192 



Discussion 



Lemberg : Are we really sure that the combination between haematin and serum albumin 

 is only through the haem carboxylic acid groups? This is certainly not so for the 

 combination of haematin a with serum albumin, in which the spectrum clearly indi- 

 cates combination with protein nitrogen. I was inclined to accept the suggestion of 

 J, Keilin for the protohaematin compound, but I feel no longer sure about it now. 



George: In considering various mechanisms that might account for haem-linked ionization 

 effects, I recently calculated the pH range over which a salt bridge would remain 

 intact, and I think this will clarify the points just raised by Orgel, Perrin, Phillips and 

 Williams. 



If the equilibrium constant for the formation of a salt bridge between, say, a 

 carboxylate group and a substituted ammonium group is K^, 



—COO- + NH3+— 



-COO-NH,+— 



and the ionization constants for the two separate groups are ATcooh and K-^n^^ 

 respectively, then in acidic solution the pH at which 50% formation occurs is given 

 by /'(A's^cooh), and in alkaline solution the corresponding pH is given by 

 p{K^Yii^lK^. As illustrated in the diagram the formation of the salt bridge results 

 in the 'titration' of the carboxyl group in a lower pH range and the amino group in 

 a higher pH range than usual — the apparent shift in the pA^ values being determined 

 by the magnitude of K^. 



The shift in the pX^ values is in an opposite sense, because in acidic solution the 

 bridge is broken by combination with H+, 



I.e. 



— COO-NH3+— + W 



— COOH + NH3+- 



(1) 



whereas in alkaline solution by the dissociation of H+, 



i.e. — COO-NH3+— ^ COO- + NHj— + H+ 



(2) 



In reaction (1) NH3+ — can be thought of as competing with H+ for the COO- group, 

 thus lowering the 'p^'; in reaction (2) the combination of H+ with NHg — giving 

 NH3+ — is favoured by the formation of the salt bridge; this has the effect of making 

 NH3+ — a weaker acid, and hence raises its pA'. 



