140 Discussion 



allowable and it is important to remember that interaction may occur among the 

 various types of excited state, resulting in shifts of their positions and donations of 

 intensity from one transition to another. 

 Williams: In his introduction George has stated that the work he described on the 

 equilibrium between two spin states was initiated in 1956. I do not wish to claim any 

 priority in the discussion of the equilibrium between two spin states as it has been 

 mentioned by a large number of authors since 1944 and extensive reference has been 

 made to the factors controlling this equilibrium both by myself and others. In par- 

 ticular, however, I would refer to a three cornered discussion between myself, George 

 and Griffith, in discussions of the Faraday Society, 1955, where I was the only one 

 to maintain this point of view. I have held this point of view consistently since 1953 

 in discussing the very compounds now examined by George and co-workers. 



The general idea that equilibrium exists between spin states has had excellent 

 experimental foundation in the study of model complexes for some years past. I 

 should add that I do not wish to detract from the importance of George's contribution 

 which sets my discussion, and that of other earlier authors, on a firm basis. 



Falk's contribution (see Orgel's paper and discussion, this volume, p. 15) should 

 be read in the light of a summary of my views in Lardy and Myrback, The Enzymes, 

 1959. There, as previously, I elaborate on spin state equilibria in the cytochromes. 

 Boardman: In their paper, George, Beetlestone and Griflith (this volume, p. 126) correct 

 their calculations for differences in the milliinolar extinction coefficients of the corres- 

 ponding ferrihaemoglobin and ferrimyoglobin derivatives. I feel that these corrections 

 are unnecessary as the extinction coefficients for the myoglobin derivatives appear 

 to be too low by a factor of approximately 1-08. 



The extinction coefficients for the myoglobin derivatives are taken from the work 

 of Hanania, who assumed a molecular weight of 17,000 for myoglobin whereas now 

 there is evidence to suggest that the molecular weight of myoglobin is above 18,000. 



A few years ago, Adair and I at Cambridge succeeded in isolating two CO-myo- 

 globins from horse-heart extracts by means of ammonium sulphate fractionation and 

 chromatography on columns of Amberlite IRC-50. The main myoglobin fraction 

 accounted for 90% of the total myoglobin. The molecular weight of the main com- 

 ponent was determined from measurements of osmotic pressure and a figure of 18,400 

 was obtained. The extinction coefficient of the ferrimyoglobin cyanide derivative at 

 542 vnn was 0-613 for a 0-1 % solution and this corresponds to a millimolar extinction 

 coefficient of 11-3, if we assume a molecular weight of 18,400. Fig. 6 of the paper by 

 George et al. (this volume, p. 113) shows a millimolar extinction maximum of 10-6 

 for ferrimyoglobin cyanide. A value of 11-3 agrees closely with the corresponding 

 value for ferrihaem.oglobin cyanide as determined by Drabkin. Theorell and Akeson 

 have concluded also that the molecular weight of horse myoglobin is above 18,000. 

 Their preparation was purified electrophoretically. Three myoglobin components 

 were obtained and the iron content of the main component was 0-297 %. This figure 

 gives a molecular weight of 18,800. 

 George : I would Uke to thank Williams for drawing attention to the early suggestion of 

 Willis and Mellor (/. Amer. chem. Soc. 69, 1237, 1947) that some co-ordination 

 compounds may be thermal mixtures of high- and low-spin forms, and to his own 

 remarks on the subject in the 1955 Faraday Discussion. 



I agree with Boardman's comments on extinction coefficients. The spectrophoto- 

 metric data employed in the calculations were obtained before the electrophoretic 

 separation of myoglobin into a major and two minor components had been demon- 

 strated. The sample used had been subjected to repeated recrystallizations from 

 ammonium sulphate and treated with strong phosphate buffer, pH 5-7, to remove 

 haemoglobin. 



For a more exact analysis of the type described in Section IV of our paper it would 

 not only be necessary to have extinction coefficients but also magnetic susceptibility 

 measurements for single components. But an analysis of this kind only gives average 

 spectra of the high-spin form and of the low-spin form for the pair of haemoproteins 

 upon which the calculations are based. Undoubtedly there will be minor variations 



