The Kinetics of Haemoglobin in Solution 



connected with, the fact that the dissociation appears to proceed 

 in two phases, a rapid one from 100 to 25 per cent 2 Hb taking 

 about 0-07 seconds and then a slow one from 25 to per cent 

 2 Hb lasting several seconds. This remarkable effect is discussed 

 in the next section. 



IOOk 



BO 



Figure 2. Rate of dissociation of 2 Hb ^ 60 

 in presence o/Na 2 S 2 4 atpH 6-8, 15-8° C £ 

 o = 100% 2 Hb at zero time, x=51% £ 40 

 2 Hb at zero time. i o 



20 



C 0-04 003 012 016 



Time in Seconds 



020 



The diphasic dissociation of oxyhaemoglobin at neutral pH — We were 

 at first led to suppose that this effect was due to one of the haems of 

 the molecule behaving in a quite different way from the others at neutral 

 pH. A much fuller investigation, however, showed great variability in 

 the percentage saturation at which the break occurred, the ' plateau ' 

 (as we have called it) varying from 33 per cent 2 Hb to per cent 

 OoHb, in the^H range 6-1 to 7-4, temperature 10-20°C. In the case 

 of the corpuscle suspensions, the plateau in three cases out of four was 

 non-existent and in the fourth case was only at ca 9 per cent O a Hb. 

 This led us to the view that the apparent ' plateau ' must be associated 

 with the formation of abnormal pigments due to the action of Na 2 S 2 4 , 

 or more probably its oxidation products, upon the haemoglobin. Such 

 compounds would clearly upset the photocolorimetric determination 

 of the per cent 2 Hb in the running fluids in the rapid reaction 

 observation tube. 



Two pieces of definite evidence were obtained in favour of this view. 

 In cases where a low ' plateau ' was observed it was found that the 

 photo-colorimetric reading obtained with a mixture of the oxyhaemo- 

 globin and buffered Na 2 S 2 4 , which had been allowed to stand for 

 30 seconds, agreed with that obtained with a similar mixture of the 

 fully reduced haemoglobin (prepared by shaking in vacuo) and the 

 Na 2 S 2 4 . On the other hand, in cases of a high ' plateau ', the reading 

 with the 2 Hb-Na 2 S 2 4 mixture differed from that of the reduced 

 Hb-Na 2 S 2 4 mixture, the former appearing to be even more ' reduced ' 

 than the latter, and in one control experiment showing appreciable 



73 



