286 VI. HEMOGLOBIN 



concentrations of oxygen and carbon monoxide during the association reac- 

 tions produce a p-fold variation in the rate and not a p"-fold variation as 

 would be expected from Hill's theory. 



The kinetic data however, differentiate less obviously between the other 

 three equilibrium equations. The fact that the ratio of the velocity constants 

 for the forward and back reactions agrees with the original Hiifner theory 

 presents a paradox (Section G.'i.'i.). Hartridge and Roughton (ll^O) deter- 

 mined the dissociation curve of a sample of the blood they were using for 

 the kinetic measurements; they were restricted by the spectroscopic method 

 they used for the measurement of the equilibrium to only four points on the 

 curve between 40 and 70%, but were able to draw a hyperbolic dissociation 

 curve through these points. 



Although the dissociation curve has been observed to be sigmoid in shape 

 in dilute solutions by a number of workers {cf. 1177,1105), their measurements 

 were not carried out in such great dilutions as were the kinetic measurements, 

 and it is possible that under the latter conditions the dissociation curve is of 

 hyperbolic shape. A second possibility is that the hemoglobin used for the 

 kinetic experiments had been altered in some way {cf. Section 5.1.6.). 



7.1.2. Reactivity of Freshly Reduced Hemoglobin. Further problems are 

 presented by some experiments which Roughton carried out on freshly 

 reduced hemoglobin {2358). The velocity of combination between carbon 

 monoxide and hemoglobin was measured with hemoglobin which had been 

 reduced some time before the commencement of the run, and with hemoglobin 

 which was prepared from oxyhemoglobin by reduction immediately prior to 

 its reaction with carbon monoxide. The velocity of the combination of 

 carbon monoxide with the two samples of hemoglobin was identical at pH 

 6.6 and 15° C, and at pH 10 and 33° C. At pH 10 and 15° C. however, the 

 reaction of carbon monoxide with the freshly reduced hemoglobin was found 

 to be twice as fast as with the "old" hemoglobin. Under these conditions, 

 high pH and low temperature, the velocity of the dissociation of oxyhemo- 

 globin is many times slower than under the conditions in which his other 

 experiments were carried out. Roughton suggested that the phenomenon 

 might be related to the dissociation of the actual molecule of hemoglobin. 

 Another possible explanation is that the reduction of oxyhemoglobin remained 

 incomplete in the experiment with freshly reduced hemoglobin. Even if the 

 saturation with oxygen is as low as 5%, this may have an effect on the rate 

 of the association, if the combination of the first heme is the rate-determining 

 step and the interaction between the hemes causes an acceleration for the 

 remaining hemes. This would suggest that the whole burden of interaction 

 between hemes as an explanation of the sigmoid equilibrium curve must be 

 placed on the association reaction.* The dissociation of oxyhemoglobin has 

 been shown to follow a logarithmic course down to 10% saturation; this is 

 most easily understood by assuming no interaction as regards the rate of 

 reaction of successive oxygens from the heme molecule. 



* Recent work by Legge, Nicholson, and Roughton {166Ha) has shown that this 

 is not the case. 



