羟基自由基氧化对牛血清白蛋白结构及水合特性的影响

doi:10.3864/j.issn.0578-1752.2017.15.015

Abstract

Abstract: 【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^-1H₂O₂). 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 H₂O₂concentration. 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 thatα-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 H₂O₂concentration-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.

Key words: Bovine Serum Albumin (BSA), hydroxyl radical (·OH) oxidizing system, protein structure, hydration properties

WANG Ce, LI Xia, DENG ShaoYing, WANG Hang, ZHANG ChunHui. Effects of Hydroxyl Radicals Oxidation on Structure and Hydration Properties of Bovine Serum Albumin[J].Scientia Agricultura Sinica, 2017, 50(15): 3013-3023.

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