Wang Xiao-Yun, Meng Fan-Guo, Zhou Hai-Meng
College of Life Science, Shandong Agricultural University, People's Republic of China.
Biochem Cell Biol. 2003 Oct;81(5):327-33. doi: 10.1139/o03-061.
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.
采用远紫外圆二色光谱（CD）、荧光光谱和酶活性测定等方法，研究了不同浓度的1,1,1,3,3,3-六氟-2-丙醇（六氟异丙醇，HFIP）诱导肌酸激酶（CK）的失活及构象变化。为避免高浓度HFIP导致CK聚集，本研究使用的HFIP浓度为0%-5%。CD光谱表明，HFIP浓度高于2.5%时会强烈诱导CK二级结构的形成。在低浓度HFIP（0%-2.5%）下未观察到明显的构象变化。用0.2% HFIP孵育10分钟后，CK失去了大部分活性。应用邹以前描述的酶活性不可逆抑制过程中底物反应的动力学理论，研究了HFIP变性过程中CK失活的动力学。用邹氏方法测定了游离酶和底物-酶复合物的失活速率常数。结果表明，低浓度的HFIP具有诱导蛋白质形成螺旋的高潜力，并且酶的活性位点位于酶分子中一个有限且灵活的区域，该区域比整个蛋白质更容易受到变性剂的影响。
