邻苯二甲醛修饰法探测中华猕猴桃蛋白酶在胍溶液中活性部位的构象变化

Palczewsski等［1］以邻苯二甲醛修饰醛缩酶活性部位的氨基和流基以形成一异蚓噪环，利用该基团的荧光特性来探测醛缩酶的活性部位构象，Weq［2］，Le［3］并成功地运用这一方法研究肌酸激酶和酵母乙醇脱氢酶的活性部位构象变化．中华猕猴桃蛋白酶的唯一游离流基（CyS-25）是催化功能团【'」，而且氨基也是活性部位的必需基因【到，符合邻苯二甲醛的反应性，所以我们借鉴Pal＿ski等的方法【1］将这一荧光基因引人中华猕猴桃蛋白酶，用以探测该酶在抓溶液中活性部位的构象变化，并与相应的活力变化以及酶的内源荧光及CD谱变化作比较．1材…

Detection of Conformational Changes at the Active Site of Actinidin in GuHCl Solutions by o-Phthaldehyde Modification

The thiol group and amino group at the active site of actinidin were modified by o-phthaldehyde to form a fluorescence group with excitation and emission wavelength maxima at 345 um and 416 nm respectively. The fluorescence group was then used to probe the conformational changes of the modified enzyme in guanidine hydrochloride (GuHCl) solution. Results were compared with the changes of activity as well as fluorescence and CD spectra of unmodified actinidin in GuHCl sloutions. It is shown that the conformational changes of the enayme active site parallel the enzyme inactivation, and both of them precede the conformatvonal changes of the enzyme as a whole revealed by fluorescence and CD spectra.