F. J. W. ROUGHTON, J. W. LEGGE and P. NICOLSON 



points, which in fact cover the first half of the reaction. The later 

 points all fall above the line, and indicate that the calculated value of 

 k' tends to rise to 30 per cent or 40 per cent above its initial value. 



Although more data are certainly necessary, those at present avail- 

 able leave little doubt that equation (1) needs to be modified at high 

 values of [XHb], both in respect of the term involving k and of that 

 involving k'. Later it will be shown that the effects so far observed 

 are just what would be expected on a simple extension of the inter- 

 mediate compound hypothesis. 



The effect of partial saturation at time zero on the kinetics of the 

 reaction — In all the previous kinetic work of Hartridge, Roughton 

 and Millikan the haemoglobin at the start of the reaction was either 

 as completely reduced or as completely saturated (with 2 or CO) as 

 possible. No tests have hitherto been made as to whether the reaction 

 velocity curves obtained with partially saturated haemoglobin at the 

 starting point agree with those found with the fully saturated or fully 

 reduced haemoglobin. A discrepancy would be of much interest, 

 especially from the standpoint of the intermediate compound 

 hypothesis. For it would imply that the concentration of the various 

 intermediates for a given percentage saturation differ appreciably 

 according to whether the system is at equilibrium or in a state of flux 

 at the time at which the particular saturation in question is reached. 

 The matter is also of physiological interest, for in vivo the state of 

 combination of the haemoglobin varies cyclically during the circulation, 

 but never reaches either complete saturation or complete reduction. 

 Finally, work of this kind might throw light on some of the anomalies 

 observed by Roughton in the reactivity of freshly-formed haemoglobin 

 compounds 7 . In 1947 we therefore carried out the following tests : — 



1 The rate of combination of CO with fully reduced sheep haemo- 

 globin (concentration 0-2-0-3 gm/lOOml) was compared with the 

 rate of combination of CO with the same haemoglobin already 

 20-30 per cent saturated with CO at the start. Neither at pH 10 

 nor at/?H 6-8 could any difference be observed in the rate of com- 

 bination, provided proper allowance was made for slight variations 

 in the concentrations of the reagents. 



2 The rate of dissociation of 2 Hb in presence of Na 2 S 2 4 was 

 measured at (a) 99-100 per cent initial saturation, (b) 50-60 per 

 cent initial saturation. AtpH 10-0, the points obtained in case (b) 

 fell within experimental error on the curve for case (a), but at 

 pH 6-8 the initial rate of dissociation in the case of the partially 

 saturated haemoglobin was somewhat faster than was to be 

 expected from the curve with the fully saturated haemoglobin 

 (Figure 2). The effect is, however, overshadowed by, and probably 



72 



