Physico-Chemical Evidence on Structure 



in which K is the equilibrium constant. This is important in connection 

 with suggestions that the combination of haemoglobin with oxygen 

 may represent an adsorption phenomenon rather than a true chemical 

 reaction. Also it shows that the molecules of myoglobin, if not in all 

 respects alike, are at least alike in their behaviour towards oxygen. 

 We may feel confident, therefore, in regarding the combination of 

 haemoglobin with oxygen as a chemical reaction, and, at least as a 

 working hypothesis, we shall assume that the haemoglobin molecules 

 are all the same in their reactions with oxygen. 



Having profited by this view of the behaviour of myoglobin, let us 

 now turn to the question of the identity of the four oxygen combining 

 centres in haemoglobin. There are two sets of observations bearing on 

 this matter, one relating to the heat of oxygenation, and the other to 

 the effect of pH on the oxygen equilibrium curves. We shall consider 

 first the pH effect (commonly known as the Bohr effect). This effect, 

 extending from pH ~ 4 to /?H ~ 9, is shown in Figure 1. In this figure 

 the value of p corresponding to half saturation of haemoglobin with 

 oxygen, which we shall call p h and which is by definition the reciprocal 

 of the oxygen affinity, is shown as a function of /?H. It will be seen 

 that the effect falls into two parts, p { having a maximum at pH 6*1. 

 Above 6-1 p i decreases, below 6-1 it increases, with increasing pH. 

 That there is any pR effect at all means of course that there are proton 

 dissociating groups in the haemoglobin molecule sufficiently close to 

 the oxygen combining groups to be affected by the uptake of oxygen. 

 The double character of the effect means that there must be one or 

 more such groups which are strengthened and one or more such groups 

 which are weakened by the reaction. Whenever therefore oxygen is 

 introduced into haemoglobin at constant pH, there must be a loss or 

 gain of protons, depending on pH. This is a reciprocal effect which is 

 well known and has been directly measured by differential titrations of 

 haemoglobin and oxyhaemoglobin. The two effects are in fact counter- 

 parts of one another and there is a fundamental relation governing 

 them which may be deduced by quite general reasoning and stated 

 as follows : 



In this equation X denotes the total negative charge on the haemo- 

 globin molecule resulting from the loss of protons. Now in all cases 

 which have been studied it is found that the equilibrium curves in which 

 Y is plotted against log p are unaffected in shape by changes of pH, 

 being subject simply to a displacement along the log p axis. This is a 

 fundamental observation and means that the right-hand member of 



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