CONFORMATIONAL-CHANGES OF CREATINE-KINASE DURING GUANIDINE DENATURATION

Abstract

The conformational changes of [rabbit muscle] creatine kinase during denaturation by guanidine hydrochloride were studied by fluorescence and UV difference spectroscopy. At low concentrations of guanidine, < 1 M, the denatured minus native difference spectra showed 2 negative peaks at 281 and 287 nm, whereas the fluorescence emission increased markedly with its maximum red-shifted from 337 to 345 nm. Control experiments showed that guanidine also increased the emission of ionized tyrosine at 345 nm. With increased concentration of guanidine, both negative peaks at 281 and 287 nm increased in magnitude to reach maximal values at 3 M guanidine and at this time a small peak appeared at 292 nm. The fluorescence maximum was further red-shifted to 355 nm, whereas the emission intensity of the main peak decreased and a small shoulder appeared at 310 nm when the guanidine concentration increased from 1 to 3 M. Further increase in guanidine concentration produced little further change in UV absorption or in fluorescence. In the native enzymes, tryptophan residues are probably partly buried and some of the tyrosine residues are in an ionized state. Guanidine below 1 M does not expose the buried tryptophan residues or affect significantly the microenvironments of the ionized tyrosine residues. At 3 M guanidine, tryptophan residues are exposed and the ionization state of tyrosine residues is also affected. At this concentration, the peptide chain seems to be fully unfolded as evidenced by the fact that 5 M guanidine produces little further change in UV absorption or fluorescence.