The Haem-Globin Linkage 



181 



probably due to a combination with a group in some denatured protein 

 present. Perhaps this group results from unmasking of the one giving the 

 maximum attachment at pH 9 in the unsplit native protein. It might also be 

 the group detected by Theorell (1942) in apohaemoglobin, with pA: of 10 

 at 0°C, not present in ferrihaemoglobin. 



The increment curves presented here should not be considered to be 

 exactly reproducible since comparison between solutions and colloidal sus- 

 pensions is being made. The increments are, however, of such magnitude 



pH 



10 12 



Fig. 3. Attachment of nickel mesoporphyrin to apohaemoglobin, apomyoglobin 

 and carboxyhaemoglobin. Soret absorbance increment curves prepared, as 

 described in the text, for nickel mesoporphyrin + apo Hb at 389 m/i 9 — • — •, 

 + apo Mb at 410 m/i O— O— O, and + CO Hb at 390 m/ii A— A— A. (Buffers, 

 pH 2-6-2 phthalate, 6-2-8-0, phosphate; 8-5-9-5, pyrophosphate; 9-9-11, 

 glycine; 11-5, phosphate; 12-5, NaOH; I = 0-05, T = 2rC.) 



(e.g. apomyoglobin increased the absorbance of nickel mesoporphyrin at 

 410 m/t from 0-22 to 0-67 at pH 10-0) that it seems legitimate to make 

 quantitative comparison. The technique should prove useful in the detection 

 and identification of linking groups in other haemoproteins. 



The Nature of the Acid Groups Linking the Haem Iron 



The likelihood of combination of haematin propionate side-chains with 

 imidazolium side-chains of horse haemoglobin called for a re-examination of 

 the evidence for the mode of attachment of the iron atom to the protein. 

 Stability curves of ferrihaemoglobin and ferrimyoglobin reported by O'Hagan 

 (1959a) suggested the possibility that groups of ^K value lower than 5-3 

 were involved. Theorell and Ehrenberg (1951), after their exhaustive study 

 of myoglobin, concluded that a group of more negative character than an 

 imidazole was responsible for iron Unkage, but gave the group a pA^ value 

 of 5-3. 



Since Coryell, Stitt and Pauling (1937) had shown that in acid ferri- 

 haemoglobin the atom of iron was joined to other atoms surrounding it by 



