Studies on Cytochrome C

Abstract

Bodo first succeeded in the crystallization of cytochrome c from the king penguin (1). We have duplicated his work by crystallizations of cytochrome c's from the more convenient mammalian sources; horse, bovine and pig heart muscle (2, 3). We have also crystallized cytochrome c from a microbial source; baker's yeast (2, 4). During purification from baker's yeast, cytochrome c in its oxidized form could be separated into several different fractions by chromatography on an Amberlite XE-64 column (2). These fractions had almost the same absorption spectrum in their oxidized and dithionite reduced forms and were reduced enzymatically at almost the same rate by lactate in the presence of yeast lactic dehydrogenase. They were also oxidized at the same rate in the presence of “Green brei” (2, 5). These facts obscured which fraction on the chromatogram was identical with the normal cytochrome c in the respiring yeast cells (native cytochrome c). Therefore, after comparison of the chromatographic patterns of cytochrome c extracted and purified by various drastic and mild procedures, one fraction of a possibly native form among the various chromatographic fractions was examined. The yeast cytochrome c crystallized from this fraction was more resistant to the digestions by trypsin and by bacterial proteinase than those of the other fractions. This is in accordance with an idea that a globular protein may be more resistant to the digestion by proteinases than one in which the secondary structure of the protein has been somewhat injured. However, native yeast cytochrome c was found to be digested much more rapidly in its oxidized than in its reduced form (2, 5, 6). During the above investigations, cytochrome c's crystallized from the mammalian sources were found to be much more resistant to the proteinase-digestion than crystalline yeast cytochrome c, though having the same absorption spectrum as that from yeast. This paper is concerned with a critical investgation of methods for purification of mammalian cytochrome c's, and with studies on some of their properties with the object of identifying native mammalian cytochrome c's.