STRUCTURAL AND FUNCTIONAL-STUDIES ON THE INTERACTION OF SODIUM DODECYL-SULFATE WITH BETA-GALACTOSIDASE
STRUCTURAL AND FUNCTIONAL-STUDIES ON THE INTERACTION OF SODIUM DODECYL-SULFATE WITH BETA-GALACTOSIDASE
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DOI：
10.1006/abbi.1993.1061
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发表时间：
1993-01-01
影响因子：
3.9
通讯作者：
BERNABEU, C
中科院分区：
文献类型：
--
作者：
MUGA, A;ARRONDO, JLR;BERNABEU, C
介绍了十二烷基硫酸钠 (SDS) 对大肠杆菌β-半乳糖苷酶的酶活性、电泳行为和构象的影响。傅里叶变换红外光谱 (FT-IR) 以前用于研究天然 β-半乳糖苷酶的结构，现已应用于检查洗涤剂对酶的影响。 20°C时，1% SDS的存在不会引起二级结构明显变化，酶活性得以保留；然而，10% SDS 会导致酶完全失活，并且 FT-IR 光谱表明构象会随之发生变化。 β-半乳糖苷酶的热变性在不存在 1% SDS 的情况下约 53°C 开始，在存在 1% SDS 的情况下约 46°C 开始热变性，表明三级结构发生变化；此外，还观察到结构（FT-IR）和功能（阿伦尼乌斯图）数据之间存在良好的相关性。热变性β-半乳糖苷酶的二级结构主要含有延伸结构，分子间相互作用产生蛋白质聚集。然而，在 10% SDS 存在的情况下，蛋白质的疏水片段被 SDS 稳定成螺旋结构，而不会发生蛋白质聚集。在 30°C、1% SDS 存在的情况下，通过凝胶电泳分离出两条蛋白质条带，其中只有一条具有活性。提出了 SDS-半乳糖苷酶相互作用的模型，根据该模型，在低表面活性剂浓度下，SDS 分子结合蛋白质的外表面，而不影响蛋白质核心。较高的去污剂浓度会产生较大的构象变化，包括酶失活和增加溶剂与蛋白质核心的可及性。在 10% SDS 存在的情况下升高温度会促进表面活性剂分子进入内部蛋白质区域并增加 β-半乳糖苷酶 α-螺旋含量。
The effect of sodium dodecyl sulfate (SDS) on enzyme activity, electrophoretic behavior, and conformation ofEscherichia coliβ-galactosidase is presented. Fourier-transform infrared spectroscopy (FT-IR), previously used to study the structure of native β-galactosidase has been applied to examine the detergent effects on the enzyme. At 20°C, the presence of 1% SDS does not cause appreciable changes in the secondary structure, and enzyme activity is preserved; however, 10% SDS produces complete enzyme inactivation and FT-IR spectroscopy indicates a concomitant change in conformation. Thermal denaturation of β-galactosidase starts at ≈53°C in the absence and at ≈46°C in the presence of 1% SDS, indicating tertiary structure changes; also, a good correlation between structural (FT-IR) and functional (Arrhenius plots) data is observed. The secondary structure of thermally denatured β-galactosidase contains mainly extended structures, and intermolecular interactions produce protein aggregation. In the presence of 10% SDS, however, the hydrophobic segments of the protein are stabilized by SDS into helical structures without protein aggregation. At 30°C, in the presence of 1% SDS, two protein bands are resolved by gel electrophoresis, only one of them being active. A model for SDS-galactosidase interaction is proposed, according to which, at low surfactant concentrations, SDS molecules bind the outer surface of the protein, without affecting the protein core. Higher detergent concentrations produce a larger conformational change involving enzyme inactivation and increased accessibility of the solvent to the protein core. Increasing temperature in the presence of 10% SDS leads to a facilitated access of surfactant molecules to the inner protein regions and to an increase of the β-galactosidase α-helical content.