H_2O_2氧化对肉牛血红素蛋白构象与氧化性质的影响
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摘要
该研究采用36月龄公牛背最长肌,提取血红素蛋白,并对蛋白质溶液建立H_2O_2氧化体系模拟体内氧化条件。采用激光拉曼光谱、傅里叶变换红外光谱分析H_2O_2处理对血红素蛋白的构象的影响,并考察其对二硫键、NO自由基和超氧化物歧化酶(superoxide dismutase,SOD)等氧化性质的影响。结果表明,血红素蛋白在使用H_2O_2氧化后,血红素卟啉环结构整体扩张,Fe原子接近卟啉环平面,α-螺旋含量下降7. 37%、β-折叠含量上升8. 77%、β-转角含量上升12. 03%、无规则卷曲含量下降9. 66%。二硫键浓度上升31. 791μmol/g,NO自由基浓度增加7. 078μmol/g,SOD活性略微上升后显著降低至11. 642 U/mg。得出H_2O_2氧化使血红素蛋白的卟啉环结构整体扩张,二级结构发生改变,进而使得二硫键含量升高,NO自由基含量上升,SOD含量降低。
In order to study the changes in the structure and oxidative properties of hemeprotein before and after oxidation,longissimus dorsi muscle from a 36-month-old ox was used to extract hemeprotein. The effects of H2 O_2 treatment on its conformation were analyzed by laser raman spectroscopy and fourier transform infrared spectroscopy( FTIR). Besides,effects of H2 O_2 on disulfide bonds,NO free radicals,and oxidative properties of superoxide dismutase( SOD) were investigated. The results showed that after oxidizing the hemeprotein with H2 O_2,the heme porphyrin ring structure was completely expanded. Moreover,the Fe atom was close to the surface of porphyrin ring,the contents of α-helix and random coil decreased 7. 37% and 9. 66%,respectively. The contents of β-fold and β-rotation angle increased 8. 77% and 12. 03%,respectively. Furthermore,the contents of disulfide bonds increased31. 791 μmol/g,and NO free radicals increased 7. 078 μmol/g. The SOD activity decreased significantly to 11. 642 U/mg after a slight increase. The results revealed that H2 O_2 oxidation resulted in a complete expansion of porphyrin ring structure in hemeprotein as well as changes in the secondary structure. Consequently,the contents of disulfide bonds and NO free radicals in hemeprotein increased,while the amount of SOD decreased.
In order to study the changes in the structure and oxidative properties of hemeprotein before and after oxidation,longissimus dorsi muscle from a 36-month-old ox was used to extract hemeprotein. The effects of H2 O_2 treatment on its conformation were analyzed by laser raman spectroscopy and fourier transform infrared spectroscopy( FTIR). Besides,effects of H2 O_2 on disulfide bonds,NO free radicals,and oxidative properties of superoxide dismutase( SOD) were investigated. The results showed that after oxidizing the hemeprotein with H2 O_2,the heme porphyrin ring structure was completely expanded. Moreover,the Fe atom was close to the surface of porphyrin ring,the contents of α-helix and random coil decreased 7. 37% and 9. 66%,respectively. The contents of β-fold and β-rotation angle increased 8. 77% and 12. 03%,respectively. Furthermore,the contents of disulfide bonds increased31. 791 μmol/g,and NO free radicals increased 7. 078 μmol/g. The SOD activity decreased significantly to 11. 642 U/mg after a slight increase. The results revealed that H2 O_2 oxidation resulted in a complete expansion of porphyrin ring structure in hemeprotein as well as changes in the secondary structure. Consequently,the contents of disulfide bonds and NO free radicals in hemeprotein increased,while the amount of SOD decreased.
引文
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[2]李向荣.抗氧化剂和自由基与血清白蛋白相互作用的微量热和谱学研究[D].新乡:河南师范大学,2014.
[3]向黎翎子.基于互信息对溶菌酶氨基酸主链二面角与侧链二面角之间关系的研究[D].长春:吉林大学,2017.
[4]周非白.氧化修饰对猪肉肌原纤维蛋白结构与功能特性的调控研究[D].广州:华南理工大学,2016.
[5]岳宏.中国肉牛产业可持续发展研究[D].长春:吉林农业大学,2011.
[6]沈月新,宋立华.冷却肉与热鲜肉、冻肉食用质量的比较研究[J].制冷技术,2000(3):43-45.
[7]刘燕德,刘涛,孙旭东,等.拉曼光谱技术在食品质量安全检测中的应用[J].光谱学与光谱分析,2010,30(11):3 007-3 012.
[8]高铮亚.水溶液中蛋白质二级结构红外分析方法的建立及c AMP受体蛋白构象研究[D].上海:复旦大学,2012.
[9]宋江宁.蛋白质二硫键结构特征与序列关系的生物信息学研究[D].无锡:江南大学,2005.
[10]王宁,王晓拓,丁武,等.辐照剂量率对牛肉脂肪和蛋白氧化及蛋白特性的影响[J].现代食品科技,2015,31(8):122-129.
[11]葛巍巍,陈静,宗良,等.氯化血红素催化氧化巯基形成二硫键[J].高等学校化学学报,2017,38(6):1 052-1 058.
[12]刘翔宇,王丽琼,吉文欣,等.几种氮氧自由基结构和性质的理论计算[J].武汉理工大学学报,2012,34(8):45-49.
[13]李华,曾勇庆,魏述东,等.猪宰后肌肉SOD与MDA的变化及其对肉质特性的影响[J].畜牧兽医学报,2010,41(3):257-261.
[14] LU X,ZHAO D,SU Z. Purification of hemoglobin by ion exchange chromatography in flow-through mode with PEG as an escort[J]. Artif Cells Blood Substit Immobil Biotechnol,2004,32(2):209-227.
[15]王丽丽.紫丁香叶多酚物质的提取及抗氧化性能研究[D].哈尔滨:东北农业大学,2007.
[16]涂宗财,谢欢,钮培佩,等.60Co辐照对卵清蛋白结构和抗氧化性的影响[J].食品工业科技,2016,37(8).
[17] LU M,ZHAO L,WANG Y,et al. Measurement of the methemoglobin concentration using Raman spectroscopy[J]. Artificial Cells Nanomedicine&Biotechnology,2013,42(1):63-69.
[18] PRIM D,CRELIER S,SEGYRA J M. Coupling of a microfluidic mixer to a Fourier-transform infrared spectrometer for protein-conformation studies[J]. Chimia,2011,65(10):815-816.
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[21] THANNHAUSER T W,KONISHI Y,SCHERAGA H A.Sensitive quantitative analysis of disulfide bonds in polypeptides and proteins[J]. Analytical Biochemistry,1984,138(1):181-188.
[22]沈高山,谷怀民,闫天秀,等.亚硝酸钠和氧合血红蛋白反应的拉曼光谱[J].中国激光,2008,35(9):1 432-1 436.
[23] WOOD B R,CASPERS P,PUPPELS G J,et al. Resonance Raman spectroscopy of red blood cells using nearinfrared laser excitation[J]. Analytical&Bioanalytical Chemistry,2007,387(5):1 691-1 703.
[24]张平丽,孟耀勇,肖军,等.显微拉曼光谱法定量检测碳氧血红蛋白饱和度[J].科学通报,2012,57(14):1 222-1 226.
[25] CAMMARATA M,LEVANTINO M,WULFF M,et al.Unveiling the timescale of the R-T transition in human hemoglobin[J]. Journal of Molecular Biology,2010,400(5):951-962.
[26] RIMAI L,SALMEEN I,PETERING D H. Comparison of the resonance Raman spectra of carbon monoxy and oxy hemoglobin and myoglobin:similarities and differences in heme electron distribution[J]. Biochemistry,1975,14(2):378-382.
[27]杨玉玲,游远,彭晓蓓,等.加热对鸡胸肉肌原纤维蛋白结构与凝胶特性的影响[J].中国农业科学,2014,47(10):2 013-2 020.
[28] ABOTT N B,ELLIOTT A. Infra-red spectrum and dichroism of crystalline acetanilide[J]. Proceedings of the Royal Society of London,1956,234(1197):247-268.
[29]赵妍,刘晓林,王若兰,等.储藏微环境对小麦蛋白质二级结构影响[J].粮食与油脂,2014(1):36-38.
[30]孙艳美.氨基酸构建肽链的转角结构研究[D].石家庄:河北科技大学,2016.
[31]王策,李侠,邓少颖,等.羟基自由基氧化对牛血清白蛋白结构及水合特性的影响[J].中国农业科学,2017,50(15):3 013-3 023.
[32]牛付阁.卵白蛋白—阿拉伯胶相互作用及其在油水界面的吸附特性[D].无锡:江南大学,2015.
[33]阮奇珺.大豆蛋白热诱导二硫键连接物的形成及巯基变化亚基水平研究[D].无锡:江南大学,2015.
[34]李学鹏,周凯,周明言,等.自由基氧化对大黄鱼肌原纤维蛋白交联和聚集的影响[J].中国食品学报,2015,15(4):13-21.
[35]陈秀敏,吕秀菊.魔芋葡甘露寡糖的制备、化学结构及对胰岛NO自由基释放量的影响[J].中国生物化学与分子生物学报,2002,18(6):741-745.
[36]孟宇竹,雷昌贵,王霞,等. SOD抗氧化作用及其在食品工业中的应用[J].中国食品添加剂,2009(1):111-114.
[37]王琨,叶继丹,常建波.镉对牙鲆幼鱼肝中超氧化物歧化酶活性及脂质过氧化物含量的影响[C].中国水产学会学术年会暨水产微生态调控技术论坛,2007.