262 LuciLE Smith and Helen Conrad 



the tissues in cold water in a Teflon homogenizer and discarding the material 

 sedimented by centrifugation at 700 rev/min for 5 min in a Servall refrigerated 

 centrifuge. 



Measurements of the Content of Cytochromes a plus ag in Preparations 



In the optically clear purified preparations the difference in absorption 

 spectrum between the oxidized preparation (nothing added) and the prepara- 

 tion reduced with sodium dithionite was measured to assay the content of 

 cytochromes a plus a^. These measurements were made either in a Beckman 

 DU spectrophotometer or in the recording spectrophotometer described by 

 Yang and Legallais (1954). The concentration of cytochrome ^3 was measured 

 by recording the absorption spectrum of the carbon monoxide compound 

 formed by gassing the dithionite-reduced preparation with carbon monoxide. 

 The concentration of cytochrome a^ was calculated using the extinction 

 coeflficient reported by Chance (1953). 



In turbid preparations, such as heart muscle particles, the difference in 

 optical density (A£) at 605 minus 630 m/< between the preparation with the 

 cytochromes reduced (anaerobic preparation containing substrate) and that 

 with the cytochromes oxidized (aerobic preparation) was used as a measure 

 of the cytochromes a plus a^ present. Cytochromes a and a^ were considered 

 to be reduced in anaerobic mitochondria and oxidized in aerobic mitochondria 

 containing substrate and phosphate acceptor (Chance and Williams, 1955). 

 With homogenates, which contained haemoglobin, a diff'erent procedure was 

 followed. Here the diff'erence in absorption spectrum was measured between 

 two samples of aerobic homogenate, one of which contained 10~^ m cyanide. 

 The observed diff'erence in optical density at 605 minus 630 m/t was multiplied 

 by 4/3 to correct for the loss of absorption of the cytochrome a-^ at 605 m// 

 in the presence of cyanide. All measurements were made in the recording 

 spectrophotometer described by Yang and Legallais (1954) or the double- 

 beam spectrophotometer designed by Chance (1951). Both instruments will 

 record small differences in optical density of turbid preparations. 



Assay of Cytochrome c Oxidase Activity 



The rate of oxidation of soluble ferrocytochrome c by the oxidase was 

 followed by recording the decrease in optical density at the a, /? or y absorp- 

 tion peak of ferrocytochrome c, as previously described by Smith and 

 Conrad (1956). The cytochrome c was prepared by the method of Keilin and 

 Hartree (1947), purified according to Margoliash (1954) and reduced with 

 hydrogen and palladium (Smith and Conrad, 1956). 



Chemicals 



Salmine sulphate, purchased from General Biochemicals, Inc., was dialyzed 

 for several hours, first against 10~^ m ethylenediamine-tetra-acetate (versene), 



