Refolding of OPTA-Iabeled creatine kinase denatured by guanidmium chloride

摘要

Although refolding of fully denatured protein molecules in vitro is not a valid model of folding of the nascent peptide chain in a biologically active protein in vivo, it can provide some useful information for protein folding. Therefore, refolding ofdenatured proteins has been extensively studied in recent years. It has been previously reported that during the refolding of guanidine or urea denatured creatine kinase, the enzymatic activity and the native conformation can be recovered fully under suitable conditions. A comparison of the rates of the refolding and reactivation course of guanidine or urea denatured creatine kinase showed that two processes are not synchronized, Although partial activity recovery occurs in reactions with nearly the same rate as those of the refolding processes, the complete reactivation can only be obtained long after any detectable conformational change reached completion. Recently, we introduced a fluorescnet probe into the active site of creatine kinase by modification with OPTA, and compared the changes of the probe fluorescence with inactivation in low concentrations of guanidinium chloride. It is shown that inactivation and exposure of the probe take place simultaneously and well before unfolding of the molecule as a whole. In this note, with the fluorescent probe of OPTA-labeled active site, the conformational changes of the active site were determined by burial of the probe during the refolding course of guanidine-denatured creatine kinase, and the above changes with the reactivation and molecular refolding were compared.