186 J. E. O'Hagan 



of the Soret peak. This finding of a stronger basic group could explain the 

 considerably lower heat of oxygenation (A//q = — 17-5kcal) found for 

 myoglobin by Theorell (1934), compared with the values of haemoglobins 

 (A/Zq '->-' — 12-5 kcal, Wyman, 1948; see also George, 1956). The greater 

 stabihty of myoglobin towards alkali (Haurowitz and Hardin, 1954) may also 

 be explainable in terms of this basic group. Change in acid strength of the 

 haem propionates would be expected to have less effect on bonds to such 

 stronger basic groups, accounting for the small Bohr effect and minor effect 

 on the shape of the oxygen dissociation curve with a change in the hydrogen 

 ion concentration. Examination of the attachment in the pH range 11-13, 

 using a hydrogen electrode, may give further information on the nature of the 

 linking group. 



German and Wyman (1937) and Wyman (1948) in discussion of the nature 

 of the group binding the haem iron, pointed out the uncertainty that the group 

 ionizing in the more acid range was imidazole, and that the second carboxyl 

 group of dicarboxylic acids should also be considered. This seems to have 

 been generally overlooked, and the matter calls for more attention in the 

 fight of the observations made here. Even a pK value of 5-3 appears low for 

 an imidazole group by comparison with its value in compounds resembfing 

 those occurring naturally. 



The strongest evidence against the hypothesis that the haem iron is linked 

 to a group other than imidazole is the ingenious experiment of Wang (1958), 

 who found that diethylprotohaem linked to l-(2-phenyl-ethyl)-imidazole in a 

 film of polystyrene, bound carbon monoxide which could be replaced by 

 oxygen. However, substitution at position 1 on the imidazole ring would 

 appear to change its character, making it unlike that presumed to occur in 

 haemoglobins. It could be that such a substitution makes the free nitrogen 

 acidic and that the acid strength of the linking groups rather than their 

 structure is the important factor. This may apply also to the imidazole haem 

 compound found by Corwin and Reyes (1956) to bind oxygen, though 

 apparently very poorly. Whatever type of linkage exists between the iron and 

 the apoprotein, whether it be imidazole, ^ or y carboxyl or an unusual type 

 not yet detected in proteins, should not materially affect the discussion which 

 follows. 



The oxygen dissociation curves of the haemoglobins can now be examined 

 in the light of the new findings. It is interesting that Barcroft (1938) likened 

 the curves to oxygen titration curves and we could perhaps visualize them as 

 representing the 'titration' by haem propionates (of changing acid strength) 

 of the weakly basic groups (haemoglobins) or more strongly basic groups 

 (myoglobins). The oxygen dissociation curve of haemoglobin could be 

 considered as representing the titration, from no combination to full combina- 

 tion, of the propionate group with the imidazolium group. The curve for 

 myoglobin would be only the upper section of the curve for the titration of 



