Physico-Chemical Evidence on Structure 



symmetrical curve for Y vs log p, as does any theory in which the 

 groups are all identically related to one another. Actually the most 

 accurate data, for example those of Forbes and Roughton on sheep 

 haemoglobin, show that the curve is in fact asymmetrical. In the 

 second place, it does not accord with recently published experiments 

 on the oxygen equilibrium of horse haemoglobin dissolved in strong 

 urea solutions, where the molecules are known to be split in two. In 

 these experiments the curve of Y vs log p was found to be exactly 

 symmetrical and corresponded to a value of n = 1-8—1*9 in equation (6) 

 (see Figure 3). From these results we may conclude that when the 

 haemoglobin molecule is split it divides into identical halves and that 

 there is a strong stabilizing interaction between the two haems present 

 in each half. To be sure any molecule containing only two groups 

 would give a symmetrical curve of Y vs log/?, but if there were two kinds 

 of such molecule the overall curve would not be symmetrical except 

 in the case of certain compensating effects which are inconsistent with 

 the assumption of the identity of the four oxygen combining centres. 

 Nor would the curve be symmetrical if there were two kinds of molecule, 

 one containing one group, the other three. It is a simple matter to 

 relate the observed value of n to the interaction constant for the case 

 of a model consisting of two identical reacting groups. For n = 1-9, 

 the required interaction constant is approximately 400, corresponding 

 to an interaction energy of 3,550 cals, though when n is so close to its 

 upper limit 2, corresponding to an infinite interaction energy, n is very 

 insensituve to the value of the interaction energy. We conclude then 

 from these experiments on haemoglobin dissolved in urea solutions 

 that unless there is a profound rearrangement accompanying splitting 

 of the molecule the four haems in the native haemoglobin molecule 

 occur in pairs. Members of the same pair are closely spaced and show 

 a strong stabilizing interaction energy of the order of 3,000-4,000 

 calories. If we adopt this interpretation, then in order to account for 

 the oxygen equilibrium of the unsplit molecule, for which n = 2-8, we 

 must postulate an additional but smaller stabilizing interaction between 

 haems belonging to different pairs. This much seems clear. It is 

 tempting, however, and perhaps not wholly useless, to go beyond, and 

 try the effect of replacing the Pauling model by a rectangle. If we do, 

 then we find that by retaining the value 400 as the interaction constant 

 for the closely spaced haems (those belonging to the same pair) and 

 assuming an interaction constant of 4 (corresponding to about 800 

 calories) for haems belonging to different pairs (there being no inter- 

 action along diagonals of the rectangle) we arrive at a formulation of 

 the equilibrium which gives a curve almost indistinguishable from 

 Pauling's. 



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