REGENERATION OF BOVINE PANCREATIC RIBONUCLEASE-A .2. KINETICS OF REGENERATION

Analysis of the experimental data of the previous paper [Rothwarf, D. M., & Scheraga, H. A. (1993) Biochemistry (first of four papers in this issue)], using the method of Konishi et al. [Konishi, Y., Ooi, T., & Scheraga, H. A.(1981) Biochemistry 20, 3945-3955; Konishi, Y., Ooi, T., & Scheraga, H. A. (1982) Biochemistry 21, 4734-4740], and a discussion of the validity of the steady-state kinetic treatment of the data analyzed here are presented. The analysis reveals that RNase A regenerates with oxidized and reduced dithiothreitol (DTT(ox) and DTT(red), respectively) through a rearrangement pathway involving one or more three-disulfide species; i.e., multiple pathways could be involved. This pathway is different from that observed when RNase A is regenerated with oxidized and reduced glutathione (GSSG and GSH, respectively). These differences result primarily from the very different characteristics of the oxidation of thiols with DTT(ox) and GSSG, respectively. In addition, the concept of multiple pathways, as applied to the regeneration of RNase A, is developed.