190 Discussion 



DISCUSSION 



Native Globin 



Drabkin: I would be glad if O'Hagan could tell us a little more about the state of his 

 apohaemoglobin as against that of his apomyoglobin. I am thinking in terms of the 

 possibility that, since myoglobin is far, far more stable toward alkali than is haemo- 

 globin, some question may be raised as to the strict validity of comparing the two 

 apoproteins. 



Lemberg: Rossi-Fanelli has found that by his method any irreversible denaturation of 

 globin from haemoglobin can be prevented, and O'Hagan followed his method rather 

 closely. Denaturation will, however, occur if the recombined haemoglobin solutions 

 contain an excess of free globin and are measured in the spectrophotometer at room 

 temperature. 



O'Hagan: Initially trouble was experienced with these apoproteins due to precipitation 

 of the denatured material at room temperature and neutral pH. The trick is to bring 

 the pH to 7-8, leave at 2rc for 1 hr, leave at O'^C for three days to remove coagulated 

 material, and centifuge for 10 min at 20,000 X g. With one preparation, which I 

 considered to be not as good as the others, on other evidence, the peak at pH 10 

 (see Fig. 3 of my paper) was higher and broader. 



George: In our work on reconstituted ferrimyoglobin, O'Hagan and I obtained data 

 similar to that of Rossi-Fanelli on reconstituted ferrohaemoglobin. In a comparison 

 with native ferrimyoglobin we found that the affinity for fluoride is scarcely altered, 

 and that the pK values of the haem-linked ionizing group associated with its Bohr 

 effect are identical to within experimental error. 



The Linkage of Iron and Protein in Haemoglobin 



Perrin : In systems such as haem, or haematin plus albumin, we have equilibria such as : 



Hm COOH ^ Hm COO- + H+ pK^ 



Alb H+ + Hm COO- ^ Hm COOH Alb K 



Alb -H H+ ^ Alb H+ pJ^T/ 



This is certainly a gross over-simplification but will serve to illustrate a difficulty in 

 using O'Hagan's spectral absorption difference approach. 



At constant albumin and total Hm concentration there are still three light-absorbing 

 species in such a system and their concentrations are governed by three unknown 

 constants, K, K^ and AT/. In addition, each of the absorbing species has its own 

 molecular extinction coefficient, so that the absorbance increment at any given pH 

 is not a simple function of the species. There is no reason to assume the A^'s are the 

 same for haem and haematin, and in fact they are most unlikely to be if any binding 

 through the iron is involved. 



I should like to ask O'Hagan how he arrives at the conclusion that 'reduction (of 

 haematin to haem) significantly decreases the acid strength of at least one of the 

 haematin propionate groups.' 



Concerning Fig. 4, 1 should like to point out that stability curves of metal complexes 

 such as ferrihaemoglobin cannot be used to obtain the pATs of the ligands. Falk, 

 Phillips and I have discussed this elsewhere (^Nature, Lond. 184, 1651, 1959). The 

 'apparent p/Ts' in such cases are, in fact, functions not only of the pAT of the ligand 

 but also of the stability constant of the metal complex and the concentration of the 

 ligand. It is quite erroneous to identify such 'apparent pATs' with the pA:'s of groups 

 such as carboxyl or imidazole. The nature of the metal-to-ligand bond does not affect 

 this conclusion: if a complex is present, there is, of course, a AG = RTXnK which 

 leads to significant changes in the thermodynamics of the system relative to the 

 proton-ligand system. 



One cannot use the A/f for haem-protein dissociation where the iron-to-protein 

 link is involved as evidence for COO- or imidazole linkage if one takes the data for 



