XeCl准分子激光辐照对溶菌酶结构的影响

摘要

利用荧光光谱、SDS-PAGE和NMR方法,考察308nm XeCl准分子激光辐照对溶菌酶结构与活性的影响.使用能量密度为0.3 mJ/mm2的激光辐照溶菌酶,脉冲数分别为25、50、100、200、600、1200、1800、3600和7200.结果表明,用低强度激光辐照(低于200个脉冲)时,溶菌酶的活性出现增高趋势.随着激光辐照脉冲数的进一步增大,溶菌酶的活性又开始逐步降低.激光辐照处理后,溶菌酶的荧光强度发生了与生物活性相对应的先增高再降低现象,说明溶菌酶的高级结构发生了显著变化.SDS-PAGE结果显示,经激光辐照后,溶菌酶出现了分子间的聚合.分析溶菌酶的1H-NMR谱发现,辐照后,溶菌酶色氨酸(Trp) 111、Trp63和Trp62的化学位移发生了变化,此结果进一步说明,激光辐照使溶菌酶的高级结构发生了变化.该实验可为激光辐照诱导蛋白质去折叠的研究提供参考.%De-activative effect of lysozyme by using 308 nm XeCI excimer laser radiation was investigated. Fluorescence spectrophotometry; SDS-PAGE and NMR techniques were employed to determine the conformation change of the protein. Lysozyme was exposed at XeCI ecimer laser radiation (energy density was 0.3mJ/mm2) of 25; 50; 100; 200; 600; 1200; 1800; 3600 and 7200 pulse. Data showed that the activity of lysozyme was improved when the radiation pulse was less than 200. Whereas the lysozyme activity was negatively regulated by fluorescence intensity when the radiation pulse was more than 200. Data of SDS-PAGE showed that lysozyme aggregated obviously after radiation; indicating that lysozyme conformation changed remarkably during laser radiation.1H-NMR analysis indicated that lysozyme's tryptophan (Try) residue displayed chemical shift after radiation; which further confirmed that laser radiation results in lysozyme conformation change. These data might provide some underlying mechanism of protein unfolding induced by laser radiation.