Ferrihaemoprotein Hydroxides 



129 



hydroxide at pH 11-0 and 5°C was recorded from 470 to 650 m,a, the optical 

 density at 540 m/t being 0-7411. The reference cuvette, which previously 

 contained buffer, was then filled with more of the hydroxide, and a baseline 

 was recorded over the wavelength range with both solutions at 5°C. The 

 solution in one cuvette was then rapidly warmed to 35°C, and maintained at 



+0.02 — 



Fig. 19. The difference spectrum in the visible region for 8-4 x 10~^ m ferrimyoglobin 

 hydroxide at 5° and 35°, plotted as id^° - ^35°). 



this temperature, by the insertion of a specially constructed hollow metal 

 heating unit through which water from a thermostat was circulated. A 

 difference spectrum was recorded, from which the curve illustrated in Fig. 19 

 was obtained after correction for the baseline. As can be seen, the effect of 

 a 30° alteration in temperature is rather small. The change in optical density 

 is at the most 2-9% at 542 m/^, while at 582 m/i it has dropped to 1-6%, 

 which accounts for the effect escaping notice in the earlier investigations. 

 Control experiments using the cyanide and fluoride derivatives showed no 

 similar effect. 



The negative regions from 480 to 520 m// and above 600 m/<, together 

 with the positive region in between which shows two well-defined bands, are 

 qualitatively consistent with an increase in the fraction of the high-spin 

 form as the temperature is increased. Some indication of its magnitude can 

 be obtained from the difference between the extinction coefficients of the 



