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邻苯二甲酸二丁酯与胰蛋白酶的相互作用

ISSN号：1006-0464
期刊名称：《南昌大学学报：理科版》
时间：0
分类：O657.3[理学—分析化学;理学—化学]
作者机构：[1]南昌大学食品科学与技术国家重点实验室,江西南昌330047
相关基金：国家自然科学基金资助项目（21167013,31460422）; 高等学校博士点基金项目（20123601110005）; 江西省自然科学基金资助项目（20142BAB204001,20143ACB20006）; 江西省科技支撑项目（20141BBG70092）; 食品科学与技术国家重点实验室基金项目（SKLF-ZZB-201305,SKLF-ZZA-201302）

关键词：邻苯二甲酸二丁酯, 胰蛋白酶, 结合特性, 分子模拟, dibutyl phosphate , trypsin , binding characteristic , molecular modeling

中文摘要：

在模拟人体生理条件下（pH值为7.4）,应用荧光光谱法、紫外光谱法和圆二色谱法（CD）并结合原子力显微镜（AFM）和分子模拟技术,研究了邻苯二甲酸二丁酯（DBP）对胰蛋白酶（Trypsin）的光谱性质、结构及催化活性的影响.结果表明,DBP通过氢键和范德华力与胰蛋白酶形成基态复合物而猝灭胰蛋白酶的内源荧光,DBP在胰蛋白酶上只有1个结合位点.同步荧光、紫外和CD光谱研究发现,DBP与胰蛋白酶的结合诱导了酶的α-螺旋、β-折叠和β-转角含量的减少,而增加了无规卷曲的含量.原子力显微镜图像显示,DBP的存在引起胰蛋白酶的表面形态发生变化,蛋白质发生了聚集.分子模拟结果表明,DBP结合于胰蛋白酶S1疏水空腔附近,与氨基酸His 57,Ser 195和Gly 193形成氢键.酶活测定结果显示,DBP的存在导致胰蛋白酶活性被抑制.

英文摘要：

The effects of plasticizer dibutyl phosphate （DBP） on the spectral properties,structure and cat- alytic activity of trypsin were investigated using fluorescence, UV-vis absorption and circular dichroism （CD） spectroscopy along with atomic force microscopy （AFM） and molecular simulation under simula- tive physiological conditions （pH 7.4）. The result of fluorescence quenching indicated that a ground state complex was formed between DBP and trypsin through hydrogen bonds and Van Der Waals forces,resul- ting in the intrinsic fluorescence quenching of trypsin. There was a single class of binding sites on trypsin for DBP. Analysis of synchronous fluorescence, UV-vis absorption and CD spectra demonstrated that the addition of DBP led to the conformational alteration of trypsin, decreases in the a-helix, ~-sheet and ~-turn contents and an increase in the random coil content. The AFM topography image showed that the binding of DBP with trypsin caused the surface morphology change of trypsin and the protein aggregation. The molecular modeling results exhibited that the binding site of DBP on trypsin was adjacent to the S1 bind- ing pocket,and three hydrogen bonds formed between the amino acid residues His 57, Ser 195 and Gly 193 of trypsin and the oxygen atom of DBP. The enzymatic activity assay indicated that the binding inter- action led to the inhibition of the trypsin activity.

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