Cytochrome bg 



561 



Figure 1 shows some mutual relationships of cytochrome b.^, flavin, and phenazine 

 methosulphate and methylene blue activities during the course of the modification of 

 the enzyme at a lower pH (Yamashita, Horio and Okunuki, unpublished). The curve 

 'cytochrome b.,' was made by dithionite-reduction of the a-absorption maximum of 

 cytochrome 60. The curve 'flavin' was measured by fluorescence with the use of 

 supernatant of an ammonium sulphate-saturated sample. Because of the use of a 

 crude enzyme preparation, I think that the amount of liberated flavin does not 



Fig. 1. Modification of bakers' yeast lactate dehydrogenase. The figure compares 

 the percentage of remaining activity with phenazine methosulphate and with 

 methylene blue after treatment of the enzyme at low pH, with the amount of 

 remaining dithionite-reducible haemoprotein (expressed as 'cytochrome ^3) and 

 the amount of liberated flavin (measured as fluorescence). 



correctly show the inter-relationship with the enzyme modification. However, it may 

 be assumed that the liberation of flavin has a certain important role in the partial 

 modification of bakers' yeast lactate dehydrogenase, as indicated by Appleby and 

 Morton (.Nature, Lond. 173, 749, 1954; Biochem. J. 73, 539, 1959). 



Compared with the enzyme, the 'cytochrome />.,' (split from the enzyme) is very 

 stable, and the cytochrome so formed can be easily crystallized. The experimental 

 conditions for direct extraction of cytochrome 63 from bakers' yeast cells, involving 

 as it does extraction at a higher pH (pH 8) and other results, clearly indicate that 

 'cytochrome b.^ does not exist independently apart from the enzyme, at least in its 

 physiologically native state. In order to investigate the mode of existence of cyto- 

 chrome 62 in the enzyme, the absorption spectra of cytochrome bo and the enzyme in 

 the visible wavelength were compared, and it was found that there is no notable 

 diff"erence above experimental error. If cytochrome c is used as a model case, variously 

 diff^erent kinds of cytochrome c's can be easily prepared : a native one having its coiling 

 structure, a modified one damaged in the coiling structure, and a proteolysed one. The 

 former two show the same absorption spectra which are remarkably difi'erent from the 

 latter. This means that the absorption spectrum of cytochrome c is not seriously 

 changed by damaging the coiling structure, but is remarkably altered by shortening 

 its amino acid composition (see Horio et al., this volume, p. 557, for references). 

 Analogously considered, the following two kinds of existence of cytochrome b^ might 

 be assumed. In (1), 'cytochrome 60' having no flavin and no enzymic activity, exists 



