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VELOCITY CONSTANTSAINS~rORTH, GIBSON ID ROL-GHTON 27 REF`EREN CES 1. Haemoglobin: a symposium based on a conference in memory of Sir Joseph Barcroft; F. J. W. Roughton and J. C. Kendrew, editors. Butterworths, London, 1949. 2. Roughton, F`. J. W.' Otis, A. B., and Lyster, R. L. l.: The determination of the individual equilibrium constants of the four intermediate reactions between oxygen and sheep haemoglobin, Proc. Roy. Soc., Series B. 144: 29, 1955. 3. Lyster, R. L. J.: Ph.D. thesis, Cambridge University, 1955. 4. Gibson, Q. H.: An apparatus for flash photolysis and its application to the re- actions of myoglobin with gases, J. Physiol. 134: 112, 1956. 5. Gibson, Q. H.: The direct determination of the velocity constant of the reaction Hb4(CO)3 + CO ~ Hb4(CO)4, J. Physiol. 134: 123, 1956. 6. Gibson, Q. H. and Roughton, F`. J. W.: The determination of the velocity constants of the four successive reactions of carbon monoxide with sheep haemoglobin, Proc. Roy. Soc., Series B. 146: 206, 1957. 7. Gibson, Q. H. and Roughton. F. J. W.: The kinetics of haemoglobin and haem compounds as models for enzyme action, Faraday Society Discussions 20: 195, 1955. 8. Gibson, Q. H. and Roughton, F`. J. W.: The kinetics of dissociation of the first oxygen molecule from fully saturated oxyhaemoglobin in sheep blood solution, Proc. :lloy. Soc., Series B. 143: 310, 1955. 9. Roughton, F. J. W.: The kinetics of haemoglobin VI. The competition of carbon monoxide and oxygen for haemoglobin, Proc. Roy. Soc., Series B. 115: 473, 1934. 10. Gibson, Q. H. and Roughton, F`. J. W.: The kinetics and equilibria of the reactions of nitric oxide with sheep haemoglobin, J. Physiol., 136: 507, 1957. 11. Gibson, Q. H. and Roughton, F`. J. W.: The kinetics of dissociation of the first ligand molecule from fully saturated carboxyhaemoglobin and nitric oxide haemoglobin in sheep blood solution, Proc. Roy. Soc., Series B. 147: 44, 1957. DISCUSSION liar Ed.;all In tile early days when T was ~ strident in (~ambrirl~e Fn~l~n~l ~.t, , ~ , in 1924 and 192S, Professors Hartridge and Roughton were carrying out the first of their studies on the reaction velocities of hemoglobin with oxygen and carbon monoxide. The progress that has been made is certainly phenom- enal. In those days the idea of detecting individual velocity constants was regarded as completely out of the range of possibility. We have some time free for discussion and there are many directions the discussion could take. Dr. [Walter Hughes: I have just one brief comment which really refers to the work of Dr. Riggs. He has measured the effect of mercurials on the dissociation constant for oxygen. I have done the reverse and measured the effects of carbon monoxide and oxygen on the association constants for methylmercury. I find a difference between the two. This is perhaps more subtle than Dr. Riggs' difference in the sense that I think most people consider carbon monoxide and oxygen to form very similar complexes when combined with hemoglobin. Dr. Riggs measured the difference between reduced hemoglobin and oxyhemoglobin. In the case of bovine hemoglobin

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28 PART I. STRUCTURE OF HEMOGLOBIN I find that methylmercury is bound about three times more tightly to oxy- hemoglobin than to carbonmonoxy hemoglobin. Dr. Roz~ghtor': May I say a word about Dr. Hughes' contribution? If ' understood him right, it was the competitive equilibrium between carbon monoxide and oxygen in hemoglobin that he measured. Dr. Hughes: It was not measured that way. These were equilibrium con- stants between methylmercury and carbonmonoxy hemoglobin on the one hand and with oxyhemoglobin on the other hand. I found the equilibrium constant for methylmercury for oxyhemoglobin was three times that for carbonmonoxy hemoglobin. I want at some time to have a discussion on sulfhydryl groups. I think there are only two sulfhydryl groups in bovine hemoglobin. Within the spread of my data (equilibrium constants on individual measurements vary by 20 per cent) the affinity constants for both seemed to be the same. Thus there does not seem to be any interaction between the two. Dr. Roughton: It does not disturb me that you find a difference between CO hemoglobin and oxyhemoglobin. It is an old creed that these two com- pounds are physico-chemically similar, but this creed must now go, for the more intimately one studies them, the more frequent are the differences that one finds. Perhaps the most dramatic instance thereof is that the velocity of combination of the first oxygen molecule with hemoglobin is independent of temperature, whereas the velocity constant for the combination of the first carbon monoxide molecule with hemoglobin has a very clearcut dependence on temperature. Dr. Edsall: Or. Riggs was, I think, the first person to observe the effect of sulfhydryl groups on oxygen dissociation. As his work has already been referred to, I would like to call on Dr. Austen Riggs. or Rim.: I believe mv original measurements) of the oxygen equilibrium I, ,~ ... . , O ~ ~ of hemoglobin in the presence of p-chloromercuribenzoate ~ PCMB ~ shed some light on the affinity of mercurials for hemoglobin. These experiments indicate that at low degrees of oxygenation PCMB greatly increases the oxygen affinity, whereas at high oxygenation levels PCMB decreases the oxygen affinity. This effect is indicated most clearly if we plot the difference in the degree of oxygenation in the presence and absence of PCMB versus the oxygen pressure. (Fig. 1~. In such a plot we see that there exist two maximum shifts: first a large increase in oxygen affinity, then a decrease, faith the peaks at about 25 and 75 per cent oxygenation. Since this appears to be a completely reversible reaction, this gives us some information about the relative affinity with which PC~B is bound. Thus, if PCMB drives oxygen off, then oxygen must decrease the affinity of hemoglobin for PCMB. This appears to be the situation at high oxygen saturations. At low oxy- genation levels PCMB increases the oxygen affinity, so that it appears that 2 j per cent oxygenated hemoglobin has a higher affinity for PCMB than . .

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DISCUSSION - , . . . -15 - +~0 - 25./. HE O1 l ,,/~ v ~ ~ O % H b O2 O- l - _ ~ ~ / _ ~ / 4. - 5 _ ~ / ~ ~ ~ / ~ 10 ~ /o _ _ \ o/ 75 % Hb O 1 ~ ' I ~ 1 ' -t.0 0.0 t O io9 Oxygen Pressure 29 FIG. 1. The effect of the mer- cu ri al, p-chloromercu ribenzoate, upon the amount of oxygen bound by hemoglobin, plotted as the increase or decrease in per cent saturation of human hemoglobin with oven versus ,, , 4, the logarithm of the oxen en ~ Hi. pressure expressed in milli- meters of mercury. The arrows hate the per cent saturation of normal hemoglobin in the absence of mercurial at the in- dicated oxygen pressures. . , . does reduced hemoglobin. These maximum changes occur in the same positions as do the changes in the dielectric constant increment obtained in the meas- urements by Takashima and Lumry upon which Dr. Edsall commented at the beginning of the conference. I believe that there is an important relation- ship here. REFEREN CE 1. Riggs, A.: Sulfhydryl groups and the interaction between the hemes in hemoglobin, J. Gen. Physiol. 36: 1 (September) 1952. Dr. Edsall: There is orate thing that struck me as I was listening to Pro- fessor Roughton's paper. I do not know whether or not this is a possible explanation of the phenomena, but I was profoundly struck by the great difference in pH effect on the different velocity constants, particularly the contrast between the 1'~ and the Z,4. This certainly suggests that there is a different kind of coupling with the heme-linked acid groups in these two cases On the other hand, if, as most of us have generally assumed, all of the heme groups are initially equivalent before any reaction has occurred, it is a little difficult for me to see offhand why this should be so. I wonder if it is possible that the fourth group is structurally different from all the rest. and may perhaps be actually completely unavailable for reaction with oxygen or CO initially, so that there has to be a structural rearrangement in the molecule to permit that group to be capable of reaction at all. But once it is opened up and is available, then it has a higher oxygen affinity than any of the others. I have not tried to think through all the implications of this view.

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30 PART I. STRUCTURE OF HEMOGLOBIN Probably Professor Roughton has considered this possibility already and may have some reason for knocking down my argument. Dr. Roughto?~: Dr. Edsall, I have, it is true, worried about your sug- gestion over the years and have tried to make quantitative deductions from it. The results have not been too promising. The difficulty is this. When any three ligand molecules combine with hemoglobin carbon monoxide, oxygen or nitric oxidethen for some reason a change in the configuration occurs which makes iron atoms behave independently. We do, indeed, now have a very considerable body of evidence which indicates that when either three or four ligand molecules are com- bined with hemoglobin, the hemoglobin then behaves like myoglobin, the iron atoms all behaving; in the same play and independently of one another This makes it seem unlikely that one out of the four iron atoms was, before combination, different from the others. What we must really seek is the mechanism of how the iron atoms become independent of one another at the particular stage when three or four of the iron atoms are combined ravish ligands. No explanation is so far available. Dr. Jacinto Steinhardt: I was very much impressed by one of the findings o ~ Professor Roughton but I want first to be sure I have understood it correctly. As I understood it, under one set of experimental procedures if part of the gas is removed from the hemoglobin, or if all of it is removed, the velocity of recombination is the same in both cases. However, with another procedure, which I believe in this case was flash photolysis, the initial velocities are very different in the two cases. I would like Professor Roughton to repeat what the differences were, and further, whether the differences in velocity persist until all the gas is recombined, or whether as more and more gas goes back on, the velocities become the same (i.e., they differ only for a particular amount taken off). Dr. Roughton: I am not sure I got your second point, but to the first point the answer is clear. The experiments in which the velocity of recombination of CO spas independent of the amount of gas dissociated by the light were or1 myoglobin and not on hemoglobin. In myoglobin there is only one carbon monoxide to come off, and you cannot in that case have the situation which was depicted in the slide for hemoglobin with its four intermedites, that is, different concentrations of the four intermediates corresponding to varying degrees of flash photolysis. Dr. Steinhardf: You have clarified that perfectly. The second point remains. If, let us say, half of the carbon monoxide is removed from the protein, and then the rate of its recombination is measured, you get one result. If you take all of it off, you get a lower velocity constant. Dr. Roughton: There you have to hammer through the mathematics, preferably by means of automatic electronic computation. When you do that,

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DISCUSSION 31 you find that the rate of the whole process is governed over such an enormous part of it by the earlier, slower velocity constants that, until the reaction has proceeded to well over 90 per cent of completion, it does not matter whether the last constant is 20 times greater than the first or a different number of times greater than the first. It is not rate-limiting at all. So, although your question is theoretically valid, yet in practice it is only possible to pick up the differences in very high ranges, e.g., over 93 per cent completion, but these are technically very difficult to study with sufficient accuracy. Dr. Steinhardt: It might be worth remarking that if you denature, let us say, half of CO hemoglobin or ferrohemoglobin, the rate at which it re- generates is considerably faster than if you denature 9j per cent of it in pre- cisely the same environment. This difference is in the velocity constant, i.e., it persists until regeneration is complete. Dr. Riggs: I would like to ask Dr. Roughton about the experiments with sodium dithionite. I have never been able to obtain the original hemoglobin with which I started by treating hemoglobin with dithionite, even with exceedingly small amounts. The spectrum is changed, and this seems to be an irreversible phenomenon. I wonder if he has any comments about this? Dr. Ro2~phton: In the first place, we use the purest dithionite we can get, and we take the greatest possible care to protect it in all stages from oxygen. We also take care to keep the dissolved oxygen content of the hemoglobin with which it is going to react as small as is compatible with nearly com- plete oxygenation of the hemoglobin. Furthermore, we do not trust the dithionite beyond a few tenths of a second. But in kinetic experiments, under these carefully controlled con- ditions, one seems to get pretty good agreement at different wave lengths. If you do not take that care, you will get quite a different answer; that is, if you use one part of the spectrum rather than another to examine the course of your reaction. I quite agree with you that after some little time has elapsed, you have a very, very poor hope indeed of getting back your original hemoglobin. Your sad experience is probably of hemo~lc~hin exnnsed to dithionite for longer than two tenths of a second. Dr. Rices: Yes. At, Dr. Edsall: In other words, dithionite for equilibrium measurement is not a very good thing to use. It is only suitable when you work in ranges of a few tenths of a second. Dr. Ro?tyhton: Even then you have to watch it all the time. Dr. Reinhold Benesch: I would like to draw attention to the possibility of using an enzyme mixture, namely glucose oxidase and catalase, for re- covering oxygen from oxyhemoglobin. This can be done very fast, and in a continuous way, almost to 90 per cent to hemoglobin. This might be useful in measurements of the kind that have just been discussed, because an arti- fact such as impure dithionite should not occur. It is possible depending,

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32 PART I. STRUCTURE OF HEMOGLOBIN of course, on the amount of the enzymeto vary the rate of reduction of oxyhemoglobin to reduced hemoglobin. It is possible to do this inside the intact red cell as long as the solution is sufficiently buffered against the gluconic acid which accumulates, since the enzyme, which is a very active one, sucks the oxygen out of the red cell without affecting its integrity. Dr. Edsall: From what tissue does this enzyme come? Dr. Benesch: It is available commercially. It contains the glucose oxidase and some catalase. In some cases the commercial preparation is deficient in catalase and then it is necessary to add it. Polarographically it is very easy to tell the proportion of catalase to glucose oxidase. I would like to make one further remark in connection with your presenta- tion, Dr. Edsall, with regard to the very cogent arguments that imidazole groups are responsible for the heme-linked ionizations. I think you will agree that one of the major arguments which Wyman brought forward to link the imidazole groups to this phenomenon was that he determined the difference in dissociation constants of these groups in hemoglobin at different temperatures. The heats of ionization derived from these data agreed very well with the imidazole groups, namely, between six and seven kilocalories. I would like to draw attention to our finding some time ago, which has also been confirmed independently by Cecil in Oxford, that the heat of dissocia- tion of the sulfhydryl group falls within the same range six to seven kilocalories. Furthermore, pK's such as the second heme-linked group, 6.8 to 7.9 and so on, would be perfectly conceivable for some sulfhydryl groups, depending on what groups were dissociating in the neighborhood of the sulfhydryl group. For example, if some very positive groups were located in the neighborhood of such a sulfhydryl group, the pK of the sulfhydryl group could fall within this range. Dr. Edsall: The possibility that the heme-linked acid groups might be sulfhydryl groups has occurred to a number of people. I know Dr. Riggs has discussed it with me once or twice. I think this is something that we cer- tainly have to bear in mind. Dr. falter Hughes: It seems very unlikely to me that the sulihydryl groups are directly linked to heme since they vary in number from species to species. In bovine hemoglobin, I find only half as many sulfhydryls as hemes, and myoglobin has no -SH groups. I would expect this variation to be reflected in different oxygen equilibria for different species and even in different absorption spectra, whereas no such differences have been observed.