Cvtochroines Cooled in Lufuul Nitrogen 



441 



observed is the appearance of a shoulder at about 430 m/^ indicative of 

 another possible absorption band. A point of interest is the appearance of 

 what have now been termed (Estabrook and Sacktor, 1958) beta prime (/5') 

 bands, a triple band structure at about 470 mfi. Although of low extinction 



ABSORBANCY 



490 520 550 



580 



Fig. 5. The effect of glycerol on the appearance of the c^2 absorption band of 

 reduced cytochrome c. Horse heart muscle cytochrome c was diluted in 0-2 m 

 phosphate buffer, pH 7-4 and reduced by the addition of Na2So04. Various 

 volumes of a mixture of 75 % glycerol, 25 % 0-2 m phosphate buffer, pH 7-4 (v/v) 

 were added to portions of the reduced cytochrome c solution to give varying 

 concentrations of glycerol. Curve A represents no glycerol, curve B was with 

 14% glycerol, and curve C contained 71 % glycerol. Optical depth of cuvettes, 

 3 mm. 



these three bands appear to be present in all the purified cytochrome haemo- 

 proteins that have been studied so far. 



Of interest is the nature of the splitting of the a- and /5-bands observed in 

 samples cooled in liquid nitrogen. One immediately asks does this imply 

 heterogeneity of the sample, thus indicating the presence of more than one 

 haemoprotein? If the split of the a- and /3-bands as observed is not due to 

 contamination by more than one haemoprotein, what is the significance of 

 this split. How may the fine band structure, as observed when samples are 

 cooled in liquid nitrogen, be modified and what factors influence these bands ? 

 It may also be asked whether the changes seen in the spectra of modified 

 samples of cytochrome c can be related to the biological activity of the 

 haemdproteins, to the protein structure itself, or to the spatial arrangement 

 of the constituent parts of the molecule ? 



G 



