Refolding and Reactivation of Liver Alcohol Dehydrogenase after Dissociation and Denaturation in 6M Guanidine Hydrochloride

Abstract

Horse‐liver alcohol dehydrogenase has been dissociated and denatured by 6 M gaunidinium hydrochloride. Removal of the denaturat under optimum conditions of the solvent leads to partial reactivation. The concentrations of the enzyme, as well as the coenzyme (NAD⁺ and Zn²⁺, affect the reactivation significantly, since high concentrations promote the formation of inactive aggregation products. Analyzing the kinetics of reactivation and reassociation, conditions far from equilibrium of dissociation‐association provide maximum yieldddd (∼ 70%). The sigmoidal kineic traces suggest a superposition of first‐order transconformation and‐second‐order association reactions; the latter are corrobosrated by the concentration dependence of thereactivation reaction. The coenzyme, NAD⁺, had no the kinetics of reactivation. Addition of Zn²⁺ leads to a significant decrease of the rate and yield of ractivation. The process of renaturation, as reflected by the regain of navive fluorescence show complex kinetics: rapid relaxations are followed by slower first‐order and secon‐order processes which parallel reactivtion.