Partition chromatography of hemins; separation of the prosthetic groups of cytochromes a and a3.

The earlier work of Keilin and Hartree (1) and the later reports of Ball and coworkers (2) and of Chance (3-5) leave little doubt that cytochromes a and a3 are two different hemoproteins. Comparison of the effect of carbon monoxide, as observed spectroscopically at 605 rnp and in the Soret region, indicates pronounced differences in the extinctions of these components and suggests either different hemin prosthetic groups or very different types of linkages between hemin and protein in cytochromes a and ~3. Other workers were concerned with the dichroic (“green”) hemin of heart muscle. Thus Negelein (6) separated a hemin which as a reduced pyridine hemochromogen possessed an absorption band at 587 rnp. Roche and Benevent (7) questioned whether this hemin was derived unchanged from the tissue, and the cryptoporphyrin prepared from the hemin was considered by Negelein (8, 9) to be an artifact. More recent work (10-12) leaves no doubt that such a “green” hemin can be derived from tissue and that it is characterized by a reduced pyridine hemochromogen having absorption peaks at 430 and 587 rnp. In addition, Dannenberg and Kiese (13) have isolated a hemin from a partially purified cytochrome oxidase with these absorption characteristics, and Person and coworkers (14) found a similar spectrum upon addition of pyridine to an oxidase preparation. The work cited implies that there is a single prosthetic group (hemin a) for cytochromes a and as. Falk and Rimington (11)) in a thorough investigation of the porphyrins derived from hemin a, demonstrated that “porphyrin CL” was labile to acid and is probably a mixture of at least four porphyrins. Thus porphyrin separation could not lead to valid conclusions as to the possible multiplicity of hemin prosthetic groups, and these investigators were unsuccessful in their attempts to purify hemin a by column chromatography. Most recently Kiese and Kurz (15) have described a chromatographic method