Physico-Chemical Evidence on Structure 



constant is of the same order of magnitude as what we have deduced 

 for the paired haems in haemoglobin. (Michaehs's semiquinone 

 formation constant is simply four times the reciprocal of our inter- 

 action constant, the factor four arising from symmetry considerations.) 

 In close analogy with haemoglobin, the two oxidizable groups of the 

 molecules investigated by Michaelis are indistinguishable and there 

 are differences of acid base properties between reductant, semiquinone, 

 and oxidant so that here also we encounter oxidation linked acid 

 groups. In contrast to haemoglobin, however, there is only one such 

 group for each oxidizable group. Owing to the compactness of the 

 molecule, there is direct interaction between these acid groups with 

 the result that the overall interaction constant of the oxidizable groups 

 is dependent on pH. This is in contrast to what is found in haemo- 

 globin, where the shape of the curve of Y vs log p or E is independent 

 ofpU. 



One final question now presents itself. This is whether the stabilizing 

 interactions operative in the oxygen equilibrium stem from the haems 

 in their uncombined state or from the haems when united with oxygen. 

 According to one hypothesis the first oxygen would enter the molecule 

 with more difficulty than the next (and so on) because of a pre-existing 

 stabilization of the uncombined haems. According to the other 

 hypothesis, the second oxygen would enter more readily than the first 

 because of a kind of attraction exerted by the presence of the first. 

 Since both interpretations lead to identical formulations of the 

 equilibrium it might seem that it would be impossible to decide be- 

 tween them. Nevertheless there is a basis for doing so, at least for 

 interactions between haems of different pairs, in the data obtained on 

 haemoglobin dissolved in urea solutions. These data show that there 

 is a decrease in p i by a factor of about 2-7 when the molecule splits 

 in two. Reflection will show that this is qualitatively just what would 

 be expected on the hypothesis that the stabilization involves un- 

 oxygenated haems, for when the molecule is split, this stabilization 

 disappears and an obstacle to oxygenation is removed. On the other 

 hand it is the opposite of what would be expected if stabilization in- 

 volved oxygenated haems. Quantitatively, the magnitude of the effect 

 is not too far from what would be expected on the basis of our rect- 

 angular model, for it is possible to show that p i should be diminished 

 by a factor equal to the square root of the interaction constant 

 involved, i.e. \/<\ = 2. All conditions (e.g. high dilution) which have 

 the effect of diminishing the value of n seem to lead to an increase in 

 the oxygen affinity (decrease of p { ). It is further suggestive that the 

 value of p h for horse myoglobin (with one haem) at 25°C and pWl is 

 about 1/20 that for horse haemoglobin. On the basis of the constants 



105 



