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

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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.