Refolding and reactivation of liver alcohol dehydrogenase after dissociation and denaturation in 6M guanidine hydrochloride.
Horse-liver alcohol dehydrogenase has been dissociated and denatured by 6 M guanidinium hydrochloride. Removal of the denaturant 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 yields (approximately 70%). The sigmoidal kinetic traces suggest a superposition of first-order transconformation and second-order association reactions; the latter are corroborated by the concentration dependence of the reactivation reaction. The coenzyme, NAD+, has no influence on the kinetics of reactivation. Addition of Zn2+ leads to a significant decrease of the rate and yield of reactivation. The process of renaturation, as reflected by the regain of native fluorescence shows complex kinetics: rapid relaxations are followed by slower first-order and second-order processes which parallel reactivation.
D000429:Alcohol Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D004789:Enzyme Activation / Q000187:drug effects; D006146:Guanidines / Q000494:pharmacology*; D006736:Horses; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D009243:NAD / Q000494:pharmacology; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D015032:Zinc / Q000494:pharmacology
D006146:Guanidines; D009243:NAD; D000429:Alcohol Oxidoreductases; D015032:Zinc