Effects of lactic acid and NaCl on creatine kinase from rabbit muscle.
The lactic acid induced unfolding and the salt-induced folding of creatine kinase (CK) were studied by enzyme activity, fluorescence emission spectra, circular dichroism spectra, and native polyacrylamide gel electrophoresis. The results showed that the kinetics of CK inactivation was a monophase process. Lactic acid caused inactivation and unfolding of CK with no aggregation during CK denaturation. The unfolding of the whole molecule and the inactivation of CK in solutions of different concentration of lactic acid were compared. Much lower lactic acid concentration values were required to bring about inactivation than were required to produce significant conformational changes of the enzyme molecule. At higher concentrations of lactic acid (more than 0.2 mM) the CK dimers were partially dissociated, as proved by native polyacrylamide gel electrophoresis. NaCl induced the molten globule state with a compact structure after CK was denatured with 0.8 mM lactic acid, and the increasing of anions led to a tight side-chain. The above results suggest that the effect of lactic acid differed from that of other denaturants such as guanidine hydrochloride, HCI, or urea during CK folding, and the molten globule state indicates that intermediates exist during CK folding.
Tang
Hong-Min
HM
Department of Chemistry, Guizhou Institute for Nationalities, Guiyang 550025, PR China.
Ou
Wen-Bin
WB
Zhou
Hai-Meng
HM
D000818:Animals; D002942:Circular Dichroism; D003402:Creatine Kinase / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D019344:Lactic Acid / Q000494:pharmacology*; D009132:Muscles / Q000201:enzymology*; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D017433:Protein Structure, Secondary / Q000187:drug effects; D011817:Rabbits; D012965:Sodium Chloride / Q000494:pharmacology*; D013050:Spectrometry, Fluorescence
D019344:Lactic Acid; D012965:Sodium Chloride; D003402:Creatine Kinase