Conformational changes of creatine kinase during guanidine denaturation.
The conformational changes of creatine kinase during denaturation by different concentrations of guanidine hydrochloride have been studied by fluorescence and ultraviolet difference spectroscopic methods. At low concentrations of guanidine, less than 1 M, the denatured minus native difference spectra showed two 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 the increase of concentrations 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 either in UV absorption or in fluorescence. From the above results, it seems that, in the native enzyme. Trp residues are partly buried and partly exposed and some of the Tyr residues are in ionized state. Guanidine below 1 M does not expose the buried Trp residues nor affects significantly the microenvironments of the ionized Tyr residues. At 3 M guanidine, Trp residues are exposed and the ionization state of Tyr 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 both UV absorption and fluorescence.
D000818:Animals; D003402:Creatine Kinase / Q000302:isolation & purification; D005453:Fluorescence; D019791:Guanidine; D006146:Guanidines; D009132:Muscles / Q000201:enzymology; D011487:Protein Conformation; D011489:Protein Denaturation; D011817:Rabbits; D013056:Spectrophotometry, Ultraviolet
D006146:Guanidines; D003402:Creatine Kinase; D019791:Guanidine