Effects of arginine on rabbit muscle creatine kinase and salt-induced molten globule-like state.
The arginine (Arg)-induced unfolding and the salt-induced folding of creatine kinase (CK) have been studied by measuring enzyme activity, fluorescence emission spectra, native polyacrylamide gel electrophoresis and size exclusion chromatography (SEC). The results showed that Arg caused inactivation and unfolding of CK, but there was no aggregation during CK denaturation. The kinetics of CK unfolding followed a one-phase process. At higher concentrations of Arg (>160 mM), the CK dimers were fully dissociated, the alkali characteristic of Arg mainly led to the dissociation of dimers, but not denaturation effect of Arg's guanidine groups on CK. The inactivation of CK occurred before noticeable conformational changes of the whole molecules. KCl induced monomeric and dimeric molten globule-like states of CK denatured by Arg. These results suggest that as a protein denaturant, the effect of Arg on CK differed from that of guanidine and alkali, its denaturation for protein contains the double effects, which acts not only as guanidine hydrochloride but also as alkali. The active sites of CK have more flexibility than the whole enzyme conformation. Monomeric and dimeric molten globule-like states of CK were formed by the salt inducing in 160 and 500 mM Arg H(2)O solutions, respectively. The molten globule-like states indicate that monomeric and dimeric intermediates exist during CK folding. Furthermore, these results also proved the orderly folding model of CK.
Ou
Wen-bin
WB
Department of Biological Science and Biotechnology, Tsinghua University, Beijing 100084, PR China.
Wang
Ri-Sheng
RS
Lu
Jie
J
Zhou
Hai-Meng
HM
D000818:Animals; D001120:Arginine / Q000737:chemistry*; D002850:Chromatography, Gel; D003402:Creatine Kinase / Q000737:chemistry*; D052283:Creatine Kinase, MM Form; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry*; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D011189:Potassium Chloride / Q000737:chemistry; D011489:Protein Denaturation; D017510:Protein Folding; D011817:Rabbits; D012965:Sodium Chloride / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods; D013056:Spectrophotometry, Ultraviolet
D007527:Isoenzymes; D012965:Sodium Chloride; D011189:Potassium Chloride; D001120:Arginine; D003402:Creatine Kinase; D052283:Creatine Kinase, MM Form