Dissociation, unfolding and inactivation of creatine kinase in urea solutions

It was previously reported that, during unfolding of creatine kinase in guanidinium chloride or urea solutions, inactivation occurred before noticeable conformational change could be detected, suggesting that the conformation at the active site is more easily perturbed and, hence, more flexible than the molecule as a whole [Tsou (1986) Trends Biochem. Sci 11, 427-429]. In the present paper, the ureagradient electrophoresis and the isoenzyme hybrid of creatine kinase has been studied. The results show that at low urea concentrations, creatine kinase is still in the dimeric state or only slightly dissociated. The dissociation and inactivation of creatine kinase during denaturation by urea are also compared. It was found that the enzyme was nearly inactivated in low urea concentrations before noticeable dissociation was detected. It therefore appears that in low urea concentrations, inactivation of creatine kinase is not due to the dissociation of the active dimer. The present result supports the hypothesis of the conformational flexibility of the active site in this enzyme.