Refolding of bovine trypsinogen and neochymotrypsinogen. Formation of early non-native disulfide bonds in intermediates and independent domain folding

Abdulkarim Mohamed Al-Obeidi, Purdue University

Abstract

Size-exclusion performance liquid chromatography was used in this thesis to study the nature of the intramolecular protein disulfide bonds that define the intermediate species that accumulate transiently during protein folding and unfolding of trypsinogen. Attention focused on the Stokes radius of both unfolding and folding at 10 min with and without sucrose, and at 60 min. The Stokes radius obtained from the rechromatography of a collection of sequential fractions of initial separation shows that only the refolded sample displays microheterogenity. The Stokes radius values of urea denatured trypsinogen differ from that of partially refolded trypsinogen. Most of the intermediates at the early stage of refolding are largely unfolded (large Stokes radius), and tend to become a more compact-like conformation with time (decreasing Stokes radius). The above indicate that disulfide formation during trypsinogen refolding was largely random, especially at the early stage of disulfide formation, and non-native disulfide bonded forms tend to predominate. In this regard, a mechanism has been proposed and some partially refolded intermediates have also been speculated. In an effort to determine whether protein folding occurs independently at domain level, the kinetic study of the regeneration of neochymotrypsinogen from reduced form has been achieved at several ratios of N- (1-146) and C-terminal (147-240) fragments by activity measurement and SDS-polyacrylamide gel electrophoresis. The refolding of the nicked protein fits first order kinetic behavior rather than second order reaction behavior. The rate constant of the reactivation shows no change (within experimental errors) in increasing either fragment concentrations. The results of this study leads to three conclusions. (1) The refolding does not necessarily begin at one end of the molecule. This is consistent with the refolding of proteolytic fragments of bovine serum albumin (Johnson et al, 1981). (2) All findings support independent domain folding and is consistent with the hypothesis that folding occurs independently in different parts of a protein molecule (Wetlaufer, 1973), and support a diffusion collision mechanism (Karplus and Weaver, 1976) in which two fragments can refold independently and then diffuse and collide to form the native structure. (3) The most probable determining step is the formation of the disulfide bond between the two domains in the final step of refolding.

Degree

Ph.D.

Advisors

Light, Purdue University.

Subject Area

Biochemistry

COinS