高压脉冲电场对HRP活性及其构象的影响和分子模拟其构象改变的研究

Effects of High Pulsed Electric Field on the Activity and Conformation of Horseradish Peroxidase and Studies on the Conformation Changes by Computer Molecular Imitate Technique

分别采用比色法、高效液相色谱法、红外光谱法、荧光光谱分析法以及分子模拟技术研究高压脉冲电场(PEF)对辣根过氧化物酶(HRP)活性及各级结构的影响,并对其构象变化进行分子模拟。分析结果表明,HRP的活性随电场强度和脉冲个数的增加而降低。当电场强度20kV/cm、20个脉冲数时,酶活下降15%;80个脉冲数时,酶活下降55%。PEF处理后HRP蛋白的一级结构未发生变化;二级结构中β-转角和无规卷曲向α-螺旋和β-折叠转化;三级结构发生变化,其荧光发生不同程度的淬灭,并在高电场条件下发生荧光峰的位移。HRP酶活性下降趋势与荧光淬灭趋势一致,呈非线性关系。两者变化过程都存在一个相似的临界场强(约10kV/cm)。对试验系统的分析表明,酶构象的改变与酶活性的变化存在密切的关系。

In the paper, colorimetry, high performance liquid chromatography, infraed spectroscopy, speetrofluorimetry and the computer molecular imitation method was adopted to analyse the effect of high pulsed electric field on the activity and conformation of horseradish peroxidase （HRP）. The results showed that the activity of HRP was decreased with the enhancing of electric field intensity and pulse numbers. When the electric field intensity was 20kV/cm, the activity of HRP was decreased 15% and 55% after the treatment by using of 20 and 80 pulses respectively. There was no change to the primary structure of HRP, hut changes occurred to the secondary structure of HRP in which the β-turn and non-regulation crimp transformed into α-helix and β-fold after the treatment of pulsed electric field. Besides, the fluorescence of HRP was quenched in different degrees based on the intensity of the pulsed electric field. The place of fluorescence intensity peak was changed with the enhancing of electric field intensity. Otherwise, both the HRP activity decline trend and fuorescence quenching trend were similar, the changes were nonlinear. There was a critical intensity for both the HRP activity decline and fluorescence quenching phenomena, which was at the same magnitude （about 10 kV/cm）. The whole experiment results indicated that the change of conformation was closely related to the inactivation of HRP.