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Some Observations on the Haemocyanin of Limulus

1927; The Company of Biologists; Volume: 5; Issue: 1 Linguagem: Inglês

10.1242/jeb.5.1.55

1477-9145

Lancelot Hogben, Kathleen F. Pinhey,

Neurobiology and Insect Physiology Research

In a previous communication it has been shown that the haemocyanins of Crustacea and 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 of Limulus is different from that of either Helix or the species of Crustacea hitherto investigated. The method employed for studying the dissociation of oxyhaemo-cyanin in this research was a colorimetric procedure. Opportunity may here be taken of describing the more elaborate colorimetric method used for this purpose, though the principle is essentially similar to that indicated as a method for class work by students in a communication by Pantin and Hogben (1925). The general similarity in behaviour of haemocyanins and haemoglobins, the extreme simplicity of the method (even in the form described below) and the importance of haemoglobin in physiological and biochemical teaching, justify the suggestion that the study of the haemocyanin system is a specially appropriate subject for laboratory work. Since a large yield can be obtained from such animals as Maia or Limulus, and since the blood of these animals, when filtered through muslin and shaken with chloroform, will keep indefinitely in the cold, no difficulty need arise in obtaining supplies through marine biological laboratories.Owing to the fact that reduced haemocyanin is colourless, whereas oxy-haemocyanin is a deep blue colour, it is possible by a colorimetric method to estimate the extent of oxidation by comparison with standards of known dilution of the blue derivative. If serum is used this may necessitate the use of a diluting solution of yellowish tint in order to match the colour of the reduced blood. With a suitable neutral pigment, this presents no difficulty. With blood of Limulus prepared as indicated 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 of Crustacea 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 removes the lipochromes which in the case of Crustacean blood usually obscure to some extent the blue tint of the haemocyanin, to which they are almost complementary.Colour standards having been prepared, the remainder of the experiment is exceedingly simple. Since it is not necessary to remove the blood from the vessel in which equilibrium is effected, it is also unnecessary to prepare gas mixtures in order to expose the sample to a particular partial pressure. By connecting the vessel with a rotary pump provided with a good manometer, the blood can be exposed to any required atmospheric pressure. Since the oxygen content of the atmosphere is constant, and the appropriate correction for the vapour pressure of water at the temperature of the apparatus is obtainable from tables, the blood can be exposed to any required partial pressure of oxygen by applying the simple formula : where p is the partial pressure of oxygen, b the atmospheric pressure, m the height of the mercury in the manometer, and v the vapour pressure of water.A convenient form of equilibrating vessel is in Fig. 1, and is made by fitting into the neck of a separating funnel of about 150 c.c. capacity a tube of uniform bore with the (carefully selected) colorimeter tubes. The other end, provided with aglass stopcock, is connected, when occasion demands, to the pump and manometer. When the pressure inside the vessel has been brought to the required value (a matter of a few seconds with a high speed pump), the stopcock is closed, and the sampling tube placed in a bath with an arrangement for shaking, such as is described by Barcroft. In these experiments an electrically driven device was used, five metal cups with spring clips to hold five sampling tubes being rotated simultaneously at high speed. Thus a five point curve can be determined at any given temperature in a few minutes. Having adjusted the pressures in the sample tubes so that the points obtained will all fall on significant parts of the curve (as determined by previous experience), a preliminary mixing in the bath is generally adopted. The tubes are then tested to ascertain whether the pressure has remained constant. The fluid is allowed to drain into the lower portion, while air is instantaneously admitted, and the original pressure restored. The object of this is to compensate for any error due to the giving off of oxygen by the blood itself, a source of error which in any case can be practically obliterated by making the air space in the manometer large. A second mixing is carried out for three minutes. The sampling tubes are removed, and, after allowing the fluid to fall into the lower tubes, are then compared with the colour standards. From start to finish, the execution of a five point dissociation curve does not require more than 20 minutes with the equipment described.The previous preparation of a large stock of blood by the chloroform method admits 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 throughout a series of tests.In an earlier paper one of us has shown that the affinity of haemocyanin for oxygen does not diminish indefinitely with increasing hydrogen ion concentration, but has a minimum value at a certain critical pH, after which it increases with further acidification. The different values of the "critical pH" found for the haemocyanins of different Crustacea by Hogben indicates that even the haemocyanins of different species of decapod Crustacea are different. This difference has been confirmed by subsequent observations of other workers. Thus for Maia the minimal affinity for oxygen was found by Hogben (1926) to be in the neighbourhood of pH 6-2. In a more recent paper Kerridge (1926) has investigated the buffering powers of the blood of Maia, and finds that it reaches a maximum in the case 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 of Maia in the neighbourhood of 6-3. If (1) as Parsons and Parsons (1923) have shown, the main buffer action is due to the respiratory protein itself; (2) the oxygen affinity is at a minimum at about this point, it follows from Le Chatelier's principle for reasons stated elsewhere (Pantin and Hogben, 1925) that the CO2 dissociation curves for reduced and oxidised blood must cross at this point. Thus the work of Kerridge is confirmatory of the conclusion that the critical pH for Maia is in the neighbourhood of 6.2. The authors have repeated previous observations on the critical pH of Maia blood, using an electrometric method with similar results. In the case of Cancer Hogben's curves give 7.0 as the critical pH. In a recent communication Stedman and Stedman (1926) confirm this : "the minimum appears, from the experiments here recorded, to occur in the neighbourhood of the neutral point."These authors appear to be under some misunderstanding. On p. 955 (loc. cit.) they state, "although the colorimetric method might be expected to give accurate results and possesses the advantage of being rapid and simple, it is evident that curves obtained by this method under different conditions of acidity will not be comparable, unless the standards are, in each case, maintained at the same degree of saturation. This condition will not be fulfilled in the case of the haemocyanin from Cancer and Homarus if the standards are in equilibrium with air and the temperature is as high as 23°. It is improbable that they were fulfilled at the slightly lower temperature of 18-7° employed by Hogben in the case of Cancer serum, unless, indeed, the salts added in the form of buffer mixtures as well as those present in the serum itself profoundly modified the shape of the curves. Whilst the colorimetric method is undoubtedly capable of indicating the general influence of pH on a particular haemocyanin, the results so obtained can have no quantitative significance unless care is taken to avoid the sources of error indicated above."We confess some surprise at this misunderstanding, because it has been explicitly stated, both in print and in personal communication to the Stedmans, that the standards were in all these experiments prepared from normal serum in equilibrium with the atmosphere at room temperature. Now the Stedmans have them-selves published data (1925) relative to the normal serum of Cancer at room temperature. On p. 547 (Biochem.Journ. xix) we find that at an oxygen partial pressure of 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 interpolation that the normal serum of Cancer is more than 95 per cent, saturated at a partial pressure 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 for the sample of blood used would betray the true state of affairs by its asymptotic character. Not only is it doubtful that the method employed by the Stedmans is more accurate : it is also a fact that in such observations as we have made, in every case using the same sample of blood throughout a whole series of experiments, many sources of interpretative error which occur in successive determinations on different samples by the more laborious method are avoided. Let it be reemphasised therefore that all the curves in any series of experiments by the colorimetric method, as used by us, have been based throughout on one sample, i.e. on a solution containing initially the same active mass of haemocyanin.In the case of Helix the authors (Hogben and Pinhey, 1926) found a minimum in the neighbourhood of pH 8-o. Redfield and Hurd (1925) in a preliminary contribution have shown that increasing CO2 tension increased the affinity of the haemocyanin of Limulus for oxygen, and it seemed at first sight that in this respect the haemocyanin of Limulus differed fundamentally from haemoglobin. However, the results obtained for Crustacea by the senior author, and subsequent studies on Helix, afforded a strong presumption to the contrary. "It is legitimate," we stated (p. 210), "to predict the likelihood on the basis of experiments on the haemocyanins of Helix and Crustacea, that with more extended observation, the haemocyanin of Limulus will not be found to contrast in behaviour in this respect with the haemocyanins so far investigated." Since these lines were written Dr Redfield has informed us that he has carried out determinations in the alkaline range which confirm our prediction : that is to say, there exists on the alkaline side of the pH of normal serum of Limulus a critical value above which increasing hydrogen ion concentration diminishes affinity for oxygen. As it was essential to our experiments on the effect of temperature and salts to locate this point, we have ourselves investigated this question; but since we understand that Dr Redfield is publishing an extensive account of his own experiments with both the analytical and colorimetric methods, we shall confine ourselves to stating the results.(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 the pH to 5-5 or 8-0 respectively does not appear to produce any marked change in the steepness of the dissociation curve." This, like the impression first gained from a study of the haemocyanin of Helix (Pantin and Hogben, 1925), is only true in so far as the haemocyanins of Helix and Limulus 'are much less profoundly influenced by hydrogen ion concentration than the haemocyanins of any Crustacea studied so far.(2) Though less marked than in the case of Helix, the shape of the dissociation curve of Limulus haemocyanin changes abruptly at the critical pH. Thus the curves for pH 8-7 and 8-45 cross one another. Subsequent experiments on Maia as well as re-examination of earlier records show that, both in the case of Crustacea and Limulus, though not perhaps to so marked an extent as in Helix, the dissociation curves for haemocyanin on the acid side of the critical pH are flatter as they approach complete saturation.In short, the phenomenon originally described by one of us for Crustacea only is a perfectly general characteristic of the family of reversibly oxidisable chromoproteins which is denoted by the term haemocyanin. The characteristic feature of the haemocyanin of Limulus is that the critical pH (about 8.5) is very high in the alkaline range—definitely though not much higher than that of Helix—whereas the critical pH of all samples of Crustacean haemocyanin so far investigated is either near the 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 influenced by changes in hydrogen ion concentration than those of Helix or Limulus. As will be seen, the influence of salts on the haemocyanins of Helix and Limulus is also of a different nature from the effect described for Crustacean haemocyanin.It is premature to say whether the haemocyanins are actually a more heterogeneous assemblage than the haemoglobins, since we know little about the physical chemistry of the latter from a comparative standpoint. However, as we have pointed out, this general property which we have described is probably characteristic of the latter as well as the former: the observations of Rona and Ÿllpo (1917) seem to indicate that beyond pH 6.0 increasing acidity increases the affinity for oxygen of mammalian haemoglobin. We cannot deny dogmatically the possibility that the critical pH corresponds to the isoelectric point, but the suggestive evidence of Rona and Ÿllpo's work, together with the new data given in a recent paper by the Stedmans, reinforce the possibility stated earlier to the effect that at the 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 imply specifically 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 for the existence of separate acid and alkaline modifications of haematin, and it would be interesting to know whether the spectroscope reveals any change in the neighbourhood of the critical point for the haemocyanins and haemoglobins.The results obtained in experiments on the effect of temperature on the dissociation of the oxyhaemocyanin of Limulus were so surprising as to necessitate some further experiments on Crustacean blood.It has been pointed out previously that if there exists a stoichiometrical relation such that l molecules of oxyhaemocyanin give rise to m molecules of reduced haemocyanin and n molecules of oxygen, then If throughout a series of observations the same sample of blood, i.e. a solution of haemocyanin of the same molecular concentration, is employed as in all our previous experiments, and if x50, x60, etc. be used to denote the oxygen partial pressure corresponding to 50, 60, etc. per cent, saturation, then by Henry's law, and If a represents a factor for solubility of oxygen at different temperatures : Applying the Van't Hoff isochore and putting tan θ for the slope of the line obtained by plotting log10ax60 against the reciprocal of the absolute temperature, we have 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 as the value per n gm. molecules.(a) Experiments with the blood of Maia. In earlier work on normal serum of Helix a value of 8000 calories was given for the reaction. For normal serum of Maia at pH 8.2 the value 9100 calories was given. The possibility that the difference was significant was left open. Experiments on Limulus described below led us to investigate the effect of temperature in the absence of salts. For dialysed serum of Maia at pH 7-7 between 13.8° C. and 36.0° C. the value of Q obtained was 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 in our previous communication; and the discrepancy is by no means surprising.Adair (p. 543 loc. cit.) gives the following values of Q for mammalian haemoglobin :This divergence he interpreted as due to different conditions of salinity and hydrogen ion concentration in experiments of different workers. If this is so, we must assume that the values of Q for the reaction between oxygen and the haemocyanin in combination with different radicals are widely divergent. Presumably, therefore, the only strictly comparable values for Q of the haemocyanins of different species would be those obtained at the isoelectric point by graphical interpolation.(b) Experiments on the blood of Limulus. Experiments on the effect of temperature on the blood of Limulus have been conducted on both the acid and alkaline sides of the critical pH. The noteworthy fact is that the effect of temperature is extraordinarily small (Figs. 4, 5, 6, 7). Even if we include the experiment indicated in Fig. 6, giving a value for Q of about 3000, which is doubtful owing to the possibility of coagulative changes occurring above 40° C., the figure is still a low one (see Fig. 7).It would almost seem necessary in the case of Limulus to postulate the existence of a complex reaction of which one phase is endothermic to account for the results obtained. But it would perhaps be premature to state that in its behaviour towards temperature alone the haemocyanin of Limulus can be differentiated from that of Helix and Crustacea.The primary object of this investigation was to ascertain the effect of neutral salts on the dissociation of the haemocyanin of Limulus. Owing to circumstances which necessitated the departure of one of the authors from America, it has not been possible to carry the investigation as far as was originally hoped.It has been shown (Hogben, 1926) that the addition of the neutral chlorides of the alkaline and alkaline earth metals increases—at least on the alkaline side of 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 is similar to that which has been described in relation to the haemoglobins hitherto investigated, certain issues of some importance are raised in this connection.In the first place it seemed desirable to ascertain whether the type of effect described for Homarus is characteristic of the haemocyanins of other species of Crustacea. As the haemocyanin of Maia is relatively less affected than that of other Crustacea investigated by changes in hydrogen ion concentration, this form is especially suitable. By the kindness of Mr Pantin we were able to obtain a supply of serum of Maia squinado prepared as indicated above.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 and magnesium was in all cases at least as great as that of 2M solutions of the chlorides of sodium, potassium and lithium, when the serum was diluted 50 per cent, with the reagent, and would suggest that it is not primarily the cation which enters into the question." These remarks apply to Homarus. The experiments epitomised in Figs. 8 and 9, and based on dialysed solutions of the blood of Maia tend to reinforce the first consideration, but since the effect of MgCl2 in half-molar concentration is apparently greater than the effect of NaCl in molar concentration, they do not necessarily indicate that the chloride ion concentration is a significant element in the influence of salts on the dissociation of haemocyanin. Furthermore, a single experiment with equivalent concentrations of KC1, KBr, and KI did not reveal such a difference between the effects of these three salts, as might be anticipated, if an explanation on the lines of oxidation-reduction potential, as put forward speculatively in the last communication (Hogben and Pinhey, 1926), were in fact admissible.As with experiments on the haemocyanin of Maia, dialysed blood of Limulus was used. Both on the acid (Fig. 10) and alkaline side of the "critical pH" addition of 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 the same standpoint. The chlorides, bromides and iodides seem to depress the affinity of the haemocyanin of Limulus for oxygen to the same extent within the limits of error which this method involves (Fig. 10), but in one or two experiments the effect of KBr was less marked than that of KC1.As to the effect of chlorides of the alkaline earths, our experiments do not lead to definite conclusions owing to the complications arising out of the impossibility of excluding the influence of differences in hydrogen ion concentration, unless relatively large quantities of buffers of alkali salts were also added.It is clear that the extent to which these various effects are due to different affinities for oxygen of the undissociated haemocyanin molecule and the dissociated salt of haemocyanin with different alkaline or acid radicals, and the possible intervention of other factors cannot be profitably discussed without knowing the isoelectric point of the haemocyanin of Limulus. For reasons stated it was not possible 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 the problem significantly, we believe, though at this stage it would be unprofitable to discuss its precise meaning. In two experiments the influence of a non-electrolyte was investigated (at pH 7-3). The substance used was urea. In both cases the addition of urea in molar concentration greatly increased the affinity of the system for oxygen. A similar effect has not been described, as far as we know, in the case of haemoglobin.Observations on the effects of salts, hydrogen ion concentration and temperature on the haemocyanin of Limulus, and some confirmatory experiments on the blood of Maia are described above.The haemocyanin of Limulus is not identical with that of Helix or that of any species of Crustacea so far investigated, but it resembles the former much more closely than the latter.The haemocyanin of Limulus, like the haemocyanins of Crustacea and Helix, has a minimal affinity for oxygen at a certain critical pH. In all cases in which this phenomenon has been investigated by the colorimetric method there is a difference in shape of the dissociation curves on either side of the critical value indicating a different type of reaction.