有机汞化合物对木瓜蛋白酶的失活机理研究  

Study of the Inactivation Mechanism of Organomercury Compound on Papain

作　　者：刘学英 王帆 何东生 李敏 陈银燕 邓燕 马靓[1,2] LIU Xueying;WANG Fan;HE Dongsheng;LI Min;CHEN Yinyan;DENG Yan;MA Liang(College of Life Sciences and Chemistry,Hunan University of Technology,Zhuzhou Hunan 412007,China;Hunan Key Laboratory of Biomedical Nanomaterials and Devices,Hunan University of Technology,Zhuzhou Hunan 412007,China)

机构地区：[1]湖南工业大学生命科学与化学学院,湖南株洲412007 [2]湖南工业大学生物医用纳米材料与器件湖南省重点实验室,湖南株洲412007

出　　处：《包装学报》2019年第4期32-38,共7页Packaging Journal

基　　金：国家自然科学基金资助面上项目（61871180）;湖南省自然科学基金资助面上项目（2017JJ2069）;青年基金资助项目（2016JJ3053）;湖南省教育厅一般资助项目（YB2016B034,16C0470,18C0498）

摘　　要：复合食品包装材料残存的痕量重金属结合物严重威胁消费者健康。为了探明重金属结合物对生物大分子的影响及作用机理,研究了一定浓度的有机汞化合物(对氯汞苯甲酸,CMBA)使木瓜蛋白酶失活前后,该木瓜蛋白酶的结构变化。结果表明,经浓度为1×10^-4mol/L的CMBA处理后,木瓜蛋白酶的二级结构变化明显,其α-螺旋结构质量分数从43.4%急剧减少至4.2%,β-折叠、β-转角和无规卷曲质量分数从12.7%、16.0%和27.2%分别提高至40.0%、19.6%和36.2%,该酶二级结构成分由α+β型绝大部分转变为全β型。CMBA分子诱导木瓜蛋白酶L-链的α-螺旋结构趋向离散,构象松散,致使其天然构象遭到破坏,木瓜蛋白酶失活。经CMBA处理后的木瓜蛋白酶,其荧光光谱最大吸收峰红移7nm,荧光强度降低显著,这是由色氨酸(Trp)残基被包埋在极性亲水环境下引起的。分子对接结果表明CMBA结合到木瓜蛋白酶的疏水活性口袋内,可与其中的甘氨酸(Gly)66形成氢键,与Trp26形成疏水键,该结果与光谱分析结果相一致。Trace amounts of heavy metal bindings in composite food packaging materials pose a serious threat to the health of consumers. In order to elucidate the effect and mechanism of heavy metal bindings on biological macromolecules, the structural changes of papain were investigated when its activity was almost inactivated as treated by chlormercuric benzoic acid (CMBA) of certain concentration. The secondary structure of the papain treated by 10^-4 mol/L CMBA presented obvious changes. The content of α-helix of the treated papain decreased sharply from 43.4% to 4.2%, the content of β-sheet,β-turn and random coil increased from 12.7%, 16.0% and 27.2% to 40.0%, 19.6% and 36.2% respectively. The secondary structural components of the treated papain were almost transformed from α+β type to full β-type. The CMBA molecule induced the stretching of α-helix and structural dispersion together with conformation loosening, these all led to the destruction of the papain natural conformation and resulted in nearly complete inactivation of papain. The maximum fluorescence spectrum absorption peak of the treated papain showed a red shift of 7 nm and the intensity was reduced almost by a half, which might be caused by the embedding of tryptophan residues in the polar hydrophilic environment. The molecular modeling indicated that CMBA could bind with papain in the hydrophobic pocket and there was a hydrogen bond formed between CMBA and glycine (Gly) 66, while a hydrophobic bond formed between CMBA and tryptophan (Trp) 26 in the ligand-binding pocket, which was consistent with the results of spectral analysis results.

关 键 词：有机汞化合物 木瓜蛋白酶 失活机理 

分 类 号：Q518.4[生物学—生物化学]