55

SOME OBSERVATIONS ON THE HAEMOCYANINOF LIMULUS

BY LANCELOT T. HOGBEN,Professor of Zoology in the University of Capetown,

AND KATHLEEN F. PINHEY,

Philip Carpenter Fellow in Zoology, McGill University.

(Received 4th February 1927.)

(With Ten Text-figures.)

(From the Department of Zoology, McGill University.)

1. INTRODUCTION.

IN a previous communication it has been shown that the haemocyanins of Crustaceaand of Helix are fundamentally different with respect to the action of neutral salts,and the pH corresponding to minimum affinity for oxygen (Hogben and Pinhey,1926). The object of this communication is to show that the haemocyanin ofLimulus is different from that of either Helix or the species of Crustacea hithertoinvestigated. The method employed for studying the dissociation of oxyhaemo-cyanin in this research was a colorimetric procedure. Opportunity may here betaken of describing the more elaborate colorimetric method used for this pur-pose, though the principle is essentially similar to that indicated as a methodfor class work by students in a communication by Pantin and Hogben (1925).The general similarity in behaviour of haemocyanins and haemoglobins, theextreme simplicity of the method (even in the form described below) and theimportance of haemoglobin in physiological and biochemical teaching, justifythe suggestion that the study of the haemocyanin system is a specially appro-priate subject for laboratory work. Since a large yield can be obtained fromsuch animals as Maia or Limulus, and since the blood of these animals, whenfiltered through muslin and shaken with chloroform, will keep indefinitely in thecold, no difficulty need arise in obtaining supplies through marine biologicallaboratories.

2. METHOD.

Owing to the fact that reduced haemocyanin is colourless, whereas oxy-haemo-cyanin is a deep blue colour, it is possible by a colorimetric method to estimate theextent of oxidation by comparison with standards of known dilution of the bluederivative. If serum is used this'may necessitate the use of a diluting solution of

LANCELOT T. HOGBEN and KATHLEEN F. PINHEY

yellowish tint in order to match the colour of the reduced blood. With a suitableneutral pigment, this presents no difficulty. With blood of Limulus prepared asindicated below it is only necessary to make the diluting solution slightly opalescent,since the reduced blood is quite colourless. The blood of Limulus, like that ofCrustacea or Helix, can be kept indefinitely in the cold, if shaken with chloroform.The latter can be removed by centrifuging before use, a procedure which removesthe lipochromes which in the case of Crustacean blood usually obscure to someextent the blue tint of the haemocyanin, to which they are almost complementary.

Colour standards having been prepared, the remainder of the experiment isexceedingly simple. Since it is not necessary to remove the blood from the vesselin which equilibrium is effected, it is also unnecessary to prepare gas mixturesin order to expose the sample to a particular partial pressure. By connecting thevessel with a rotary pump provided with a good manometer, the blood can be exposedto any required atmospheric pressure. Since the oxygen content of the atmosphereis constant, and the appropriate correction for the vapour pressure of water at thetemperature of the apparatus is obtainable from tables, the blood can be exposedto any required partial pressure of oxygen by applying the simple formula:

p = o-2i (b — m— v),

where p is the partial pressure of oxygen, b the atmospheric pressure, m theheight of the mercury in the manometer, and v the vapour pressure of water.

A convenient form of equilibrating vessel is in Fig. i, andis made by fitting into the neck of a separating funnel of about150 c.c. capacity a tube of uniform bore with the (carefullyselected) colorimeter tubes. The other end,provided with aglassstopcock, is connected, when occasion demands, to the pumpand manometer. When the pressure inside the vessel has beenbrought to the required value (a matter of a few seconds witha high speed pump), the stopcock is closed, and the samplingtube placed in a bath with an arrangement for shaking, suchas is described by Barcroft. In these experiments an electricallydriven device was used, five metal cups with spring clips tohold five sampling tubes being rotated simultaneously at highspeed. Thus a five point curve can be determined at any giventemperature in a few minutes. Having adjusted the pressuresin the sample tubes so that the points obtained will all fallon significant parts of the curve (as determined by previousexperience), a preliminary mixing in the bath is generallyadopted. The tubes are then tested to ascertain whether thepressure has remained constant. The fluid is allowed to draininto the lower portion, while air is instantaneously admitted,and the original pressure restored. The object of this is tocompensate for any error due to the giving off of oxygen by theblood itself, a source of error which in any case can be practically obliterated by

•WatertightRubber Cap

Fig.

—Rubber

Colouri-Standard*

Tubes

IZcms.1. EquilibratingApparatus.

Some Observations on the Haemocyanin of Limulus 57

making the air space in the manometer large. A second mixing is carried out forthree minutes. The sampling tubes are removed, and, after allowing the fluid to fallinto the lower tubes, are then compared with the colour standards. From start tofinish, the execution of a five point dissociation curve does not require more than20 minutes with the equipment described.

The previous preparation of a large stock of blood by the chloroform methodadmits of a large number of experiments being done on the same sample of blood,i.e. a solution with uniform concentration of haemocyanin may be used throughouta series of tests.

3. THE EFFECT OF HYDROGEN ION CONCENTRATION.

In an earlier paper one of us has shown that the affinity of haemocyanin foroxygen does not diminish indefinitely with increasing hydrogen ion concentration,but has a minimum value at a certain critical pH, after which it increases withfurther acidification. The different values of the "critical pH" found for thehaemocyanins of different Crustacea by Hogben indicates that even the haemo-cyanins of different species of decapod Crustacea are different. This differencehas been confirmed by subsequent observations of other workers. Thus for Maiathe minimal affinity for oxygen was found by Hogben (1926) to be in the neigh-bourhood of pH 6-2. In a more recent paper Kerridge (1926) has investigated thebuffering powers of the blood of Maia, and finds that it reaches a maximum in thecase of reduced blood at 6-39 and in the case of oxidised blood at 6-205. The CO2

dissociation curves of reduced and oxidised blood do in fact cross in the case ofMaia in the neighbourhood of 6-3. If (1) as Parsons and Parsons (1923) haveshown, the main buffer action is due to the respiratory protein itself; (2) the oxygenaffinity is at a minimum at about this point, it follows from Le Chatelier's principlefor reasons stated elsewhere (Pantin and Hogben, 1925) that the CO2 dissociationcurves for reduced and oxidised blood must cross at this point. Thus the workof Kerridge is confirmatory of the conclusion that the critical pH. for Maia is inthe neighbourhood of 6*2. The authors have repeated previous observations onthe critical pH. of Maia blood, using an electrometric method with similar results.In the case of Cancer Hogben's curves give yo as the critical pH. In a recentcommunication Stedman and Stedman (1926) confirm this: "the minimum appears,from the experiments here recorded, to occur in the neighbourhood of the neutralpoint."

These authors appear to be under some misunderstanding. On p. 955 (loc. cit.)they state, "although the colorimetric method might be expected to give accurateresults and possesses the advantage of being rapid and simple, it is evident thatcurves obtained by this method under different conditions of acidity will not becomparable, unless the standards are, in each case, maintained at the same degreeof saturation. This condition will not be fulfilled in the case of the haemocyaninfrom Cancer and Homarus if the standards are in equilibrium with air and thetemperature is as high as 230. It is improbable that they were fulfilled at theslightly lower temperature of 18-7° employed by Hogben in the case of Cancer

58 LANCELOT T. HOGBEN and KATHLEEN F. PINHEY

serum, unless, indeed, the salts added in the form of buffer mixtures as well asthose present in the serum itself profoundly modified the shape of the curves.Whilst the colorimetric method is undoubtedly capable of indicating the generalinfluence of pH on a particular haemocyanin, the results so obtained can have noquantitative significance unless care is taken to avoid the sources of error indicatedabove."

We confess some surprise at this misunderstanding, because it has been ex-plicitly stated, both in print and in personal communication to the Stedmans, thatthe standards were in all these experiments prepared from normal serum in equili-brium with the atmosphere at room temperature. Now the Stedmans have them-selves published data (1925) relative to the normal serum of Cancer at roomtemperature. On p. 547 (Biochem.Journ. xrx) we find that at an oxygen partial pressureof 21-7 mm. the blood is 88-i per cent, saturated; at 80 mm. it is 100 per cent,saturated; and from curves on pp. 548-9, it can be seen by graphical interpolationthat the normal serum of Cancer is more than 95 per cent, saturated at a partialpressure equivalent to one quarter of the atmospheric partial pressure of oxygen.Even if we had not this evidence, the shape of the dissociation curve obtained forthe sample of blood used would betray the true state of affairs by its asymptoticcharacter. Not only is it doubtful that the method employed by the Stedmans ismore accurate: it is also a fact that in such observations as we have made, in everycase using the same sample of blood throughout a whole series of experiments,many sources of interpretative error which occur in successive determinations ondifferent samples by the more laborious method are avoided. Let it be re-emphasised therefore that all the curves in any series of experiments by the colori-metric method, as used by us, have been based throughout on one sample, i.e. ona solution containing initially the same active mass of haemocyanin.

In the case of Helix the authors (Hogben and Pinhey, 1926) found a minimumin the neighbourhood of pH 8-o. Redfield and Hurd (1925) in a preliminarycontribution have shown that increasing CO2 tension increased the affinity of thehaemocyanin of Limulus for oxygen, and it seemed at first sight that in this respectthe haemocyanin of Limulus differed fundamentally from haemoglobin. However,the results obtained for Crustacea by the senior author, and subsequent studieson Helix, afforded a strong presumption to the contrary. " I t is legitimate," westated (p. 210), "to predict the likelihood on the basis of experiments on thehaemocyanins of Helix and Crustacea, that with more extended observation, thehaemocyanin of Limulus will not be found to contrast in behaviour in this respectwith the haemocyanins so far investigated." Since these lines were written DrRedfield has informed us that he has carried out determinations in the alkalinerange which confirm our prediction: that is to say, there exists on the alkaline sideof the pH of normal serum of Limulus a critical value above which increasinghydrogen ion concentration diminishes affinity for oxygen. As it was essential toour experiments on the effect of temperature and salts to locate this point, wehave ourselves investigated this question; but since we understand that DrRedfield is publishing an extensive account of his own experiments with both

Some Observations on the Haemocyanin of Limulus 59

the analytical and colorimetric methods, we shall confine ourselves to stating theresults.

(1) We cannot wholly confirm the statement made by the Stedmans that"addition of acid or alkali to the dialysed serum in sufficient amount to bring thepH to 5-5 or 8-0 respectively does not appear to produce any marked change inthe steepness of the dissociation curve." This, like the impression first gainedfrom a study of the haemocyanin of Helix (Pantin and Hogben, 1925), is only truein so far as the haemocyanins of Helix and Limulus 'are much less profoundlyinfluenced by hydrogen ion concentration than the haemocyanins of any Crustaceastudied so far.

(2) Though less marked than in the case of Helix, the shape of the dissociationcurve of Limulus haemocyanin changes abruptly at the critical pH. Thus the curvesfor pH. 8-7 and 8-45 cross one another. Subsequent experiments on Maia as wellas re-examination of earlier records show that, both in the case of Crustacea andLimulus, though not perhaps to so marked an extent as in Helix, the dissociationcurves for haemocyanin on the acid side of the critical pH are natter as theyapproach complete saturation.

In short, the phenomenon originally described by one of us for Crustacea onlyis a perfectly general characteristic of the family of reversibly oxidisable chromo-proteins which is denoted by the term haemocyanin. The characteristic feature of thehaemocyanin of Limulus is that the critical pH (about 8*5) is very high in the alkalinerange—definitely though not much higher than that of Helix—whereas the criticalpH of all samples of Crustacean haemocyanin so far investigated is either nearthe neutral point (Cancer) or on the acid side of it {Homarus, Maia, Palinurus).Further, the haemocyanins of the Crustacea as a group are much more influencedby changes in hydrogen ion concentration than those of Helix or Limulus. As willbe seen, the influence of salts on the haemocyanins of Helix and Limulus is alsoof a different nature from the effect described for Crustacean haemocyanin.

It is premature to say whether the haemocyanins are actually a more hetero-geneous assemblage than the haemoglobins, since we know little about the physicalchemistry of the latter from a comparative standpoint. However, as we havepointed out, this general property which we have described is probably charac-teristic of the latter as well as the former: the observations of Rona and Yllpo(1917) seem to indicate that beyond pH 6-0 increasing acidity increases the affinityfor oxygen of mammalian haemoglobin. We cannot deny dogmatically the possi-bility that the critical pH corresponds to the isoelectric point, but the suggestiveevidence of Rona and Yllpo's work, together with the new data given in a recentpaper by the Stedmans, reinforce the possibility stated earlier to the effect that atthe critical pH there is an abrupt change in the character of the oxidation system,not directly related to changes in the ionisation of the protein. This does not implyspecifically that the haemocyanin of a given species exists in different tautomers,as at one time suggested by us. But it does suggest a possible reason forthe existence of separate acid and alkaline modifications of haematin, and itwould be interesting to know whether the spectroscope reveals any change

6o L A N C E L O T T . H O G B E N and K A T H L E E N F . P I N H E Y

in the neighbourhood of the critical point for the haemocyanins and haemo-globins.

4. THE EFFECT OF TEMPERATURE.

The results obtained in experiments on the effect of temperature on the disso-ciation of the oxyhaemocyanin of Limulus were so surprising as to necessitate somefurther experiments on Crustacean blood.

It has been pointed out previously that if there exists a stoichiometrical re-lation such that / molecules of oxyhaemocyanin give rise to m molecules of reducedhaemocyanin and n molecules of oxygen, then

(Cyf)'»(O2)»

(cyoyIf throughout a series of observations the same sample of blood, i.e. a solution ofhaemocyanin of the same molecular concentration, is employed as in all ourprevious experiments, and if x^, %,, etc. be used to denote the oxygen partialpressure corresponding to 50, 60, etc. per cent, saturation, then by Henry's law,

and n (9 log x^)t = 9 log K.

If a represents a factor for solubility of oxygen at different temperatures:

n . d log ax^o = d log K.

Applying the Van 't Hoff isochore

and putting tan 6 for the slope of the line obtained by plotting logyjOX^ against thereciprocal of the absolute temperature, we have

-- = 2 x 2-303 . tan0.

This gives as stated (Hogben, p. 230) a value for Q per gm. molecule of oxygen.Opportunity may here be taken to correct an error overlooked on p. 238 (Hogben)and pp. 206 and 214 (Hogben and Pinhey) where the value of Q is referred to asthe value per n gm. molecules.

(a) Experiments with the blood of Maia. In earlier work on normal serum ofHelix a value of 8000 calories was given for the reaction. For normal serum ofMaia at/>H 8-2 the value 9100 calories was given. The possibility that the differ-ence was significant was left open. Experiments on Limulus described below ledus to investigate the effect of temperature in the absence of salts. For dialysedserum of Maia at pH 7-7 between i3-8° C. and 36-0° C. the value of Q obtainedwas about 5000. Above 36-0° the temperature coefficient increases abruptly—possibly due to coagulative changes, as shown in Fig. 2.

This shows that no importance is to be attached to the difference recorded inour previous communication; and the discrepancy is by no means surprising.

Some Observations on the Haemocyanin of Limulus 61

Adair (p. 543 loc. cit) gives the following values of Q for mammalian haemo-globin :

Du Bois Raymond ... ... ... 13,000-19,000Berthelot ... ... ... ... 11,600Barcroft ... ... ... ... 27,000Adolph and Henderson ... ... 10,000Adair 13,600Brown and Hill ... ... ... 11,500-15,000

This divergence he interpreted as due to different conditions of salinity andhydrogen ion concentration in experiments of different workers. If this is so, we

MAIA p H - 7 7 -Dialysed Serum

10 20 30 40 50 60 70 80 »Oxygen partial pressure (mm.)

Fig. 2.

£15"

MAIA pH 7-7Dialysed Serum

350 345 340 335 330 325

Fig- 3-

must assume that the values of Q for the reaction between oxygen and the haemo-cyanin in combination with different radicals are widely divergent. Presumably,therefore, the only strictly comparable values for Q of the haemocyanins of differentspecies would be those obtained at the isoelectric point by graphical interpolation.

(b) Experiments on the blood of Limulus. Experiments on the effect of tem-perature on the blood of Limulus have been conducted on both the acid andalkaline sides of the critical pH. The noteworthy fact is that the effect of tem-perature is extraordinarily small (Figs. 4, 5, 6, 7). Even if we include the experi-ment indicated in Fig. 6, giving a value for Q of about 3000, which is doubtfulowing to the possibility of coagulative changes occurring above 400 C , the figureis still a low one (see Fig. 7).

It would almost seem necessary in the case of Limulus to postulate the existenceof a complex reaction of which one phase is endothermic to account for the resultsobtained. But it would perhaps be premature to state that in its behaviour towards

62 LANCELOT T. HOGBEN and KATHLEEN F. PINHEY

temperature alone the haemocyanin of Limulus can be differentiated from that ofHelix and Crustacea.

Oxygen partial pressurefmm)

10 20 30 40Oxygen partial pressure (mm.)

Fig. 6.

10 20 30Oxygen partial pressure (mm.)

Fig- 5-

Fig. 7 .

S. THE EFFECT OF NEUTRAL SALTS.

The primary object of this investigation was to ascertain the effect of neutralsalts on the dissociation of the haemocyanin of Limulus. Owing to circumstanceswhich necessitated the departure of one of the authors from America, it has notbeen possible to carry the investigation as far as was originally hoped.

Some Observations on the Haemocyanin of Limulus 63

It has been shown (Hogben, 1926) that the addition of the neutral chloridesof the alkaline and alkaline earth metals increases—at least on the alkaline sideof the "critical point"—the affinity of the haemocyanin of the lobster for oxygen.In the case of Helix the reverse was found to be the case (Hogben and Pinhey,1926). As the type of effect which has been described in the case of Homarus issimilar to that which has been described in relation to the haemoglobins hithertoinvestigated, certain issues of some importance are raised in this connection.

In the first place it seemed desirable to ascertain whether the type of effectdescribed for Homarus is characteristic of the haemocyanins of other species ofCrustacea. As the haemocyanin of Maia is relatively less affected than that ofother Crustacea investigated by changes in hydrogen ion concentration, this formis especially suitable. By the kindness of Mr Pantin we were able to obtain a supplyof serum of Maia squinado prepared as indicated above.

s 100r

20

10

— 0-'5MSrClz O -pH. 735

M NaCl ApH. 7-9

— Dialysed Serum 0 —pH. 80S

MAIA

10 20 30 40 50 60 70Oxygen partial pressure (mm.)

Fig. 8.

90

80

70

60

50

40

30

20

10

_0SMgCl2pH 76

— M. KCL V-pH 8-26

— Dialysed Serum Q-pH 8-26

MAIA t=i9-5°c.10 20 30 40 50 60 70Oxygen partial pressure (mm.)

Fig. 9.

In a previous communication it was tentatively stated by one of us (Hogben,1926) that "the effect of molar solutions of chlorides of calcium, strontium andmagnesium was in all cases at least as great as that of 2M solutions of the chloridesof sodium, potassium and lithium, when the serum was diluted 50 per cent, withthe reagent, and would suggest that it is not primarily the cation which enters intothe question." These remarks apply to Homarus. The experiments epitomised inFigs. 8 and 9, and based on dialysed solutions of the blood of Maia tend to reinforcethe first consideration, but since the effect of MgCl2 in half-molar concentrationis apparently greater than the effect of NaCl in molar concentration, they do notnecessarily indicate that the chloride ion concentration is a significant element inthe influence of salts on the dissociation of haemocyanin. Furthermore, a singleexperiment with equivalent concentrations of KCl, KBr, and KI did not revealsuch a difference between the effects of these three salts, as might be anticipated,

64 LANCELOT T . HOGBEN and KATHLEEN F. PINHEY

if an explanation on the lines of oxidation-reduction potential, as put forwardspeculatively in the last communication (Hogben and Pinhey, 1926), were in factadmissible.

As with experiments on the haemocyanin of Maia, dialysed blood of Limuluswas used. Both on the acid (Fig. 10) and alkaline side of the "critical pH" additionof chlorides of sodium and potassium depress the dissociation of the oxyhaemo-cyanin of Limulus. In this respect the haemocyanin of Limulus more closely re-sembles that of Helix than that of the Crustacean genera studied so far from thesame standpoint. The chlorides, bromides and iodides seem to depress the affinityof the haemocyanin of Limulus for oxygen to the same extent within the limits oferror which this method involves (Fig. io), but in one or two experiments theeffect of KBr was less marked than that of KC1.

90

80

70

60

50

40

30

20

10

11

1

///

_/1

. —

DiaJysed SerumpH 78

M KBr. pH 7-48-MKCL. pH 7-45

MKl pH 75

LIMULUS t-i9-5°c

0

•v -A

10 20 30 40 50 60 70Oxygen partial pressure (mm.)

Fig. 10.

As to the effect of chlorides of the alkaline earths, our experiments do not leadto definite conclusions owing to the complications arising out of the impossibilityof excluding the influence of differences in hydrogen ion concentration, unlessrelatively large quantities of buffers of alkali salts were also added.

It is clear that the extent to which these various effects are due to differentaffinities for oxygen of the undissociated haemocyanin molecule and the dissociatedsalt of haemocyanin with different alkaline or acid radicals, and the possible inter-vention of other factors cannot be profitably discussed without knowing the iso-electric point of the haemocyanin of Limulus. For reasons stated it was notpossible to make this determination, as it is hoped to do later, by one of us.But there is an item of new information which bears on the complexity of theproblem significantly, we believe, though at this stage it would be unprofitable todiscuss its precise meaning. In two experiments the influence of a non-electrolyte

Some Observations on the Haemocyanin of Limulus 65

was investigated (at pH 7-3). The substance used was urea. In both cases theaddition of urea in molar concentration greatly increased the affinity of the systemfor oxygen. A similar effect has not been described, as far as we know, in the caseof haemoglobin.

6. CONCLUSIONS.

1. Observations on the effects of salts, hydrogen ion concentration and tem-perature on the haemocyanin of Limulus, and some confirmatory experiments onthe blood of Maia are described above.

2. The haemocyanin of Limulus is not identical with that of Helix or that ofany species of Crustacea so far investigated, but it resembles the former muchmore closely than the latter.

3. The haemocyanin of Limulus, like the haemocyanins of Crustacea andHelix, has a minimal affinity for oxygen at a certain critical pH. In all cases inwhich this phenomenon has been investigated by the colorimetric method there isa difference in shape of the dissociation curves on either side of the critical valueindicating a different type of reaction.

7. REFERENCES.ADAIR (1925). Jowrn. Biol. Chem. 63.HOGBEN (1926). This Journal, 3.HOGBEN and PINHEY (1926). This Journal, 4.KERRIDGE (1926). Jowrn. Pkytiol. 62.PANTIN and HOGBEN (1925). Joitrn. Marine Biol. Atsoc. 13 (n. 5).PARSONS and PARSONS (1923). Joitrn. Gen. Phytiol. 6.REDFIELD and HURD (1925). Proc. Nat. Acad. Sci. 11.RONA and YLLPO (1917). Biochem. Zeit. 76.STEDMAN and STEDMAN (1925). Biochem. Journ. 19.

(1926). Biochem. Journ. 20.

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