Electrometric and Other Studies of Cytochrome c 



375 



groups from Hb or Mb would be expected. The cytochrome c was then 

 concentrated with TCA as in the final stage of the Keilin and Hartree 

 procedure. This material had an iron content of 0-32% and a trace only of 

 zinc was present. Prior to TCA-treatment no absorption at 630-635 mfi due 

 to metmyoglobin or methaemoglobin could be detected and subsequently the 

 absorption spectra of the oxidized forms before and after resin chroma- 

 tography were identical (Fig. 3). No fore-running non-cytochrome fraction 

 containing protohaem could be detected on resin column chromatography as 

 is the case with TCA-extracted samples. However, increased absorption, 

 particularly on the longer wave-length slope of the a-band is apparent in the 

 reduced form of the material prior to resin treatment (Fig. 3) and the band 

 ratios differ considerably (Table 4). Inhibition of the copper-catalysed 

 oxidation of ascorbic acid was similar to that of the usual '0-34% Fe' content 

 preparations, indicating combination with copper. A sample which had 

 been treated with copper under the conditions described in Fig. 2, was given 

 a repetition of the final TCA precipitation of the Keilin and Hartree pro- 

 cedure. It was found that this material would then again take up a similar 

 quantity of copper. It appeared therefore, as in fact would be expected, that 

 the copper originally combined was removed under the acid conditions. 



Table 4. Comparison of optical-density ratios of several ox-heart 

 cytochrome c samples 



Combination of Copper with Globin 



Myoglobin was prepared from horse-heart by a combination of the methods 

 of Theorell (1932) and Lawrie (1951) and globin from this by the procedure 

 of Theorell and Akeson (1955). Globin was added in varying concentration 

 to constant copper and ascorbate concentration as for the cytochrome c 



