绿原酸与蛋白质氨基酸残基共价互作效率研究

Study on covalent interaction efficiency between chlorogenic acid and protein amino acid residues

中文关键词:  绿原酸  氨基酸  共价互作  循环伏安  反应效率

英文关键词:chlorogenic acid  amino acid  covalent interaction  cyclic voltammetry  reaction efficiency

中文摘要:

      目的 探究绿原酸(chlorogenic acid, CGA)氧化形成的CGA醌与氨基酸侧链基团在不同pH条件下的反应效率, 为CGA与蛋白质共价互作反应位点的调控提供理论依据。方法 利用循环伏安法研究了酸性(pH 5.0)、中性(pH 7.0)和碱性(pH 8.0)条件下CGA与氨基酸侧链基团的反应效率, 采用超高效液相色谱-四极杆飞行时间串联质谱法(ultra performance liquid chromatography-quadrupole time of flight-tandem mass spectrometry, UPLC-QTOF-MS/MS)对反应产物进行结构鉴定。结果 在pH 7.0、扫描速率10 mV/s, 0.20 mmol/L CGA与10.00 mmol/L封闭α-氨基的氨基酸的反应效率依次为Nα-乙酰-L-半胱氨酸(~100.00%)≈Nα-乙酰-L-色氨酸(~100.00%)>Nα-乙酰-L-酪氨酸(39.20%±2.19%)>Nα-乙酰-L-赖氨酸(10.25%±0.83%)>Nα-boc-L-组氨酸(~0.00%)≈Nα-乙酰-L-精氨酸(~0.00%); 在其他反应条件相同的情况下, 缩小扫描电压范围至?0.4~0.4 V, CGA与Nα-乙酰-L-半胱氨酸、Nα-乙酰-L-色氨酸及Nα-乙酰-L-酪氨酸的共价互作效率分别为~100%、12.83%±1.16%及~0%; pH 7.0或8.0时, GCA与氨基酸残基的共价互作效率高于pH 5.0; CGA醌与Nα-乙酰-L-赖氨酸、Nα-乙酰-L-精氨酸的反应产物以氧化态的醌-氨基酸形式存在, 而CGA醌与Nα-乙酰-L-半胱氨酸、Nα-boc-L-组氨酸、Nα-乙酰-L-色氨酸的反应产物以还原态的酚-氨基酸形式存在。结论 CGA氧化形成的CGA醌可与蛋白质中半胱氨酸、色氨酸、赖氨酸、组氨酸及精氨酸残基发生反应, 其中半胱氨酸残基是CGA醌与蛋白质共价互作的主要位点, 可通过调整食品体系的pH和绿原酸醌浓度实现绿原酸-蛋白质共价互作位点的调控。

英文摘要:

      Objective To investigate the reaction efficiency of amino acid side chain groups and CGA quinone formed by oxidation of chlorogenic acid (CGA) under different pH conditions to provide a theoretical basis for the regulation of sites for CGA reaction with protein. Methods The reaction efficiency of CGA with amino acid side chain groups under acidic (pH 5.0), neutral (pH 7.0) and alkaline (pH 8.0) conditions was studied by cyclic voltammetry, and the structures of the reaction products were identified by ultra performance liquid chromatography-quadrupole time of flight-tandem mass spectrometry (UPLC-QTOF-MS/MS). Results At pH 7.0 and scan rate of 10 mV/s, the efficiency of 0.20 mmol/L CGA reaction with 10.00 mmol/L α-carbon amine group blocked amino acids followed the order: Nα-acetyl-L-cysteine (~100.00%)≈Nα-acetyl-L-tryptophan (~100.00%)> Nα-acetyl-L-tyrosine (39.20%±2.19%)>Nα-acetyl-L-lysine (10.25%±0.83%)>Nα-boc-L-histidine (~0.00%)≈Nα-acetyl-L-arginine (~0.00%). By narrowed down the scanning potential range to ?0.4~0.4 V, covalent interaction efficiencies of CGA with Nα-acetyl-L-cysteine, Nα-acetyl-L-tryptophan and Nα-acetyl-L-tyrosine were ~100%, 12.83%±1.16% and ~0%, respectively. The efficiency of covalent interaction efficiencies between GCA and amino acid residues was higher at pH 7.0 or 8.0 than at pH 5.0. The reaction products of CGA quinone with Nα-acetyl-L-lysine and Nα-acetyl-L-arginine existed as quinone-amino acids (oxidized state), while the reaction products of CGA quinone with Nα-acetyl-L-cysteine, Nα-boc-L-histidine, and Nα-acetyl-L-tryptophan existed as phenol-amino acids (reduced state). Conclusion CGA quinone formed by oxidation of CGA can react with cysteine, tryptophan, lysine, histidine and arginine residues in proteins, among which cysteine residues are the main sites of covalent interaction between CGA quinone and proteins, and the regulation of chlorogenic acid-protein covalent interaction sites can be achieved by adjusting the pH value and chlorogenic acid quinone concentration of food system.