The Denaturation‐Renaturation of Chicken‐Muscle Triosephosphate Isomerase in Guanidinium Chloride

Publisher Website
Google Scholar
Abstract
The process of denaturation of the chicken muscle dimeric enzyme triosephosphate isomerase on addition of guanidinium chloride was studied at pH 7.6, the pH at which the recovery of activity is optimal (100%) on removal of denaturant. Determinations of the sedimentation coefficient, intrinsic viscosity, MW (by sedimentation equilibrium studies) and the absorption coefficient at 280 nm in various concentrations of guanidinium chloride concurred in showing a single, sharp transition at about 0.7 M guanidinium chloride at a protein concentration 1-5 mg/ml from the native enzyme to the dissociated, unfolded chains of the monomer. Relative fluorescent intensity measurements revealed a single transition at about 0.4 M guanidinium chloride at enzyme concentrations of about 0.05 mg/ml. The process of denaturation in different guanidinium chloride concentrations was 1st order with respect to enzyme and about 6th order with respect to denaturant. The rate of attainment of equilibrium during the renaturation obeyed 2nd-order/1st-order reversible kinetics. It was concluded that the rate-determining step in renaturation at pH 7.6 must be the association of 2 subunits.