The Location of Cytochromes in Escherichia Coli 



219 



centrifugation (Tissieres et a/., 1959). It consisted then of 90-95% of 

 RNP particles and 5-10% of respiratory granules. 



Reduced diphosphopyridine nucleotide (DPNH) oxidase activity was 

 estimated according to Slater (1950) using the reaction mixture given by 

 Tissieres et al. (1957). Protein and ribonucleic acid (RNA) were determined 

 by the biuret (Gornall, Bardawill and David, 1949) and orcinol (Dische, 

 1953) methods respectively. 



A Zeiss microspectroscope fitted to a microscope, as described by Keilin 

 and Hartree (1946) was used to examine the absorption bands of cytochromes. 

 Fractions of small granules were isolated in a Model L Spinco ultra-centrifuge. 

 The centrifugal forces given were calculated for the middle of the centrifuge 

 tube. Analytical ultra-centrifugation was done with a Model E Spinco 

 centrifuge, with schlieren optics. 



Tris buffer was Sigma 7-9 biochemical buffer from Sigma Chemical 

 Company, St. Louis, Missouri. The pH of tris solutions was adjusted to 

 7-3 by addition of 0-1 n HCl. Deoxyribonuclease was obtained from 

 Worthington Biochemical Corp., Freehold, New Jersey, and lysozyme and 

 DPNH from Sigma Chemical Company. 



EXPERIMENTAL 



1 . Cytochromes in the Various Particulate Fractions 



The typical cytochrome components of E. coli were observed in cell debris 

 and granules of all sizes, with the exception of purified RNP particles: the 

 a-absorption bands of reduced cytochromes ^3, a^ and b^, characteristic of 

 E. coli (Keilin and Harpley, 1941), were seen under the microspectroscope. 



0-2 



0-1 



-0-1 



-0-2 



550 



600 650 



Wavelength, m/j 



Fig. 1. DifTerence spectrum, oxidized minus reduced, of a small granule fraction, 



The base line, which corresponded to an optical density of at 750 m/<, was somewhat 



sliifted up at shorter wavelengths. 



H.E. — VOL. I — Q 



