Regeneration of bovine pancreatic ribonuclease A. 3. Dependence on the nature of the redox reagent.

Monothiol reagents such as oxidized and reduced glutathione (GSSG and GSH, respectively) form stable mixed disulfides with protein thiols while dithiol reagents such as oxidized and reduced dithiothreitol (DTTox and DTTred, respectively) do not. This large difference in the stabilities of the mixed disulfides is reflected in much greater rates of formation and reduction of protein disulfide bonds with GSSG/GSH than with DTTox/DTTred. With dithiothreitol, the concentrations of intermediate species in the steady state depend on the redox potential, i.e., on the [DTTox]/[DTTred] ratio, and not on the absolute concentrations of these reagents. With glutathione, the redox potential and hence the concentrations of intermediate species in the steady state depend on the [GSSG]/[GSH]2 ratio; hence, with glutathione, in contrast to dithiothreitol, the absolute values of the concentrations do affect the steady-state concentrations. Consequently, the regeneration pathways of bovine pancreatic ribonuclease A depend on the nature of the redox reagent as well as the redox potential at which they are used. The use of GSSG/GSH favors multiple regeneration pathways, while the use of DTTox/DTTred favors regeneration through fewer pathways. These concepts are also illustrated by an analysis of literature data for the regeneration pathways of bovine pancreatic trypsin inhibitor.