The effects of various alcohols, ethers, and amides on the absorption spectrum of cytochrome c have been investigated. A hemoundecapeptide, isolated from a peptic hydrolysate of cytochrome c, was similarly studied, (a) Increasing the concentration of organic solvent normally caused an initial increase in the intensity of the Soret peak, which subsequently decreased with further increases in solvent concentration. The maximum slope of a graph of absorbance change against mole per cent of organic solvent, Sm, was used as the criterion for comparing the effectiveness of the organic solvents, (b) The nature of the effects was similar in cytochrome c and the hemopeptide, but the magnitude of the change was usually two to three times greater in the hemopeptide. If the increase in Soret absorbance reflects denaturation, this result implies that the structure of the hemopeptide in aqueous solution is not completely random, (c) The addition of electrolytes significantly enhanced the Sm, presumably due to the encouragement of the disruption of ionic bonds in solutions of higher ionic strength, (d) The presence of urea similarly enhanced the Sm. (e) An increase in the length of the hydrocarbon chain of the alcohols and ethers increased Sm. Branching, however, produced a decrease. Hydrophobicity is thus important for conformational changes but accessibility of the solvent hydroxyl group or the interior of the polypeptide chain is also important, (f) The importance of hydrophobicity is also evident from the decrease in Sm which occurs on replacing a carbon by an oxygen or on inserting an oxygen into the chain. When a hydroxyl group replaces a hydrogen atom, the loss of effectiveness is even greater, (g) When chain length and oxygen content were simultaneously increased by adding a carbinol group, the above opposing effects balanced each other, (h) Hydrogen bond disrupting capacity is also important, as determined by comparison of formamide and dimethylformamide. On a volume per cent basis these are of roughly equal effectiveness in enhancing the Soret peak. Thus the increase in hydrophobicity only just balances the loss of hydrogen bonding capacity. The above results are interpreted as qualitative evidence for contribution of hydrophobic, hydrogen, and ionic bonds to the stability of the native structure of both cytochrome c and the hemopeptide. © 1969, American Chemical Society. All rights reserved.
