Inactivation and conformational changes of creatine kinase at low concentrations of hexafluoroisopropanol solutions.
Using the methods of far-ultraviolet circular dichroism (CD) spectra, fluorescence spectra, and enzyme activity assays, the inactivation and conformational changes of creatine kinase (CK) induced by 1,1,1,3,3,3-hexafluoro-2-propanol (hexafluoroisopropanol (HFIP)) of different concentrations were investigated. To avoid the aggregation of CK that occurs with high HFIP, concentrations of 0%-5% HFIP were used in this study. The CD spectra showed that HFIP concentrations above 2.5% strongly induced the formation of secondary structures of CK. No marked conformational changes were observed at low concentrations of HFIP (0%-2.5%). After incubation with 0.2% HFIP for 10 min, CK lost most of its activity. The kinetic theory of the substrate reaction during irreversible inhibition of enzyme activity described previously by Tsou was applied to study the kinetics of CK inactivation during denaturation by HFIP. The inactivation rate constants for the free enzyme and the substrate-enzyme complex were determined by Tsou's method. The results suggested that low concentrations of HFIP had a high potential to induce helices of protein and that the active site of the enzyme was situated in a limited and flexible region of the enzyme molecule that was more susceptible to the denaturant than was the protein as a whole.
Wang
Xiao-Yun
XY
College of Life Science, Shandong Agricultural University, People's Republic of China.
Meng
Fan-Guo
FG
Zhou
Hai-Meng
HM
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000465:Algorithms; D000818:Animals; D020134:Catalytic Domain / Q000187:drug effects; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003401:Creatine / Q000737:chemistry / Q000378:metabolism; D003402:Creatine Kinase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D007700:Kinetics; D008956:Models, Chemical; D009132:Muscles / Q000201:enzymology; D009391:Nephelometry and Turbidimetry; D020005:Propanols / Q000737:chemistry* / Q000494:pharmacology; D011487:Protein Conformation; D011489:Protein Denaturation; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D006845:Hydrocarbons, Fluorinated
D000255:Adenosine Triphosphate; D003401:Creatine; D003402:Creatine Kinase; D004791:Enzyme Inhibitors; D020005:Propanols; C001337:hexafluoroisopropanol; D006845:Hydrocarbons, Fluorinated