Evidence for the Existence of Two Functionally Distinct Forms of Cytochrome c Monomer at Alkaline pH

The work of Margoliash (I) has clearly shown that cytochrome c preparations, obtained by most of the known methods, contain in addition to the monomers, quantities of polymerized material, the proportions of which vary with the method of isolation.The conditions governing the interconversion of the various forms have been described by Margoliash and Lustgarten (2) while Margoliash, Frohwirt, and Wiener (3) have demonstrated that only the monomer exhibits maximal electron transfer activity in various enzyme systems derived from the terminal electron transfer chain.Schejter et al. ( 4) have reassigned the extinction coefficients for monomeric and dimeric cytochrome c and have noted that the band at 695 rnp, first described by Theorell and Akesson (5) in 1939, appears to be peculiar to the monomer.The 695 mp absorption band of cytochrome c has been shown to be sensitive to temperature and to protein denaturants by Schejter and George (6), who conclude as a result of their studies that it reflects protein conformational changes not directly associated with the heme group chromophore.The results reported in this paper purport to show that the monomeric species can apparently exist in two forms in solution on the alkaline side of neutrality.Only one of the forms appears to be capable of reacting with ascorbate or tetrachlorohydroquinone. EXPERIMENTAL PROCEDURECytochromx c Monomer -This was prepared according to the method of Margoliash (7) starting from salt-free, equine heart cytochrome c obtained from Calbiochem.Amberlite IRC-50 used in the purification was obtained from Mallenchrodt.The mesh size was reduced by grinding and settling as described (7) above.Stopped Flou, Kinetic Experiments-These were performed in the apparatus described by Gibson and Milnes (8) outfitted with a 2-cm light path cell.For experiments at 695 rnp the Bausch and Lomb small grating monochromator was supplemented with a red filter glass, Corning CS 2.58, to remove stray and second order light.All measurements, unless otherwise reported, were made at 20".