IMIDAZOLE HYPOTHESIS 233 



be bound to two "hemaffine" groups in the protein, one of these groups 

 being displaced on oxygenation. He pointed out the improbabihty 

 of the entry of oxygen affecting the dissociation of the carboxyi 

 groups of the porphyrin and suggested that the oxylabile hydrogen 

 ion might originate from the displaced "hemaffine" group. Although 

 Conant spoke only of the probability that the histidine groups were 

 involved, the recent developments of the imidazole hypothesis suggest 

 that both of Conant's "hemaffine" groups are imidazoles. The imi- 

 dazole hypothesis has been generally accepted, but recently a number 

 of serious objections have been put forward. We shall consider these 

 after presenting the evidence for the theory. 



3.2.2.2. Present Status of the Imidazole Hypothesis. The 



investigations of van Slyke and co-workers {21IfO) had established the 

 existence of an "oxylabile" group in hemoglobin whose dissociation 

 in the region of pH 7 was increased when the pigment combined with 

 oxygen. In 1937 Cohn, Green, and Blanchard (^6^) titrated crystal- 

 line horse carboxyhemoglobin and pointed out that between pH 5 

 and pH 9 the results could be accounted for by postulating thirteen 

 histidines with apparent /jK values of 5.7 and twenty histidines 

 with apparent pK values of 7.5. The number of histidines indicated 

 on the basis of the titration was identical with the value of 33 found 

 analytically by Vickery and Leavenworth (2877). Below pH 4 and 

 above pH 11 the ionizable groups found by titration do not agree so 

 well with the analytical figures, perhaps due to denaturation. The 

 groups, however, which are involved in the changes in acidity on 

 oxygenation, are certainly present in native protein. German and 

 Wyman (988), by using a wider pH range than had van Slyke, 

 showed that between pH 4.5 and 6.1 oxyhemoglobin is a weaker acid 

 than hemoglobin, while between pH 6.1 and 9.0 it is a stronger acid. 

 Outside this limit, there was no difference between the two proteins. 

 These data fitted well with the two pK values at which Cohn and 

 co-workers had shown the histidines to dissociate. 



Wyman next analyzed the heat of dissociation of hydrogen ions 

 from oxyhemoglobin and its variation with pH and confirmed the 

 conclusions drawn from the electrometric titrations (3131^). The 

 heats of dissociation found at pH less than 4.5 were characteristic of 

 carboxyi groups. Between p\\ 6 and /;H 8 a heat of dissociation of 

 6200 cal. was observed; this is of the right order for imidazole ioni- 

 zation; the dissociation of the hydrogen ion from the imidazole in 

 histidine and histidylglycine requires 6900 and 7500 cal., respectively 



