Dissociation and denaturation equilibriums and kinetics of a homogeneous human immunoglobulin Fab fragment

Abstract

The conformational equilibria and the kinetics of the approach to equilibrium of an immunoglobulinG1 myeloma (Wes) Fab fragment (SSFab) and its mildly reduced and S-carboxyamidomethylated derivative (RAFab) were studied as a function of guanidine hydrochloride concentration. The unimolecular denaturation of SSFab, the bimolecular denaturation of RAFab, and the denaturation of Wes L [light] chain reported previously are interpreted in terms of the domain structure and evaluated in terms of the thermodynamic stability of the protein and the covalent and noncovalent interactions among its subunits. The Fd-L interactions are extremely strong and are maintained at concentrations of denaturant sufficient to denature the individual domains. A 2 region structure for Fab is probable, 1 composed of the variable region (V)L and VH domains, and the other composed of the constant region (C)L and CH domains, so that there are 2 sites of noncovalent Fd-L interactions. One region, identified as the c region, is 102-104 .times. more stable than the other; this difference in stability is attributed largely to a stronger and more extensive interaction between the domains of this region. The kinetics of the approach to equilibrium are extremely slow in the center of the transitions, requiring up to a week for equilibration for RAFab, and several months for SSFab. This unusual kinetic behavior is due to the strong Fd-L interaction under conditions where the monomeric domains are unstable.