Rate-determining folding and association reactions on reconstitution pathway of porcine skeletal muscle lactic dehydrogenase after denaturation by guanidine hydrochloride

Abstract

Reactivation of tetrameric porcine skeletal muscle lactic dehydrogenase after dissociation and extensive unfolding of the monomers by 6 M guanidine hydrochloride (Gdn.cntdot.HCl) is characterized by sigmoidal kinetics, indicating a complex mechanism involving rate-limiting kinetics, indicating a complex mechanism involving rate-limiting folding and association steps. For analysis of the association reactions, chemical cross-linking with glutaraldehyde may be used. The formation of a dimeric intermediate is determined by a first-order folding reaction of the monomers with ki = (8.0 .+-. 0.1) .times. 10-4 s-1. The rate constant of the association of dimers to tetramers, which represents the second rate-limiting step on the pathway of reconstitution after guanidine denaturation, was then determined by reactivation and cross-linking experiments after dissociation in 0.1 M H3PO4 containing 1 M Na2SO4. The rate constant for the dimer association (which is the only rate-limiting step after acid dissociation) was k2 = (3.0 .+-. 0.5) .times. 104 M-1 s-1. On the basis of the given 2 rate constants, a kinetic model is described for the complete reassociation pattern of porcine lactic dehydrogenase after dissociation and denaturation in 6 M Gdn.cntdot.HCl.