Scheme 1. Regeneration of RNase A (25 °C, pH 8) Proceeds via the Successive Oxidation of Its Cysteines to Form Disulfide Bonds.

Intermediates (1S–4S) differing in their degree of oxidation (number of disulfide bonds) populate the regeneration chromatogram, and each nS “intermediate” is an ensemble of isomers containing n disulfide bonds. Native protein (N) is formed via the oxidation of two three-native-disulfide-containing 3S* intermediates that accumulate from their 3S isomers by conformational folding. At temperatures <15 °C, two dead-end three-disulfide-bond-containing structured species also populate the oxidative folding landscape of RNase A.