A monomeric cytochrome c containing an intramolecular disulfide bond linking residues 20 and 102 was obtained from bullfrog hearts, as previously reported by Chan et al. [Chan, S. K., Walasek, O. F., Barlow, G. H., & Margoliask, E. (1967) Fed. Proc., Fed. Am. Soc. Exp. Biol. 26, 723]. The stability of the native globular conformation of this protein is enhanced relative to that of other cytochromes c; e.g., it is stable to 0.01 N HCl in the absence of neutral salts at 25 degrees C, and its denaturation transition in guanidine at neutral pH and 25 degrees C is centered at 3.8 M. Guanidine-denatured from cytochrome c renatures in two kinetic phases whose time constants differ by about 2 orders of magnitude as observed when using other cytochromes c. However, the absolute values for the time constants of the frog protein are notably smaller. We note that the time constants for cytochromes c are inversely related to the midpoints of their guanidine transitions, suggesting that within a homologous series of proteins the more stable the conformation the faster it folds.

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in which the longest decay component could be unambiguously determined, these measurements represent an important check on the validity of the conclusions drawn from the anti-DNS studies. Furthermore, the measurements were carried out on an antibody population with a different specificity. Thus, Fab segmental flexibility is probably generally present in IgG molecules. 19 homoarginine residues are distributed over a range of about 1 ppm and include two discrete single guanido carbon resonances. Distinct changes in the 13C NMR spectrum are observed upon complexation of guanidinated ferricytochrome c with ferrihexacyanide, upon conversion of the guanidinated ferricytochrome c to its alkaline isomer, and upon reduction to guanidinated ferrocytochrome c. Analysis of the changes in the visible absorbance spectrum accompanying guanidination of ferricytochrome c with O-methylisourea indicates that 18 lysine residues are guanidinated about 3 times more rapidly than the single remaining lysine residue, most likely lysine-79. The guanido carbon of this residue was preferentially enriched with l3C-enriched O-methylisourea. Analysis of the Ferricytochrome c is known to form discrete complexes which facilitate electron exchange with electron acceptors such