450 



R. W. ESTABROOK 



Dr. Garfinkel (Garfinkel, 1957) from two different sources were investigated. 

 As shown in Fig. 14 there is a difference in the magnitude of the b^^^ absorp- 

 tion band when comparing the spectrum of the two samples. These samples 

 of cytochrome b^ were reduced enzymatically by a specific DPNH-cytochrome 

 Dr. Garfinkel. The variation observed would 



CYTOCHROME B= 



490 520 550 580 



Wavelength (rriAi) 



Fig. 14. A comparison of the spectral properties of cytochrome 6,,, purified from 

 two different sources. Samples of purified cytochrome Zjj were diluted in 01 m 

 phosphate buffer, pH 7-4 and a purified reduced diphosphopyridine nucleotide 

 (DPNH)-cytochrome h-^ reductase and DPNH were added. The sample was then 

 diluted with an equal volume of glycerol and the spectrum recorded. Curve A 

 represents cytochrome b^ purified from rabbit liver microsomes while curve B is 

 that obtained using pig liver as the source of microsomes. The dotted curve 

 represents the spectrum of reduced heart muscle cytochrome c. Optical depth 

 was 1 mm. Condition II. 



indicate that the splitting of the a-band seen with cytochrome b^ may be due 

 to the presence of more than one pigment, the haemoprotein contributing to 

 the b-^i-hand having been partially removed during the course of purification. 

 If this hypothesis is correct, i.e. that more than one haemoprotein con- 

 tributes to the spectrum of cytochrome b^, then a preferential reduction of 

 one or the other pigment might be expected when the system is titrated with 

 DPNH. As shown in Fig. 15 this is not the case. Addition of small amounts 

 of DPNH and then rapid cooling of the sample (see below) shows that both 

 b^^i and br^^.y appear together. Tliis indicates cither that the two bands 

 observed are associated with a single pigment or that two pigments are being 

 reduced simultaneously to the same extent when a substrate such as DPNH 

 is added. 



