336 M. Morrison and E. Stotz 



and cryptohaemin a are so closely related that either could have been the 

 prosthetic group of either cytochrome a or a^. 



The cytochromes a and a^ have never been isolated and separated from each 

 other. An attempt (Connelly, Morrison and Stotz, 1959) to achieve such a 

 separation by electrophoresis was made in our laboratory using a purified 

 cytochrome c oxidase preparation as a starting material. This effort was 

 unsuccessful, although an active cytochrome c oxidase was separated from 

 an inactive haemoprotein. This inactive haemoprotein retained the spectral 

 properties of cytochromes a and a^ and even the ability of cytochrome a^ 

 to combine with carbon monoxide. The inactive preparation could not be 

 reactivated and the haemins present in the preparation were identical with 

 those of the active enzyme. 



In at least one micro-organism, however, nature has already performed 

 the separation of cytochromes a and ^3. Smith (1954, 1955) has shown 

 that Staphylococcus albus contains only cytochrome a, at least insofar as it 

 can be defined by spectrophotometric studies. The haemins from this 

 organism were extracted by procedures that had been developed in our 

 laboratory (Morrison and Stotz, 1955, 1957; Connelly, Morrison and Stotz, 

 1958). The haemins were separated and studied by chromatographic and 

 spectral techniques with results showing that haemin a was the prosthetic 

 group of the cytochrome a of S. albus. This added a second criterion to the 

 already cited spectrophotometric work which more firmly establishes the 

 cytochrome component in S. albus as cytochrome a, comparable to that of 

 mammalian tissue. It is also an interesting demonstration of the use of a 

 neglected tool for the characterization of cytochromes ; that is, the characteri- 

 zation of the prosthetic groups of cytochromes by chromatographic methods. 

 No cryptohaemin was identified in the haemins extracted from the S. albus. 



Although there is no comparable source for observing cytochrome ^3 

 free of cytochrome a, we did turn to Bacillus subtilis, an organism in which the 

 concentration of cytochrome ^3 is 2-3 times (Smith, 1954, 1955) the concentra- 

 tion of cytochrome a. We were able to isolate haemin a from this organism 

 as well as some cryptohaemin a, but the ratio of cryptohaemin a to haemin a 

 was less than that found for the haemins extracted from heart muscle 

 (Morrison and Stotz, 1955; Morrison, Connelly and Stotz, 1958). Providing 

 that the components in the bacteria are strictly comparable to those in heart 

 muscle, these results do not support the suggestion that cryptohaemin is the 

 prosthetic group of cytochrome a^. 



When a cytochrome oxidase preparation is allowed to stand at 4°C for a 

 prolonged period, it loses its enzymic activity. This loss is shown in Fig. 1, 

 and is paralleled by a loss in haemin, as judged by the extinction at 605 m/<. 

 After standing two weeks at 4°C, the spectrum of the solution is altered 

 markedly. In the reduced form, the aged protein preparation has httle absorp- 

 tion in the visible region and the Soret peak has shifted from 444 m/n to 



