羟基自由基氧化对牛血清白蛋白结构及水合特性的影响

Effects of Hydroxyl Radicals Oxidation on Structure and Hydration Properties of Bovine Serum Albumin

【目的】研究羟基自由基(·OH)氧化对蛋白结构及其水合特性的影响,探讨氧化介导的关于蛋白质和水分相互作用的变化情况。【方法】将牛血清白蛋白(bovine serum albumin,BSA)溶解在含15 mmol·L^(-1)的PIPES缓冲溶液中(pH 6.0),并在不同浓度H_2O_2(0、0.5、1.0、5.0、10.0和20.0 mmol·L^(-1))的铁-抗坏血酸-过氧化氢(FeCl_3-Vc-H_2O_2)羟基自由基氧化体系孵育12 h,采用氨基酸自动分析仪分析BSA不同程度氧化的氨基酸含量变化;通过测定羰基含量、巯基含量分析蛋白的氧化程度;通过测定BSA表面疏水性、离子键、氢键,考察BSA氧化不同程度后水合能力的变化情况;通过测定Zeta电位值来分析蛋白氧化后表面电荷的变化;采用傅里叶红外光谱法分析蛋白二级结构的变化。【结果】随着羟基自由基氧化体系中H_2O_2浓度的增加,氨基酸含量呈下降趋势,但不同种类氨基酸对·OH氧化敏感性不同,在1 mmol·L^(-1) H_2O_2处理浓度下,苏氨酸(Thr)、赖氨酸(Lys)含量显著降低,较对照组(未经H_2O_2处理组)分别下降了4.30%、4.22%;当H_2O_2浓度升高到20 mmol·L^(-1)时,较对照组蛋氨酸(Met)、酪氨酸(Tyr)、组氨酸(His)这3类氨基酸的含量下降程度最大,分别为35.32%、19.19%、11.15%,其次为异亮氨酸(Ile)、甘氨酸(Gly)、半胱氨酸(Cys)。随H_2O_2浓度的增加,BSA的羰基含量显著增加(P<0.05),20 mmol·L^(-1) H_2O_2处理组比对照组羰基含量升高了57.52%;巯基含量显著降低(P<0.05),20 mmol·L^(-1) H_2O_2处理组比对照组巯基含量降低了74.04%,蛋白氧化程度显著加剧;BSA经氧化处理后,蛋白二级结构发生变化,α-螺旋、无规卷曲含量显著降低(P<0.05),β-折叠、β-转角含量显著增加(P<0.05),多肽链中α-螺旋结构伸展变成线性结构,蛋白表面疏水性随着氧化程度的加剧而显著增加(P<0.05),蛋白持水力降低;同时氧化作用降低了蛋白表面的净电荷数,Zeta电位的绝对值随着羟基自由基氧化体系中H_2O_2浓度的增加而显著降低(P<0.05),离子键和氢键作用力显著减弱,降低了蛋白-水分子相互作用力,蛋白持水力降低。【结论】BSA在羟基自由基氧化体系中,存在显著的浓度效应,随着氧化程度加剧,蛋白表面的净电荷显著降低,离子键和氢键作用力显著减弱,蛋白二级结构发生了变化,蛋白-水分子相互作用力降低。

【Objective】The effects of oxidation on protein structure and its hydration properties were investigated.The purpose of this study was to explore the changes in protein and water interactions mediated by oxidation.【Method】Ferric ion-ascorbic acid-hydrogen peroxide（FeCl3-Vc-H2O2） hydroxyl radical oxidation system was used in this study.Bovine Serum Albumin（BSA） was suspended in 15 mmol·L（-1） piperazine-1,4-bisethanesulfonic acid（PIPES） buffer（pH 6.0） and incubated at 4℃ for 12 h with ferric ion（Fe3＋） and ascorbic acid（Vc） at different concentrations of hydrogen peroxide（0,0.5,1.0,5.0,10.0,and 20.0 mmol·L（-1） H2O2）.Amino acid content was measured by automatic amino acid analyzer.Carbonyl content and sulfhydryl content were detected to evaluate the degree of proteins oxidation.The changes of hydratability of BSA was evaluated by measuring the surface hydrophobicity,the content of ionic bond and the content of hydrogen bond.Electrochemical change rule under different oxidation levels was studied by measuring Zeta potential.In addition,the protein secondary structure was analyzed by Fourier Transform Infrared（FT-IR） spectroscopy.【Result】The content of amino acids in BSA declined with the increasing H2O2 concentration.However,the selective effects of ·OH on amino acids were observed.Compared to the control group（0 H2O2 treatment）,threonine（Thr） and lysine（Lys） decreased significantly by 4.30% and 4.22%,respectively,at 1 mmol·L（-1） of the H2O2 concentration.When the concentration of H2O2 increased to 20 mmol·L（-1）,the contents of methionine（Met）,tyrosine（Tyr） and histidine（His） showed the highest reduction,which was reduced by 35.32%,19.19% and 11.15%,respectively.Then the content of leucine（Ile）,glycine（Gly） and cysteine（Cys） also showed their higher reduction.It was observed that the carbonyl content increased significantly（P〈0.05） with increasing H2O2 concentration,while sulfhydryl content significantly declined（P〈0.05）,i.e.Carbonyl content in 20 mmol·L（-1）H2O2 treatment group（0.723 nmol·mg-1 protein） increased by 57.52% when it was compared to control（0.459 nmol·mg-1 protein）,and sulfhydryl content in 20 mmol·L（-1） H2O2 treatment group（19.853 nmol·mg-1 protein） decreased by 74.04% when it was compared to control（76.471 nmol·mg-1 protein）.Both indicated that BSA protein oxidation became more severe with the extension of H2O2 concentration.The result of secondary structure analysis indicated that α-helix,random coil decreased significantly with the increase of ·OH（P〈0.05）,while β-sheet,β-turn increased significantly（P〈0.05）,which demonstrates BSA oxidation induced the α-helix structure become a linear structure.The exposure of hydrophobic residues led to the significant increase of protein surface hydrophobicity（P〈0.05）.The absolute value of Zeta potential was significantly decreased（P〈0.05） with the increase of H2O2 concentration,which demonstrated that the protein oxidation could significantly affect the surface charge of protein.Chemical interactions results showed that hydrogen bond and ionic bond were weakened significantly（P〈0.05）,with reduction of interaction between protein and water molecules and the protein hydration capacity.【Conclusion】BSA oxidation is H2O2 concentration-dependent in hydroxyl radical oxidation system.Protein surface electrostatic charge decreased and protein secondary structure changed significantly as affected by protein oxidation,the interaction between protein and water molecules were reduced.