Physico-Chemical Evidence on Structure 



In the second place, from these values of the two assumed constants, 

 it is possible, by means of equation (3.1) in integral form, to predict 

 relative values of p± as a function of pH. When this is done the agree- 

 ment with the directly measured values of p± is satisfactory. The 

 degree of success of these two checks will be seen from Figures 1 and 2.* 



0-6 



Figure 2. Difference of charge 

 between haemoglobin and oxy- 

 haemoglobin. Smooth curve is 

 calculated from constants given 

 in Table I. 



0-4 



•\ 02 



0-2 



10 



pH 



We now turn to the effect of temperature on the oxygen equilibrium. 

 This is exactly like the effect of pH : altering the temperature simply 

 shifts the curve of Y vs log p without changing its shape. From this, 

 by a type of reasoning analogous to, though somewhat more compli- 

 cated than, that given above and involving the concept of the thermo- 

 dynamic heat as defined by the van 't Hoff equation, we arrive at the 

 conclusion that the heat absorbed is the same for each successive 

 molecule of oxygen introduced into the haemoglobin molecule, and 

 is given, on a mole basis, by 



AH= - 2-303 jgj»/gig|&) 



pH 



(4) 



Since this heat includes the heat of dissociation of the protons split off 

 as a result of oxygenation, it indicates that this component of the heat 

 is the same for each oxygen combining centre. This in turn indicates 

 once more that the same pair of acid groups is linked with each haem. 

 It is possible to go beyond this. By making differential titrations at 

 different temperatures, which in effect give us the variation of pi with 

 pH at each of the temperatures and make it possible to determine 

 AH as a function of pH from its value at any one/?H, we can obtain 

 the actual value of the heat of dissociation of the oxygen linked acid 



* In a sense these two checks are not independent, for it follows from equation (3) 

 that if the hypothesis accounts for the titration data it must account also for the oxygen 

 affinity data. What we have done really is to apply this hypothesis separately to two quite 

 different sets of experimental results. 



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