Regeneration of ribonuclease A from the reduced protein. Isolation and identification of intermediates and equilibrium treatment

Reduced RNase A was reoxidized, and the incorrectly formed disulfide bonds were reshuffled to the native ones by oxidized and reduced glutathiones, as described in the first paper of this series. The intermediates in the regeneration of the disulfide bonds were trapped without any chemical modification and were fractionated on a carboxymethylcellulose column at pH 3.5 with a salt gradient. The elution curves of the partially regenerated RNase A from the carboxymethylcellulose column were obtained by measurement of the absorption at 275 nm and by determination of the SH content (of cysteine residues) and consisted of 11 fractions, G8, G7, G6, G5, G4, G3, G2, G1, G0, N, and F. Some of the fractions were isolated, and their measured molecular weights were consistent with those of monomeric RNase A. Fraction F had a molecular weight between that of the monomer and dimer, so that this fraction could not be identified. The regeneration pathway could be represented in terms of two simple reactions, RNase A(-SH) + GSSG in equilibrium or formed from RNase A(-SSG) + GSH and RNase A(-SH-SSG) in equilibrium RNase A(greater than S2) + GSH, which produced 24 monomeric intermediates (not counting the fully reduced and the native species), which differed from each other in their amino acid composition. These 24 intermediates, plus the fully reduced protein, were assigned to fractions G8--G0 (as indicated in the last column of Table I), with the aid of data from amino acid analysis, SH content, and the elution position on the carboxymethylcellulose column chromatogram. Since the regeneration reaction rapidly reached a preequilibrium among the intermediates and the fully reduced RNase A prior to the rate-limiting steps, i.e., the relative concentrations of the intermediates and fully reduced RNase A became constant with reaction time, the populations of some of the intermediates in preequilibrium were estimated by curve fitting of the elution pattern from the carboxymethylcellulose column chromatogram. The equilibrium constants among the intermediates were calculated from their populations at preequilibrium. These equilibrium constants were "extrapolated" to other intermediates whose populations could not be estimated by curve fitting, and the relative populations of all of the possible intermediates at preequilibrium were thereby represented as a function of the concentrations of reduced and oxidized glutathiones. The regeneration process was also restarted from several of the isolated intermediates, and the resulting distribution of intermediates was consistent with that from which the equilibrium constants were determined, supporting the representation of the regeneration pathways in terms of two simple reactions. Thus, the equilibrium treatment of the regeneration pathways was useful to characterize the preequilibrium state, i.e., to identify the intermediates prior to the rate-limiting steps in the pathways and to estimate their stabilities at preequilibrium at various concentrations of reduced and oxidized glutathiones.