盐酸胍诱导的淀粉液化芽孢杆菌α-淀粉酶去折叠过程的研究  

Unfolding of Bacillus Amyloliquefaciens α-Amylase Induced by Guanidine Hydrochloride

作　　者：郑会娟[1] 边六交[1] 董发昕[2] 郑晓晖[1] 

机构地区：[1]西北大学生命科学学院,西安710069 [2]西北大学化学系,西安710069

出　　处：《化学学报》2009年第8期786-794,共9页Acta Chimica Sinica

基　　金：陕西省自然科学基金(No.2001K10-G3-(3))资助项目

摘　　要：分别用内源荧光光谱法、荧光相图法、荧光探针法、荧光猝灭法、蛋白质电泳法以及体积排阻色谱法研究了盐酸胍诱导的淀粉液化芽孢杆菌α-淀粉酶的去折叠过程.内源荧光光谱和荧光相图结果表明,当变性液中盐酸胍浓度约为1.0mol/L时,芽孢杆菌α-淀粉酶的去折叠过程中出现一个部分折叠中间体,其去折叠过程符合"三态模型";荧光探针结果表明,在溶液中盐酸胍浓度约为1.0mol/L时,中间态芽孢杆菌α-淀粉酶分子中存在着能够与探针分子1-苯胺基-8-萘磺酸(ANS)结合的稳定的疏水区域;荧光猝灭研究给出了不同程度变性的淀粉液化芽孢杆菌α-淀粉酶中的Trp的分布情况,结果表明中间态芽孢杆菌α-淀粉酶分子中能够被碘化钾猝灭的位于分子表面的色氨酸残基数目达到最大的8个;蛋白电泳和体积排阻色谱结果表明,在盐酸胍诱导的芽孢杆菌α-淀粉酶分子的整个去折叠过程中,不会以共价键或非共价键形式形成芽孢杆菌α-淀粉酶分子之间的集聚体或集聚体沉淀.在此基础上,对盐酸胍诱导的淀粉液化芽孢杆菌α-淀粉酶的去折叠过程进行了描述.The unfolding of Bacillus amyloliquefaciens α-amylase induced by guanidine hydrochloride was studied by using its intrinsic fluorescence spectroscopy, fluorescence phase diagram, fluorescence probe, fluorescence quenching, protein electrophoresis and chromatography. The results of its intrinsic fluorescence spectroscopy and fluorescence phase diagram showed that when the guanidine hydrochloride concentration in denaturation solution was about 1.0 mol/L, there existed a partially folded intermediate of Bacillus amyloliquefaciens α-amylase during its unfolding procedure, which followed a three-state model; the result of its fluorescence probe showed that when the guanidine hydrochloride concentration in denaturation solution was about 1.0 mol/L, there existed some stable hydrophobic regions, which could interact with a hydrophobic reagent 8-anilino-1-naphthalene sulfonic acid （ANS）, in the partially folded intermediate of Bacillus amyloliquefaciens α-amylase; and the results of fluorescence quenching using acrylamide and potassium iodide as quenchers showed the distribution of Trp residues in Bacillus amyloliquefaciens α-amylase in different denaturation solution, with the maximum number （8） of tryptophan residues in a partially folded intermediate Bacillus amyloliquefaciens α-amylase molecule could be quenched by potassium iodide; and the results of their protein electrophoresis and SEC showed that no aggregate or aggregate precipitation of Ba-cillus amyloliquefaciens α-amylase formed during the whole unfolding procedure of Bacillus amyloliquefaciens α-amylase induced by guanidine hydrochloride. And finally, a suggested unfolding procedure of Bacillus amyloliquefaciens α-amylase induced by guanidine hydrochloride was presented.

关 键 词：淀粉液化芽孢杆菌α-淀粉酶 去折叠 折叠中间态 盐酸胍 

分 类 号：Q51[生物学—生物化学]