132 P. George, J. Beetlestone and J. S. Griffith 



approximations, consisting of average values of the extinction coefficient 

 appropriate to the two individual high-spin forms and the two individual 

 low-spin forms. The new spectra in Fig. 20, based entirely on data for one 

 haemoprotein, are therefore more valid. 



THE SPECTRA OF FERRIMYOGLOBIN DERIVATIVES IN 

 HEAVY WATER 



The interplay of structural and electronic factors necessary for a ferri- 

 myoglobin derivative to exist as a mixture of high- and low-spin forms is 

 evidently so critical that the ligands most closely related to the hydroxyl 

 group in chemical type give predominantly, or entirely, high-spin or low-spin 

 complexes. On the basis of spectroscopic data, or magnetic data, or both, it is 

 clear that the complexes with phenol, and presumably ethanol, i.e., 

 Fejib+++ — OCgHg and Fe]^+++ — OC2H5 come in the former category; 

 whereas the complexes with the sulphur analogues, hydrogen sulphide, ethyl 

 mercaptan and thiophenol, i.e., Fejib'^++ — SH, Fe]yii,+++ — SC2H5 and 

 Fejnj+++ — SCgHg, come in the latter (George, Lyster and Beetlestone, 1958; 

 Coryell and Stitt, 1940; Keilin, 1933; Heussenstam and Coryell, 1954). The 

 least drastic of all substitutions that can be achieved, with the exception of 

 employing HaO^^, is the replacement of hydrogen by deuterium, and the 

 spectrum of the alkaline form in heavy water, which should accordingly 

 have the structure Feniij+++ — OD, has therefore been studied. In the prelimi- 

 nary experiments, reported below, the highest mole ratio of DgO to H2O 

 that could be attained was 134: 1. Hence, although the affinities of the iron 

 atom for OH~ and 0D~ also have to be taken into consideration because 

 they determine the relative amounts of Fe]ynj+++ — OH and FejyQj+++ — OD 

 formed, it is unlikely that in pure D2O the effect observed would be very much 

 enhanced. 



A very concentrated solution of acidic ferrimyoglobin in ordinary water 

 was used, so that only 0-02 ml in a total of 3 ml was required to give optical 

 density values of about 0-7 at the band maxima in the visible region. Solutions 

 of the alkaline form were prepared in the following way. Tiny quantities of 

 caustic soda solution were added to acidic ferrimyoglobin (0-02 ml stock 

 solution -f 2-98 ml ordinary water) from a micro-syringe until the pH was 

 11-0. The same volume of caustic soda was added to a corresponding solution 

 of acidic ferrimyoglobin made up in heavy water. Difference spectra were 

 then recorded with the heavy water solution in the reference cuvette for the 

 alkaline form, and, as controls, for the acidic form and the cyanide derivative, 

 which was prepared by adding a little solid KCN. 



With the cyanide derivative no difference could be detected and with the 

 acidic form there was scarcely any change. But with the alkaline form a well- 

 defined difference spectrum was obtained, very similar to that in Fig. 20, and 

 the optical density differences were about the same in magnitude. The 



