enzymes: peroxidase I or paraperoxidaseand peroxidase II or true peroxidase. Accordingto Theorell the characteristic properties of theseperoxidases are as follows:

Peroxidase I or paraperoxidase is present in horse-radish roots in variable but always much lower con-centrations than those of true peroxidase. It showsan absorption spectrum of the parahaematin typewith two bands at 583 and 548 m,u. which on re-duction with Na2S204 are replaced by typicalhaemochromogen bands lying at 565 and 535 m,.Peroxidase I is precipitated with picric acid and oncataphoresis atpH 7 5 it moves towards the cathode.Its peroxidatic activity on a haemin basis is of thesame magnitude as that of true peroxidase. Itcatalyses the oxidation of dihydroxymaleic aciddirectly and this reaction is almost completely in-hibited by 10-5M-KCN.

Peroxidase II or true peroxida8e shows a met-haemoglobin-like spectrum with bands at 640 and

VoI. 49 95

D. KEILIN AND E. F. HARTREE

498 m,.; it is not precipitated by picric acid and oncataphoresis at pH 7-5 it moves towards the anode.For oxidation of dihydroxymaleic acid it requiresa carrier such as quinol and this reaction is notaffected by cyanide.

These views were extended by Theorell (1942a) inhis main paper on the subject and in a general review(Theorell, 1943). It was, however, partly withdrawnby Theorell & Akeson (1942) in a paper dealing withthe microdetermination of amino-acids in peroxi-dase and catalase. Unfortunately this withdrawalwas lost in a paper the subject matter of which hadno direct bearing on the problem, with the result thatthe supposed existence in horse-radish roots of twoperoxidases has reached books and reviews. More-over, even in his more recent review, Theorell (1947)considers the so-called paraperoxidase as only'possibly a derivative of peroxidase' and what isimportant, in none of his papers or reviews does herefer to his earlier statement that true peroxidase isunable to catalyse directly the aerobic oxidation ofdihydroxymaleic acid.

Personal ob8ervations. In our experience withmany preparations of horse-radish peroxidase wehave always obtained only a typical true peroxidasewith the characteristic methaemoglobin-like ab-sorption spectrum and even the most purifiedfractions catalysed the oxidation of dihydroxy-maleic acid without the addition of quinol. How-ever, on several occasions we found that when asolution of highly purified true peroxidase was leftfor a considerable time in an ice chest its methaemo-globin-like absorption spectrum turned graduallyinto that of a parahaematin. The paraperoxidasethus obtained showed the following properties:

(1) Instead of the normal brown colour the solu-tion was reddish-brown and showed two absorptionbands in the visible region lying at about 582 and543 mu.

(2) On addition of Na2S204 a haemochromogen-like spectrum rapidly appeared but with the bandslying nearer to the red end of the spectrum thanusual, i.e. at about 565 and 535 m,. This spectrumwas, however, soon replaced by that of typicalreduced true peroxidase with a wide, strong band at556 and a narrow, faint band at 594 m,u.

(3) On addition of KH2PO4 and NaF the solutionturned greenish and the band of fluoride peroxidaseappeared at 612 mu. while the bands of para-haematin disappeared.

(4) On addition of KCN the solution turned redand showed the characteristic cyan-peroxidaseabsorption spectrum.

(5) On addition of H202 the solution showed inturn the absorption spectra of peroxidase-peroxidecompounds II and III.

(6) The peroxidatic activity of the solution whenthe parahaematin spectrum was fully developed was

only 38% lower than that of the original peroxidasesolution.

All this clearly shows that paraperoxidase is atransformation product of true peroxidase. What isimportant, however, is that both compounds reactwith fluoride, cyanide and a reducer (Na2S204) toyield similar derivatives. It is therefore conceivablethat paraperoxidase can, under certain conditions,revert to the original product and that its formationis due to a partial and reversible denaturation of theprotein. Should this be the case, then the change toparaperoxidase is in all probability due to a replace-ment of the water molecule occupying the sixth co-ordination position by a group in the protein, so thatin paraperoxidase two of the six iron valencies aresatisfied by the protein. Although paraperoxidase isnot a natural product, its properties and the condi-tions of its formation deserve further study. Theterm paraperoxidase should therefore be retained forthis interesting derivative.

In this connexion, it is of interest to note thatsimilar spectroscopic changes may also occur insolutions of catalase. Thus a solution of horse-livercatalase of Kat.f. = 43,000 was left in a refrigeratorfor about 2 weeks during which time its activityremained constant. At the end of this period, how-ever, its absorption was considerably modified, theband in the red at 622 m,. being greatly diminishedin intensity while two strong bands appeared at 575and 537 m,. This absorption spectrum was notaffected on treating the solution with either Na2S2O4or K3Fe(CN)*, but on addition of NaN3 the two newbands disappeared and a typical azide-catalasespectrum was formed. On passing a current of airthrough the solution of modified catalase for severalhours the absorption spectrum reverted to that ofnormal catalase. So far no explanation can beoffered as to the nature of these changes in catalasesolutions.

Reaction of catala8e with hydrogen peroxidePeroxidatic activity of catalase. The rapid decom-

position of H202 by tissue extracts has been knownfor more than a century (Thenard, 1818). It wasconsidered at first as a general property of tissuesshared only with finely divided metals and later as aproperty common to all enzymes. It was eventuallyrecognized by several workers and definitely demon-strated by Loew (1901) that this reaction is catalysedby one specific enzyme which he named catala8e.From that time the decomposition of H202 to mole-cular oxygen and water was believed to be the solefunction of catalase. However, we have shown(Keilin & Hartree, 1936b) that in addition to thisreaction catalase is also capable of catalysing thecoupled oxidation of the lower alcohols by means ofH202 produced in certain primary oxidation re-actions (e.g. those catalysed by xanthine oxidase,

96 I95I

PEROXIDASE, CATALASE AND METHAEMOGLOBIN

uriease, D-amino-acid oxidase and glucose oxidase).Although for this reaction catalase can also use freeH202, provided it is added slowly and continuously,yet the primary oxidation reaction is by far the mostefficient mechanism of supply of H202 since nearlyevery molecule of H202 thus formed can be utilizedin the oxidation of alcohols. In all other methods ofsupply of H202 only a small fraction is utilized inperoxidatic oxidation, the main portion being de-composed catalatically (Keilin & Hartree, 1945 b).These observations confirmed our earlier conclusionthat 'the physiological function of catalase would bemainly peroxidatic and only in exceptional casesa catalatic one' (Keilin & Hartree, 1936b). Theperoxidatic activity of catalase has been confirmedand extended by other workers. To the six alcohols:methanol, ethanol, n-propanol, isobutanol, f-aminoethanol and ethylene glycol, which we pre-viously found acting as hydrogen donors in peroxi-datic oxidations catalysed by catalase, Chance(1947, 1948) has added formaldehyde, formic acid,ascorbic acid and peroxides, while Heppel &Porterfield (1949) have added nitrite. What is,however, of special interst is that among hydrogendonors in peroxidatic oxidation by catalase Chance(1948, 1949d) includes H202 itself. In other words,H202may act both as substrate and hydrogen donor,the reaction consisting in the oxidation of onemolecule of H202 by another. This would bring eventhe catalatic decomposition of H202 into the cate-gory of peroxidatic reactions.

Absorption spectra of catalase-peroxide compoundsWe have seen that when H202 is added to methae-

moglobin or peroxidase the colour and the absorp-tion spectrum of these haemoproteins undergomarked changes (cf. pp.88 and 93). Onthe otherhand,when H2O2 is added to catalase, the explosive decom-position of H202 to 02 and water is not accompaniedby any obvious changes in the colour and absorptionspectrum of the solution that could be observed inthe usual way. Only by applying the rapid flowmethod and a very sensitive spectrophotometer tothe study of this problem was Chance (1947, 1949b)able to observe the formation of the primarycatalase-peroxide complex, which can be recognizedby a slight decrease and a shift towards the red of theSoret band as well as by the presence of a band atabout 670 m,u. (Chance, 1950). He was also able toshow that the formation of this complex involvedonly one of the four haematin nuclei of mammaliancatalase. Moreover, he considers that 'the attach-ment of more than one molecule of hydrogen per-oxide to catalase results in an as yet undetected com-plex which rapidly decomposes'. In other words it isthe catalatic activity of the enzyme that limits thesaturation value of the primary catalase H202 Com-plex to 1 molecule of H202 per molecule of catalase.

Biochem. 1951, 49

With H202 continuously generated by an oxi-dizing system such as notatin, glucose and oxygen,Chance (1950) observed that the rapidly formedprimary catalase-H202 complex (I) changes gradu-ally into a secondary complex (II) which shows twoabsorption bands in the visible region: a, 572 andf, 536 mj. He has studied the kinetics of formationof complex II under various conditions and hassuggested that the compound with two absorptionbands at 565 and 535 m,u. which is formed whenascorbic acid is added to catalase is not a catalase-ascorbic complex, as was believed by Lemberg &Foulkes (1948), but is in fact the catalase-peroxidecomplex II, the H202 arising from autoxidation ofascorbic acid. The variation in the position of ab-sorption bands of complex II with the experimentalconditions led us to further studies of the reactions ofcatalase with continuously generated H202. Themain result of this work was the characterization of anew complex which can be described as catalase-H202 III.

Experiments with catalase, notatin and glucose. Toa small vertical glass tube, 9 mm. diameter, with afused optical bottom, was added 0-4 ml. catalase(0-184 mm with respect to haematin), 0 4 ml. 0-2M-acetate buffer pH 5.0, 0-1 ml. 0-5M-glucose and atrace of octyl alcohol. The tube was stood above thecondenser of a microscope and the reaction followedwith a microspectroseope. On adding 0-1 ml.notatin solution (0-1 %) and passing a fine stream ofair bubbles through the mixture for a few seconds,the colour of the solution turned from brown to redand the absorption bands of free catalase were re-placed by two bands: oc 568 and : 536 m,t. Thepositions of these bands were confirmed with aHartridge reversion spectroscope. If aeration wascontinued the a-band tended to spread towards thered and thus become asymmetric, while eventually avery feeble band appeared at 585 myt. On additionof 0-2 ml. 0-1 N-H202 to the aerated solution, theabsorption bands of complex II were replaced bytwo bands lying nearer to the red: oc, 585 and fi,545 my. which we ascribe to the complex III. BothII and III revert on standing to free catalase.Experiments at different pH showed that the abovereactions proceeded rapidly only within the range4-0-5-5.

Experiments with catalase and ascorbic acid. It caneasily be demonstrated that the changes in colourand absorption spectrum of catalase that arebrought about by ascorbic acid are due to theformation of catalase-H202 II and not to a compoundbetween catalase and ascorbic acid. Thus the re-action requires the presence of oxygen, it is greatlyaccelerated by the addition of copper salts andthe compound obtained is spectroscopically in-distinguishable from that observed on aeration ofcatalase + notatin + glucose.

VoI. 49 97

D. KEILIN AND E. F. HARTREE

The reaction was carried out in the vertical glasstubes using 0 4 ml. catalase (as above), 0-4 ml.buffer, 0-1 ml. neutralized ascorbic acid (0.5M) andvarious quantities of CUS04 (0-1 ,umole) in 0-1-0-2 ml. water. In absence of copper, aeration of thismixture within the pH range 3-9-6-8 caused nochange in absorption spectrum during 5 min. In anexperiment at pH 5 0 (01 M-acetate buffer) theaddition of 01 ,umole CUS04 during aeration had noeffect, whereas 044,mole gave rise immediately tocomplex II with bands at 568 and 536 m,. When thesolution was allowed to stand for a few minutes theac-band widened towards the red and appeared tohave a maximum at 572 m. At this stage thesolution was aerated again and a further 0-6 ,umoleCuSO4 was added. The complex II was immediatelyand completely transformed into complex III withbands at 586 and 546 m,. At pH 3 9 a very similarresult was obtained, but more ascorbic acid wasrequired for the complete formation of complex III.Under similar conditions at pH 5 the very sparinglyionized copper glycine gave rise to complex II plus atrace of III while even the water-insoluble copperacetylacetone catalysed the slow production ofcomplex II. In all experiments involving ascorbicacid the formation of complexes with H202 wasaccompanied by precipitation of catalase. No pre-cipitate was formed if either ascorbic acid or copperwas omitted. Nevertheless, even after precipitationthe spectra reverted on standing to that of freecatalase.From the above results it is clear that the ab-

sorption bands of catalase-H202 II are: ac, 568 and#, 536m,., while those of complex III are: ac, 585 andf, 545 m,. The ac-band at 572 m,u. previously re-corded (Chance, 1950) is probably that of complex IIat a stage where it has undergone a slight conversionto III.

Reaction of methaemoglobin (Methb)with hydrogen peroxide

It has been established (Keilin & Hartree, 1935;Haurowitz, 1935) that methaemoglobin forms withH202 and with ethyl hydroperoxide well defined com-pounds analogous to those which it forms withcyanide, fluoride, sulphide and azide. The formationof Methb-peroxide requires 1 molecule of peroxideper iron atom of Methb. The Methb-hydrogenperoxide (abbreviated to Methb-OOH) is red incolour and shows two bands: ac, 589 and fi, 545 m,.The P-band is stronger than the oc-band which isdiffuse, asymmetric and may even appear to havetwo maxima. The complex nature of this band aswell as the fact that both peroxidase and catalaseform with H202 several spectroscopically recogniz-able compounds have prompted us to re-examinemore closely the spectroscopic changes whichaccompany the reaction between Methb and H202.

A preliminary account of this work has alreadyappeared (Keilin & Hartree, 1950b).For this purpose Methb was obtained from

catalase-free crystalline horse oxyhaemoglobin bytreatment with NaNO2 or with K3Fe(CN)6 andsubsequent dialysis. The experiments were carriedout in the small vertical tubes described above andthe reactions were followed with a microspectro-scope. The solution of Methb used in these experi-ments was 0-384 mm with respect to haematin andwas made up in 0-2M-phosphate buffer pH 6-8.A 6 mm. depth of this solution clearly showed theabsorption band in the red of Methb. The quantityofH202 added to Methb will be expressed in terms ofthe molar ratio (R) of H202 to haematin. When R = 1the formation of Methb-OOH was 80% complete(see also Keilin & Hartree, 1935). When R= 2 5-5,Methb-OOHwas completely formed, but on standingit decomposed liberating acid Methb. During thisprocess part of the Methb underwent destruction.As the concentration of H202 was increased the

double appearance of the a-band became moremarked, the band showing two maxima at 578 and592 m,u. When R = 25 the oc-band was distinctlydouble, its two components being of almost equalintensity. When R= 100 the band at 578 m,u. wasmuch stronger than the band at 592 m,. When Rreached 250-500 the band at 578 m,L. predominatedand after a short time the band at 592 m,t. becamecompletely invisible. Thus in presence of an excessof H202 the Methb-OOH was replaced by anothercompound which could easily be recognized asoxyhaemoglobin (HbO2). In fact its absorptionspectrum was indistinguishable from that of HbO2.On treatment with Na2S204 it gave deoxygenatedhaemoglobin (Hb) and in presence of CO it formedHbCO. The same sequence of events can be observedby using the notatin system to supply H202 asdescribed above for the study of catalase-H202compounds. On treating a mixture of Methb,notatin and glucose at pH 6-8 with a stream of airMethb-OOH is rapidly formed, but does not becomeappreciablyreduced to HbO2. Since at the same timethe concentration of H202 remains very low andthere is consequently no appreciable destruction ofhaematin, the maximum at 592 m/,. stands out veryclearly with only a faint shading at 578 m,. repre-senting a trace of HbO2.

In experiments with H202 added as a solution it isof special interest that bubbles of 02 deriving fromthe decomposition of H202 do not appear until thea-band of HbO2 becomes discernible, and activedecomposition of H202 takes place only when thebands of HbO2 are already very distinct. Thereaction between Methb and H202 therefore con-sists in the formation of the Methb-OOH complexwhich, on further reaction with H202, brings aboutits decomposition to molecular 02 and water, a

98 I95I

PEROXIDASE, CATALASE AND METHAEMOGLOBINreaction which is accompanied by the reduction ofMethb to Hb and the oxygenation of the latter toHbO2. The greater the excess ofH202 added from thebeginning the more rapid and complete is the forma-tion of HbO2 and the more vigorous is the decompo-sition of H202. But however great is the excess ofH202, the appearance ofHbO2 is always preceded bya transient stage when the total Methb is convertedinto Methb-OOH. Onadditionofasuitablehydrogendonor, such as ascorbic acid, which can be oxidizedperoxidatically, to a solution showing the absorptionbands ofHbO2 and Methb-OOH, the latter is rapidlydecomposed liberating Methb while HbO2 remainstemporarily unchanged. The catalatic decompositionof H202, together with the appearance of HbO2, aresoon followed by a destruction of the haem nucleusas shown by disappearance of absorption bands ofthe solution which becomes colourless. Since theabsolute extinction of the ac-band of HbO2 is muchgreater than that of Methb-OOH when the relativeconcentrations of these two compounds in thesolution are equal, the c-band ofHbO2 appears to bemuch stronger than that of Methb-OOH.

All the above reactions have been observed withboth recrystallized methaemoglobin and metmyo-globin at pH's varying between 5-5 and 9-2. AtpH below 5 the reaction follows a different course.Thus with great excess of H202 the Methb-OOHformed is rapidly replaced by HbO2, whereas withsmaller concentration of H202 the Methb-OOH isgradually replaced by a new compound with a wideabsorption band at 590 mpc. and showing red-greendichroism. The formation of this compound is pre-ceded by an intermediate stage devoid of anydistinct bands in the visible region. However, theaddition of NaN3 or NaF at this stage yields thecorresponding Methb derivative, which can readilybe identified spectroscopically, while Na2S204 givesrise to Hb. The haematin prosthetic group, or atleast a considerable proportion of it, is thus intact.If the minimum ofH202 is used the compound show-ing the 590 m,. band is stable for an hour or more.These conditions are obtained by adding 0-1 ml.0-05N-H202 to 7-5 ml. Methb (0-077 mM with respectto haematin) in 0-2M-acetate buffer pH 4-5. The590 mp. band reaches full intensity after about8 min. The addition of pyridine and Na2S204 givesrise to a strong protohaemochromogen with twovery faint bands nearer to the red, but the addition ofNaF causes only a slow appearance of fluoride-Methb in very poor yield. These results indicate anoxidation of the porphyrin ring, possibly at themethine bridges, without an opening of the tetra-pyrrol structure. This view is confirmed by thefinding that the Soret band persists, but is much lessintense (E410 = 40,00 as compared with e40=163,000 for acid Methb).

Since the peroxidatic and catalatic activities of

Methb, although much lower than those of per-oxidase and catalase, are nevertheless very distinct,Methb can be considered as a suitable model for thestudy of the reactions catalysed by these twoenzymes. As the catalatic decomposition ofH202 byMethb involves the formation of Methb-OOH andsubsequent valency changes of Methb iron, it isreasonable to assume that the similar decompositionof H202 by catalase is also accompanied by valencychanges of catalase iron, a view which is, moreover,supported by the study of azide-catalase (Keilin &Hartree, 1936a, 1945a).

DISCUSSION

Properties of peroxidaBeAs the quantitative study of different properties,including the absolute absorption spectra, of per-oxidase required highly purified enzyme prepara-tions a comparatively simple method was developedfor isolation and purification of peroxidase fromhorse-radish roots. Our most active preparation hada P.N. of about 1220. Its haemin content was1-61 % which corresponds to a minimum molecularweight of 40,500 (± 600) while sedimentation-diffusion studies carried out by Dr A. G. Ogston andDr R. Cecil (see Addendum, p. 105) gave the figure39,800.Although the haemin content of our purest per-

oxidase preparation (1.61 %) is higher than thevalue (1-48%) given by Theorell (1942a), it shouldperhaps be mentioned that the haemin content ofone of his crystalline preparations, of which heobtained 2-8 g., was actually 1-62 %. He concluded,however, that this preparation 'still contained somehemin that did not belong to the peroxidase mole-cule, which according to the determination ofmolecular weight should contain 1-48% haemin'.The high haemin value (1-62 %) he ascribed to theomission of electrodialysis which should remove theextraneous haematin. The fact that a haemincontent of 1-48% was obtained by Theorell afterelectrophoresis 'performed at a much too high pHvalue', which reduced the yield of the enzyme from2-8 to 0-5 g. suggests the possibility that the enzymeof this preparation may have undergone a changeinvolving loss of haematin.

Although our values of haemin content and ofmolecular weight differ from those of Theorell byonly 8-9% far greater discrepancies appear in thefigure for P.N. Whereas our most active preparationshad a P.N. of about 1220, Theorell (1942a) regardsP.N. 930 as the activity of the pure enzyme andascribes his occasional higher values (1100) tocatalytic impurities. We have confirmed the findingsof previous workers (summarized by Ettori, 1949)namely, that the values of P.N. obtained in a series ofestimations on the same sample under apparently

7-2

VoI. 49 99

D. KEILIN AND E. F. HARTREEidentical conditions can vary appreciably. In anattempt to make the P.N. estimation more reliableSumner & Gjessing (1943) carried out the reaction ina small volume of buffer solution. This method wascarefully examined by Ettori who also devised amanometric method based upon C02 productionduring the oxidation of pyrogallol but, according tohim, neither method is free from the fluctuations ofthe normal test. In our experience, these fluctua-tions can be reduced to a large extent by the use ofcarefully purified pyrogallol (see Methods). Samplesof pyrogallol which have not been so purified giverise to P.N. values whichmay be not only too high butalso too low. Under these circumstances the dis-crepancies between the results of estimations ofP.N. in different laboratories are in no way sur-prising.One of the main objects of the present investiga-

tion was to determine the absolute absorptionspectra of peroxidase and its derivatives both forthe visible and the violet or Soret regions of thespectrum. It is important to note that in haemo-proteins the most significant changes which are inone way or another associated with the iron atom aremore clearly reflected in the general pattern of theabsorption spectrum in the visible region than in theSoret band. In fact it was through the study of suchchanges in the visible region that the haematinnature of peroxidase was established and the forma-tion of peroxidase-peroxide, in other words of anenzyme-substrate complex, was demonstrated forthe first time (Keilin & Mann, 1937). It is also theexistence of absorption bands in the visible regionthat makes possible the direct spectroscopic studyof peroxidase in crude tissue extracts as well as inslices of horse-radish root, a property which will bevaluable for the study of the physiological functionof peroxidase.

Freezing and cooling to liquid air temperature ofperoxidase and its derivatives in solutions or inslices ofhorse-radish greatly intensifies and sharpensthe absorption bands. In this respect peroxidasebehaves like other haematin compounds and manyother pigments (Keilin & Hartree, 1949, 1950a). Inslices cooled with liquid air even the band in the redof free peroxidase can easily be seen. The fact thatsimilar cooling of alkaline peroxidase causes itsabsorption spectrum to change to that of the neutralform has been interpreted by us as indicating thatthe sixth co-ordination position around the haematinFe is occupied by a molecule of water and not by a

hydroxyl group as was postulated by Theorell &Paul (1944).

There is no evidence that the 'paraperoxidase' ofTheorell (1940) occurs naturally in horse-radishroots, although in fact it may be formed in a solutionof pure peroxidase which has been allowed to stand.It appears to be a reversible derivative ofperoxidase

in which the water molecule attached to the Fe atomis probably replaced by a group belonging to theprotein.

Peroxidase forms with hydrogen peroxide onegreen (I) and two red (II and III) compounds. Sincethe green compound is the first to appear whenperoxidase is treated with a small amount of H202 inabsence of hydrogen donors, Theorell (1942c, 1947)suggested that this compound is the only catalytic-ally active complex, i.e. the only one of the threewhich can react directly with hydrogen donors toyield oxidized donors and free peroxidase. However,this has not been confirmed by Chance (1949c) in thecourse of his study of the kinetics of reactionscatalysed by peroxidase. Chance has been able toshow that the rate of decomposition of the primarygreen compound (I) is proportional to the rate offormation and decomposition of the secondary redcompound (II). The velocity of transition from I toII is greatly accelerated by the presence of ahydrogen donor and can even reach the velocity offormation of the primary compound. In the cata-lytic activity ofperoxidase the complex II is thus therate-determining Michaelis intermediate and duringcatalytic activity the enzyme is liberated not fromthe primary (I) but from the secondary (II) complex.This indicates that both complexes I and II areinvolved in the catalytic activity of the enzyme.

COMPARISON OF PEROXIDASE, CATALASEAND METHAEMOGLOBIN

In comparing these three haemoproteins the termmethaemoglobin will be used in the widest sense tocover the oxidized or ferric forms of all haemo-globins. Catalase will also be used without distinc-tion as to origin. On the other hand, the termperoxidase will be applied only to the enzymeobtained from horse-radish roots, since the know-ledge so far available of other peroxidases, especiallyof their prosthetic groups, is far from complete.We shall examine separately (a) the properties

which are common to these haemoproteins and(b) the properties in which they differ.

(a) Properties common to peroxida8ae,catalae and methaemogtobin

(1) They are haemoprotein compounds having anidentical prosthetic group, protohaematin, united todifferent proteins. (2) Their iron is in the trivalentstate. (3) The colour and the general pattern oftheirabsorption spectra are those of acidmethaemoglobin(Fig. 5). (4) All three haemoproteins react reversiblywith KCN, H2S, NaF, NaN3, NH20H, NO, H202 andROOH (alkyl peroxides) forming spectroscopicallywell defined compounds, the formation of whichrequires 1 molecule of each of these substances periron atom. (5) The cyanide and fluoride derivatives

100 I95I

PEROXIDASE, CATALASE AND METHAEMOGLOBIN

of the three haemoproteins show very similar ab-sorption spectra (Figs. 1, 6 and 7). (6) On denatura-tion or treatment with pyridine and Na2S204 theygive the same haemochromogen. They also yield thesame haemin and the same porphyrin (proto-porphyrin IX).

(b) Difference8 in propertie8 of peroxida8e,catala8e and mehaemoglobin

(1) Although the absorption spectra of thesehaemoproteins are ofthe same general pattern, thereare certain interesting differences in the position andin the relative heights of their bands (Figs. 1-3, 5-7,also Table 2). The absorption spectrum of catalase inthe visible region is represented in Fig. 5 by twocurves obtained (i) from liver catalase, which con-tained a certain amount of verdo-haematin, and(ii) crystalline bacterial catalase, which is com-pletely free from verdo-haematin (from the data ofHerbert & Pinsent, 1948). As shown in Fig. 5, theband in the red (a) of catalase is much higher thanthose of methaemoglobin and peroxidase, the latterbeing the lowest of the three and lying nearer thered end of the spectrum. On the other hand, therelative heights of their Soret bands (y) follow the

r

1-2

1.0

08 -\ /

C XI

6

1-6

1-4-

1-2

10

10 ,*~~%~/\*~~ Methaemci ~~~~globin

018 f* s16-

/0'6 Catalase -124 ~ (liver)

~- Peroxidase 8

. Proxidase0 0

0-

06w

10

700 660 620 580 540 500 460 420 380Wavelength (m,t.)

Fig. 5. Absolute absorption spectra of methaemoglobin,peroxidase and liver catalase (present investigation)together with the corresponding curve for bacterialcatalase from the paper by Herbert & Pinsent (1948). Fordefinition of units see Fig. 1.

I 540 5Wavelength (mrn*)

Fig. 6. Absolute absorption spectra at pH 6 of methaemoglobin (prepared from crystalline horse oxyhaemoglobin) andof its fluoride (F-), cyanide (CN-), azide (N-) and H202 derivatives. For definition of units see Fig. 1.

VoI. 49 101

D. KEILIN AND E. F. HARTREE

order: Methb> catalase> peroxidase. The ratios ofabsorption coefficients EyI/a for these three haemo-proteins are: Methb 39, peroxidase 31 and catalase9-14-2. So far no explanation can be offered for themuch stronger absorption oflight by catalase and itsderivatives in the visible region compared with thoseof peroxidase and Methb.

18 l l

1 6

CN-

14 F

1 2

unaffected by this reducing agent. In this respectcatalase differs from all knownhaematin compounds.

(4) Methb (Fig. 6) and peroxidase (Fig. 1) ontreatment with azide turn from brown to red andtheir absorption spectra are drastically changed.Catalase (Fig. 7), on the other hand, becomes some-what more greenish and retains the same general

I,Z

!/

t _ ~~~~~~~~~16-0Catalase

C N- 10 o

'I~~~~~~~~~~~~CII~~~~~~~~~~~~~-

700 660 620 580 540 500 460 420 380Wavelength (m,L.)

Fig. 7. Absolute absorption spectra at pH 6 of catalase (from horse liver) and of its fluoride (F-), cyanide (CN-) andazide (N-) compounds. For definition of units see Fig. 1.

(2) On making a solution of Methb alkaline itscolour changes from brown to red and its character-istic absorption spectrum is replaced by two widebands at 545 and 575 m,. and a narrow band at600 m,. Methb behaves as an indicator betweenpH 6-8 and 9 5 with pK=8-1. Peroxidase, likeMethb, shows changes in colour and absorptionspectrum with changes of pH. At pH 11 a reddishalkaline peroxidase is obtained with bands at 574and 545 m,. (Fig. 2). On the other hand, the colourand absorption spectrum of catalase are onlyslightly affected by changes in pH.

(3) Methb and peroxidase (Fig. 2) are rapidlyreduced by Na2S2O4 from the ferric to the ferrousstate as is shown by changes in their colours andabsorption spectra, whereas catalase is completely

spectral pattern, the only noteworthy change beingthat the band in the red becomes reinforced andshifted slightly towards the blue.

(5) Azide-catalase differs from azide-Methb andazide-peroxidase not only in its optical propertiesbut also in its reaction with H202. Thus H202, whenadded to azide-Methb or azide-peroxidase, partiallyreplaces the azide and forms the correspondingperoxide complexes in which the Fe remains tri-valent. On the other hand, H202 reacts with azide-catalase to reduce its Fe to the divalent state,foriming a compound which reacts both with oxygenand with CO. It was the study of this reaction whichsuggested that the catalatic decomposition of H202by free catalase is also accompanied by valencychanges ofthe iron (Keilin & Hartree, 1936a, 1945 a).

.I95I102

Vol. 49 PEROXIDASE, CATALASE AND METHAEMOGLOBIN

Table 3. Wavelengths (m1i.) of absorption bands in the vi8ible region of complexesformed reversibly by peroxidase, catalase and Methb with H202

PeroxidaseCatalaseMethb

Complex I657670 (?)Not known

Complex II

a p555* 527568 536Not known

Complex III

a x583 546585 545592* 545

103

* These bands are asymmetric and the figures given represent the wavelengths of maximum absorption and not thecentres of the bands.

(6) The three haemoproteins form with H202different complexes which, according to theirabsorption spectra in the visible region, can beclassified as shown in Table 3. Thus peroxidase andcatalase give three spectroscopically recognizablecomplexes of which peroxidase I and II and catalaseI appear to be active in reactions which are so farknown to be catalysed by these enzymes. Methbforms only one recognizable complex which corre-sponds to the type III given by peroxidase andcatalase. On further reaction with H202 this Methbcomplex undergoes reduction to Hb and oxygena-tion to HbO2, a reaction which is accompanied bycatalatic decomposition of the H202.

(7) The most striking differences in the propertiesof these three haemoproteins are found in themagnitude of their catalytic activities. WhereasMethb shows only very slight peroxidatic andcatalatic activities, peroxidase and catalase are verypowerful catalysts.

For a long time the catalytic activities of peroxi-dase and catalase were considered to be funda-mentally different, peroxidase utilizing H202 for theoxidation of various substances and catalase beingcapable only of decomposing H202 into oxygen andwater. However, this difference lost all significancewhen catalase was shown to be capable of utilizingH202 for the peroxidatic oxidation of alcohols(Keilin & Hartree, 1936b, 1945b). The list ofhydrogendonors for peroxidatic activity of catalase has sincebeen extended (see p. 97) and now includes H202itself (Chance, 1948, 1949d). The difference in thecatalytic activities of peroxidase and catalase istherefore an expression of their specificity towardsfiydrogen donors undergoing peroxidatic oxida-tion.

Although the kinetics of the reactions of catalaseand peroxidase with their substrate, and also withhydrogen donors and inhibitors, have been ex-tensively studied the structures of the variousenzyme-substrate intermediates remain obscure.Furthermore, in spite of the extensive investigationsto which these enzymes have been subjected, workwhich has led to their complete purification andto a considerable knowledge of their constitution,very little is known of their role in cellular meta-bolism.

SUMMARY

1. A comparatively simple method is describedfor the isolation and purification of peroxidase fromhorse-radish roots.

2. The preparation contained 13 2% nitrogenand 1- 61 % haemin. The mean value of its activity interms of Purpurogallin Number was 1220 and themolecular weight was about 40,000 (see Addendum,p. 105).

3. Absorption curves in the visible and Soretregions are given for peroxidase, for its alkaline andreduced forms, and for its compounds with carbonmonoxide, cyanide, azide, fluoride and hydrogenperoxide.

4. The absorption bands in the visible region givea better indication than those in the Soret region ofchanges in the Fe linkages of peroxidase and itsderivatives. These bands may be observed in crudeenzyme preparations and even in slices of horse-radish root.

5. No evidence was found that 'paraperoxidase'exists as such in horse-radish roots, but it couldbe obtained as a transformation product of pureperoxidase.

6. Peroxidase catalyses the oxidation of di-hydroxymaleic acid directly without the need forquinol or any other 'carrier'.

7. The effect of liquid air temperature on theabsorption spectra of peroxidase and methaemo-globin suggests that the sixth co-ordination positionaround the iron atom in these haemoproteins, at aneutral or slightly acid pH, is a water moleculerather than a hydroxyl group.

8. Peroxidase gives rise successively on additionof hydrogen peroxide to three complexes which areeasily recognized spectroscopically. Their absorptionbands in the visible region are: I, approx. 657 m,.;II, 555 and 527 m,u.; III, 583 and 546 mi,. Only Iand II appear to take part in the normal catalyticactivity of the enzyme. The band which appears at670 m,. as a result of prolonged treatment withhydrogen peroxide is probably that of a degradationproduct of peroxidase rather than a complex withhydrogen peroxide.

9. Catalase also forms with hydrogen peroxidethree spectroscopically recognizable complexes with

104 D. KEILIN AND E. F. HARTREE I95Ithe following bands: I, approx. 670 m,u.; II, 568 and536 mpu.; III, 585 and 545 m/i. Complex I, whichcan only be detected by the rapid-flow technique,appears to be the only one that participates in theknown reactions of this enzyme. Complex II can beobtained when hydrogen peroxide is suppliedcontinuously by an oxidizing system; it can be con-verted to III by further reaction with hydrogenperoxide.

10. Methaemoglobin forms only one complexwith hydrogen peroxide. The bands are at 592 and545 m,u. and it thus corresponds to type III ofperoxidase and catalase. The reaction of thiscomplex with further hydrogen peroxide results inthe reduction of methaemoglobin to haemoglobin,which can be detected as oxyhaemoglobin, andsimultaneous liberation of oxygen from hydrogenperoxide.

11. Peroxidase resembles catalase and methae-moglobin in structure (all three being protein-

protohaematin compounds), colour, general patternofabsorption spectra and formation ofreversible andspectroscopically recognizable compounds withcyanide, sulphide, azide, fluoride, nitric oxide andperoxides.

12. Peroxidase and methaemoglobiii, unlikecatalase, can be reduced with sodium dithionite andshow marked changes in colour and absorptionspectrum with pH or on combining with azide.Azide-catalase, however, is spectroscopically verysimilar to free catalase and is reduced to a ferrousderivative by hydrogen peroxide.

13. There are no fundamental differences in thenature of the catalytic activities of peroxidase andcatalase since the latter has been shown to catalyseperoxidatic oxidation of alcohols and other sub-stances. However, the peroxidatic activity ofcatalase shows maximum efficiency only whenhydrogen peroxide is continuously supplied by asystem which reduces oxygen to hydrogen peroxide.

REFERENCES

Bourquelot, E. (1898). C.R. Soc. Biol., Paris, 50, 381.Chance, B. (1947). Acta chem. scand. 1, 236.Chance, B. (1948). Nature, Lond., 161, 914.Chance, B. (1949a). Science, 104, 204.Chance, B. (1949b). Arch. Biochem. 21, 416.Chance, B. (1949c). Arch. Biochem. 22, 224.Chance, B. (1949d). J. biol. Chem. 180, 947.Chance, B. (1950). Biochem. J. 46, 387.Coryell, C. D., Stitt, F. & Pauling, L. (1937). J. Amer. chem.

Soc. 59, 633.Elliott, K. A. C. & Keilin, D. (1934). Proc. roy. Soc. B, 114,

210.Ettori, J. (1949). Biochem. J. 44, 35.Harned, H. S. & Embree, N. D. (1934). J. Amer. chem. Soc.

56, 1050.Haurowitz, F. (1935). Hoppe-Seyl. Z. 232, 159.Heppel, L. A. & Porterfield, V. T. (1949). J. biol. Chem. 178,

549.Herbert, D. & Pinsent, J. (1948). Biochem. J. 43, 193.Keilin, D. (1927). C.R. Soc. Biol., Paris (Reunion plenniere),

97, 39.Keilin, D. (1933). Proc. roy. Soc. B, 113, 393.Keilin, D. (1936). Proc. roy. Soc. B, 121, 165.Keilin, D. & Hartree, E. F. (1935). Proc. roy. Soc. B, 117, 1.Keilin, D. & Hartree, E. F. (1936a). P-oc. roy. Soc. B, 121,

173.Keilin, D. & Hartree, E. F. (1936b). Proc. roy. Soc. B, 119,

141.Keilin, D. & Hartree, E. F. (1945a). Biochem. J. 39, 148.Keilin, D. & Hartree, E. F. (1945b). Biochem. J. 39, 293.Keilin, D. & Hartree, E. F. (1949). Nature, Lond., 164, 254.

Keilin, D. & Hartree, E. F. (1950a). Nature, Loud., 165,504.Keilin, D. & Hartree, E. F. (1950b). Nature, Lond., 166,

513.Keilin, D. & Mann, T. (1937). Proc. roy. Soc. B, 122, 119.Keilin, D. & Wang, Y. L. (1946). Biochem. J. 40, 855.Kuhn, R., Hand, D. & Florkin, M. (1931). Hoppe-Seyl. Z.

201, 255.Lemberg, R. & Foulkes, E. C. (1948). Nature, Lond., 161,131.Linossier, G. (1898). C.R. Soc. Biol., Paris, 50, 373.Loew, 0. (1901). U.S. Dep. Agric. Wash. Report no. 68, 1.Markham, R. (1942). Biochem. J. 36, 790.Sch6nbein, C. F. (1863). J. prakt. Chem. 98, 323.Sumner, J. B. & Gjessing, E. C. (1943). Arch. Biochem. 2,291.Sumner, J. B. & Howell, S. F. (1936). Enzymologia, 1, 132.Thenard, L. J. (1818). Ann. Chim. (Phys.) 9, 314.Theorell, H. (1940). Ark. Kemi Min. Geol. 14B, no. 20.Theorell, H. (1941). Enzymologia, 10, 250.Theorell, H. (1942a). Ark. Kemi Min. Geol. 16A, no. 2.Theorell, H. (1942 b). Ark. Kemi Min. Geol. 15B, no. 24.Theorell, H. (1942 c). Ark. Kemi Min. Geol. 16A, no. 3.Theorell, H. (1943). Ergebn. Enzymforsch. 9, 278.Theorell, H. (1947). Advanc. Enzymol. 7, 265.Theorell, H. & Akeson, A. (1942). Ark. Kemni .Mmin. Geol.16A, no. 8.

Theorell, H., Bergstrom, S. & Akeson, A. (1943). Ark. KemiMin. Geol. 16A, no. 13.

Theorell, H. & Paul, K. G. (1944). Ark. Kemni Mimi. Geol.18A, no. 12.

Warburg, 0. (1928). Naturwissenschaften, 16, 345.Willstatter, R. & Pollinger, A. (1923). LiebigsAnn. 430,269.Willstatter, R. & Stoll, A. (1918). Liebigs Ann. 416, 21.

105

ADDENDUM

Determination of Sedimentation and Diffusion Constantsof Horse-Radish Peroxidase

BY R. CECIL AND A. G. OGSTONDepartment of Biochemi8try, Univeraity of Oxford

(Received 13 September 1950)

Samples of peroxidase (fraction H), provided byProf. D. Keilin and Dr E. F. Hartree were examinedin a Svedberg oil-turbine ultracentrifuge and in theGouy diffusiometer (Coulson, Cox, Ogston &Philpot, 1948). The buffer was NaCl, 0-2M; KH2PO4,0-046M; Na2HPO4, 0-01OM.

1)

Fig. 1. Sedimentation diagrams: 40, 65 and 80 min. afterreaching full speed; a and c by refraction, and b byabsorption.

The sedimentation constant was first determinedin a solution containing 7-7 mg./ml. at 1000 rev./sec.; observations were made alternately by thediagonal-schlieren method of Philpot (1938) at600-640 m,u., and by absorption (Philpot, 1939) at540-570 m,. Fig. 1 shows refractometric andabsorptiometric diagrams. In Fig. 2 the positions ofthe main boundary at successive times are shownplotted as S(Ax) against S(At') (Cecil & Ogston,1948): the positions obtained from absorption lieclosely on the same curve as those obtained re-fractometrically, showing that the absorbingmaterial sediments exactly with the main refractingcomponent. Of the total refracting material 92%forms the main sedimenting boundary (Cecil &

Ogston, 1948), the remaining 8% being probablyaccounted for by a small amount of heterogeneousmaterial. A second determination of the sedimenta-tion constant, at a concentration of 3-4 mg./ml., wasmade for comparison with the diffusion constantdetermined at the same concentration.

Diffusion was observed by the Gouy method,using light of 622 m,. (from a mercury lamp). At6-9 mg./ml. the optical extinction of the solution was0-37 and this proved to be too great for a reallysatisfactory record to be obtained: however, theresult indicated that the material is nearly homo-

117Ev

X

S (At')Fig. 2. Plot of S(Ax) in cm. measured on the record against

S(At') in units of 1012: 0, by refraction; *,,by absorption.

geneous with respect to diffusion. At 3-4 mg./ml.(extinction = 0-18) a satisfactory record was ob-tained. The theory of the interference of light in theGouy method (tegeles & Gosting, 1947) appliesstrictly only to the case where no absorption of lighttakes place: elementary theory (which may not bereliable) suggests that a moderate degree of ab-sorption should not result in a large shift ofthe inter-ference minima, and the results have been inter-preted on this assumption.The sedimentation and diffusion constants

(corrected to their values for water at 200) and otherquantities are given in Table 1. The value of thepartial specific volume was measured by Keilin &Hartree (1951) and agrees moderately well with thevalue 0-690 given by Theorell (1942).

106 R. CECIL AND A. G. OGSTON I95I

Table 1. Sedimentation and diffusion constaats of peroxidaBeConcen- Proportiontration S20 (corr.) D20 (corr.) in main

(mg./ml.) ( X1013) ( x 107) V M flfo boundary7-7 3.47 - 0-923.4 3-48 7*05 0*699 39,800 1-36

Theorell (1942) has reported sedimentation anddiffusion measurements on peroxidase: our value forS20 (corr.) is about 10% lower than his (3-85 x 10-13)(cf. Cecil & Ogston, 1948), while our value for D20(corr.) agrees fairly well with his value of 6 84 x 1o-7.

Our value for the molecular weight is lower thanTheorell's (44,100), but is in agreement with thatcalculated by Keilin & Hartree (1951) from thehaematin content; the chief uncertainty in ourquantity probably lies in the value of V.

REFERENCES

Cecil, R. & Ogston, A. G. (1948). Biochem. J. 43, 592.Coulson, C. A., Cox, J. T., Ogston, A. G. & Philpot, J. St L.

(1948). Proc. roy. Soc. A, 192, 382.Kegeles, G. & Gosting, L. J. (1947). J. Amer. chem. Soc. 69,

2516.

Keilin, D. & Hartree, E. F. (1951). Biochem. J. 49, 88.Philpot, J. St L. (1938). Nature, Lond., 141, 283.Philpot, J. St L. (1939). Biochem. J. 33, 1707.Theorell, H. (1942). Ark. Kemi Min. Geol. B, 15, no. 24.

Studies on the Enzymic Synthesis of Glutamine

BY W. H. ELLIOTTBiochemical Laboratory, Univer8ity of Cambridge

(Received 5 September 1950)

Earlier reports described the discovery ofan enzymesystem in sheep brain which synthesizes glutaminein the presence of L-glutamic acid, adenosinetri-phosphate (ATP) and ammonia (Elliott, 1948;Elliott & Gale, 1948). The present paper deals withthe purification and detailed study of this enzymesystem and the results of a distribution survey.A summary of this work was presented at the FirstInternational Congress of Biochemistry at Cam-bridge in August 1949 (Elliott, 1949). Speck(1947, 1949a, b) has independently reported a studyof the glutamine-forming system present in pigeonliver.

MATERIALS

ATP was prepared as described by Needham (1942) exceptthat in the final stages the method of Kerr (1941) wasadopted to produce the monobarium salt. This was convertedto the Na salt by dissolving it in x-HCI and adding a slightexcess of Na2SO4. After centrifuging, the supernatant wasneutralized to pH 6-8 and stored at -10°. The solution usedcontained 0.1 mg. inorganic P/ml. and 4-3 mg. of P hydro-lysed by boiling N-HC1 in 10 min. (acid-labile P). Thesolution was analysed enzymically by the method of Bailey(1949). The percentages of the acid-labile P hydrolysed bymyosin, by myosin+myokinase, and by inorganic pyro-phosphatase were 45-6, 91-2 and 2-6 respectively.

Adenosinediphosphate (ADP) was prepared from ATP bythe method of Bailey (1942). The Ba salt was treated in thesame way as that for ATP. The solution used contained3-16 mg. of total P/ml., of which 50% was hydrolysed by

boiling N-HCI in 10 min. The percentages of the totalP split by myosin alone, by myosin+myokinase, and byinorganic pyrophosphatase were 1*0, 47 and-02 respectively.

Adenylic acid (AA) was supplied by the Armour Labora-tories, Chicago.

L-Glutamic acid hydrochloride, hydroxylamine hydro-chloride and hydrazine sulphate were commercial samplestwice recrystallized from water. All solutions were neutral-ized before use.

Methylamine hydrochloride was prepared free fromNH4C1 by the method of Francois (1907). A large excess ofyellow HgO was used to make sure that all traces of NH3were removed.

DL-Methionine oxide was prepared by the method ofToennies & Kolb (1939).

D-Glutamic acid, N-carbobenzyloxy-L-glutamic acid, L-i8oglutamine (L-glutamic acid oc-amide), and L-glutaminewere kindly given by Dr A. C. Chibnall.

Phosphate buffers were mixtures of Na2HPO4 andKH2PO4.

Glyoxaline buffers were prepared by adjusting the pH ofsolutions of the base with HCI.

METHODSIncubations were usually performed in open tubes at 300 andsamples taken as necessary for estimations.

Chemical determinationsHydroxamic acid was estimated by the method ofLipmann

& Tuttle (1945) except that determinations were done on4.5 ml. samples as described previously (Elliott & Gale,1948).