Cytochromes Cooled in Liquid Nitrogen 



451 



A point worthy of mention is the possible confusion which may arise 

 because of the similarity of absorption band maxima of reduced cytochrome 

 q and b^^2- This situation reaffirms the contention that spectral studies alone 

 may be misleading and must be coupled with enzymic studies employing 



ABSORBANCY 

 INCREMENT 

 = 0.10 



I I I I I I I I ' I I ' I 

 490 520 550 580 610 

 WAVELENGTH (m>j) 



Fig. 15. Spectra showing the titration of cytochrome b^ of rat liver microsomes 

 by DPNH. Samples of rat liver microsomes were suspended in 0- 1 m phosphate 

 buffer, pH 7-4. One portion was removed and placed in the reference cuvette. 

 To the remainder were added small amounts of DPNH. The sample was then 

 cooled by plunging the cuvettes into liquid nitrogen and the difference spectrum 

 recorded. Curve A represents the spectrum obtained when DPNH sufficient to 

 reduce 27% of the cytochrome be, was added, curve B was the spectrum for 54% 

 reduction, curve C for 81 % reduction while curve D was the spectrum obtained 

 with a large excess of DPNH. Optical depth of the cuvette was 3 mm. 



various specific inhibitors or substrates in order to distinguish clearly one 

 pigment from another. 



PARTICLE-BOUND PIGMENTS 



As is apparent from the above, low temperature studies are at present 

 purely descriptive and of principal importance for identification of pigments. 

 The technique has therefore been extended to a study of the respiratory 

 pigment complement of particle-bound haemoproteins from a variety of 

 tissues. Among the first samples to be investigated were the particulate heart 

 muscle preparations; these studies v/ere carried out in collaboration with 

 Dr. Mackler (Estabrook and Mackler, 1957). As shown in Fig. 16 one 



