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



high- and low-spin forms, calculations give the following percentages for the 

 various haemoproteins : 



Myoglobin : High - 70 %, Low - 30 % 

 Haemoglobin : High - 50 %, Low - 50 % 

 Peroxidase: High- 7%, Low -93% 



From the extinction coefficients of the hydroxides the spectra of the high- 

 and low-spin forms have been obtained over the range 250 to 950 m^. 

 Major absorption bands, or shoulders, occur at about the following wave- 

 lengths, with extinction coefficients having the approximate values given in 

 brackets : 



High-spin form: 830 (1-2), 740 (0-9), 600 (11), 540 (8), 490 unresolved 

 band (10), 405 (116), 350 shoulder (44). 



Low-spin form: 575 (10), 545 (12), 417 (104), 360 (20), 340 (14). 



The calculation procedure has certain inherent limitations, nevertheless these 

 absorption bands correspond so closely to those which distinguish high- from 

 low-spin derivatives that the assumption of a thermal mixture can be regarded 

 as entirely consistent with the spectroscopic and magnetic data. 



THE EFFECT OF TEMPERATURE ON THE SPECTRUM AND ON 

 THE MAGNETIC MOMENT OF FERRIMYOGLOBIN HYDROXIDE 



As suggested previously, if the ferrihaemoprotein hydroxides are thermal 

 mixtures of high- and low-spin forms, then changing the temperature would 

 be expected to influence the equilibrium. 



High-spin hydroxide ^^ Low-spin hydroxide (12) 



and a change should therefore be observable in the magnetic moment and in 

 the absorption spectrum. The magnitude of the change would depend on the 

 value of AT/ for reaction (12), since this determines the variation of equili- 

 brium constant with temperature according to the van't Hoff Isochore. But, 

 since a close balance between the energies of the two forms is to be anticipated, 

 A// is likely to be small, and, as a consequence, K^ to have a small temperature 

 dependence resulting in only slight changes in magnetic moment and absorp- 

 tion spectrum. This is borne out by the observation that there were no 

 noticeable variations in the optical densities of ferrimyoglobin or ferri- 

 haemoglobin hydroxide solutions at 582 m// and 578 m/t respectively over the 

 temperature range 7-5° to 37°C in experiments carried out to obtain thermo- 

 dynamic data for the ionization reactions (George and Hanania, 1952, 1953). 

 However, further experiments have now been made using a sensitive 

 recording spectrophotometer, and a temperature effect has been detected. 

 The absorption spectrum of a concentrated solution of ferrimyoglobin 



