没食子酸与牛血红蛋白相互作用的研究
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摘要
牛血红蛋白(bovine hemoglobin,BHb)与人血红蛋白高度同源,且易获得,常用于血红蛋白与小分子化合物结合的研究。没食子酸(gallic acid,GA)作为一种多酚类小分子化合物,可被用作小分子药物模型,研究其与BHb的相互作用,可为其在医药领域的应用提供理论依据。运用紫外-可见光谱(UV-vis spectroscopy)、荧光光谱(fluorescence spectroscopy)、傅里叶变换红外(Fourier transform infrared,FTIR)光谱、圆二色光谱(circular dichroism,CD)等手段,在pH 7.0条件下,研究GA与BHb的相互作用。紫外-可见光谱的结果表明,GA对BHb的氧合状态没有影响,不会使氧合血红蛋白脱氧。结合荧光光谱和相关公式计算结果可知,GA与BHb相互作用发生了荧光猝灭,且猝灭常数随着温度的升高而增大,说明GA与BHb相互作用的猝灭类型为动态猝灭;通过计算得到了不同温度下GA与BHb相互作用的结合常数,分别为K_a~(298)=7.941×10~3L/mol,K_a~(308)=10.478×10~3L/mol; GA与BHb之间主要靠疏水作用力结合,可自发发生反应;色氨酸和酪氨酸残基所处微环境受到扰动,色氨酸残基的荧光吸收峰强度比酪氨酸残基变化大,表明GA与BHb分子的结合位点更接近于色氨酸残基。FTIR光谱和CD的检测结果显示,与GA作用前后,BHb均以α-螺旋结构为主,即GA对BHb分子的二级结构影响较小。
Bovine hemoglobin( BHb) is highly homologous to human hemoglobin and obtained easily,so it is often used in the study of binding of hemoglobin to small molecule compounds. Gallic acid( GA) is a polyphenolic small molecule compound,which can be used as drug model. The study on interaction between GA and BHb can provide a theoretical basis for the application of GA in the field of medicine. The interaction between GA and BHb was investigated at pH 7.0 by ultraviolet-visible( UV-vis) spectroscopy,fluorescence spectroscopy,Fourier transform infrared( FTIR) spectroscopy and circular dichroism( CD). The results of UV-vis spectroscopy showed that GA had no effect on oxygenation state of BHb,and did not deoxidize the oxygenated hemoglobin. Combined fluorescence spectra with calculation results of related formulas,it could be known that the interaction between GA and BHb resulted in fluorescence quenching,and the quenching constant increased with the increase of temperature,indicating that the quenching type of the interaction between GA and BHb was dynamic quenching. Then the binding constants of GA and BHb at different temperatures were calculated,which were K_a~(298)= 7. 941 × 10~3L/mol,K_a~(308)= 10. 478 × 10~3L/mol,respectively. And the binding process was a spontaneous molecular interaction procedure, in which hydrophobicinteractions played major roles. The results also demonstrated that the microenvironment of tryptophan and tyrosine residues had been disturbed,under the same condition,variations of fluorescence absorption peak intensity of the tryptophan residue were greater than that of the tyrosine residue,indicating that the binding site of GA and BHb was closer to the tryptophan residue. The results of FTIR spectroscopy and CD showed that the secondary structures before and after the interaction between BHb and GA were both dominated by α-helices,it meant that GA had little influence on the secondary structure of BHb molecules.
Bovine hemoglobin( BHb) is highly homologous to human hemoglobin and obtained easily,so it is often used in the study of binding of hemoglobin to small molecule compounds. Gallic acid( GA) is a polyphenolic small molecule compound,which can be used as drug model. The study on interaction between GA and BHb can provide a theoretical basis for the application of GA in the field of medicine. The interaction between GA and BHb was investigated at pH 7.0 by ultraviolet-visible( UV-vis) spectroscopy,fluorescence spectroscopy,Fourier transform infrared( FTIR) spectroscopy and circular dichroism( CD). The results of UV-vis spectroscopy showed that GA had no effect on oxygenation state of BHb,and did not deoxidize the oxygenated hemoglobin. Combined fluorescence spectra with calculation results of related formulas,it could be known that the interaction between GA and BHb resulted in fluorescence quenching,and the quenching constant increased with the increase of temperature,indicating that the quenching type of the interaction between GA and BHb was dynamic quenching. Then the binding constants of GA and BHb at different temperatures were calculated,which were K_a~(298)= 7. 941 × 10~3L/mol,K_a~(308)= 10. 478 × 10~3L/mol,respectively. And the binding process was a spontaneous molecular interaction procedure, in which hydrophobicinteractions played major roles. The results also demonstrated that the microenvironment of tryptophan and tyrosine residues had been disturbed,under the same condition,variations of fluorescence absorption peak intensity of the tryptophan residue were greater than that of the tyrosine residue,indicating that the binding site of GA and BHb was closer to the tryptophan residue. The results of FTIR spectroscopy and CD showed that the secondary structures before and after the interaction between BHb and GA were both dominated by α-helices,it meant that GA had little influence on the secondary structure of BHb molecules.
引文
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[24] Temboot P,Usman F,Ul-Haq Z,et al.. Biomolecular interactions of amphotericin B nanomicelles with serum albumins:A combined biophysical and molecular docking approach[J].Spectrochim. Acta A,2018,205:442-456.
[25] Wang C,Liu B,Bian G,et al.. Investigation on the interaction of glipizide with bovine hemoglobin by spectroscopy and molecular docking[J]. Spectrosc. Lett.,2017,50(9):476-481.
[26] Tunc S,Cetinkaya A,Duman O. Spectroscopic investigations of the interactions of tramadol hydrochloride and 5-azacytidine drugs with human serum albumin and human hemoglobin proteins[J]. J. Photoch. Photobio. B,2013,120:59-65.
[27] Yu X,Jiang B, Liao Z, et al.. Study on the interaction between 21-(Ph-NN)-NCTPP and bovine serum albumin by spectroscopic techniques[J]. Spectrochim. Acta A,2015,142:260-265.
[28] Zhou J,Wu X,Gu X,et al.. Spectroscopic studies on the interaction of hypocrellin A and hemoglobin[J]. Spectrochim.Acta A,2009,72:151-155.
[29] Bani-Yaseen A D. Spectrofluorimetric study on the interaction between antimicrobial drug sulfamethazine and bovine serum albumin[J]. J. Lumin.,2011,131(5):1042-1047.
[30] Azimipour S,Ghaedi S,Mehrabi Z,et al.. Heme degradation and iron release of hemoglobin and oxidative stress of lymphocyte cells in the presence of silica nanoparticles[J]. Int.J. Boil. Macromol.,2018,118:800-807.
[2]段纪俊,严亚琼,杨念念,等.中国恶性肿瘤发病与死亡的国际比较分析[J].中国医学前沿杂志,2016,8(7):17-23.
[3]张亮亮,汪咏梅,徐曼,等.植物单宁化学结构分析方法研究进展[J].林产化学与工业,2012,32(3):107-116.
[4] Demas J N,Addington J W. Luminescence quenching of dicyanobis(1,10-phenanthroline)ruthenium(II)by cupric ion in aqueous solutions. Dynamic and static processes[J]. J. Am.Chem. Soc.,1974,96(11):3663-3664.
[5]张雅丽,李建科,刘柳,等.五倍子没食子酸研究进展[J].食品工业科技,2013,34(10):386-390.
[6]柯发敏,张开莲.没食子酸的研究进展[J].泸州医学院学报,2011,34(4):440-442.
[7]梁爽.没食子酸抗肿瘤作用研究进展[J].广西医学,2017,39(7):1068-1072.
[8] Liu Y,Lin J,Chen M,et al.. Investigation on the interaction of the toxicant,gentian violet,with bovine hemoglobin[J].Food Chem. Toxicol.,2013,58:264-272.
[9] Shanmugaraj K,Anandakumar S,Ilanchelian M. Exploring the biophysical aspects and binding mechanism of thionine with bovine hemoglobin by optical spectroscopic and molecular docking methods[J]. J. Photoch. Photobio. B,2014,131:43-52.
[10] Chi Z,Liu R,Yang B,et al.. Toxic interaction mechanism between oxytetracycline and bovine hemoglobin[J]. J. Hazard.Mater.,2010,180(1-3):741-747.
[11] Xiao J B,Huo J L,Yang F,et al.. Noncovalent interaction of dietary polyphenols with bovine hemoglobin in vitro:Molecular structure/property-affinity relationship aspects[J]. J. Agric.Food Chem.,2011,59(15):8484-8490.
[12] Teng Y,Liu R,Yan S,et al.. Spectroscopic investigation on the toxicological interactions of 4-aminoantipyrine with bovine hemoglobin[J]. J. Fluoresc.,2010,20:381-387.
[13] Wang L,Liu R,Chi Z,et al.. Spectroscopic investigation on the toxic interactions of Ni2+with bovine hemoglobin[J]. Spectrochim. Acta A,2010,76(2):155-160.
[14] Yan X,Liu B,Chong B,et al.. Interaction of cefpiramide sodium with bovine hemoglobin and effect of the coexistent metal ion on the protein-drug association[J]. J. Lumin.,2013,142:155-162.
[15] Rashidipour S,Naeeminejad S,Chamani J. Study of the interaction between DNP and DIDS with human hemoglobin as binary and ternary systems:Spectroscopic and molecular modeling investigation[J]. J. Biomol. Struct. Dyn.,2016,34(1):57-77.
[16] Su Y, Qiu B, Chang C, et al.. Separation of bovine hemoglobin using novel magnetic molecular imprinted nanoparticles[J]. RSC Adv.,2018,8(11):6192-6199.
[17] Siddiqui G A,Siddiqi M K,Khan R H,et al.. Probing the binding of phenolic aldehyde vanillin with bovine serum albumin:Evidence from spectroscopic and docking approach[J].Spectrochim. Acta A,2018,203:40-47.
[18] Tang J,Yang C,Zhou L,et al.. Studies on the binding behavior of prodigiosin with bovine hemoglobin by multi-spectroscopic techniques[J]. Spectrochim. Acta A,2012,96:461-467.
[19]王欢,杨小玲.光谱法研究茜素黄GG与牛血清蛋白的相互作用[J].光谱实验室,2012,29(4):2291-2295.
[20] Chen T,Zhu S,Cao H,et al.. Studies on the interaction of salvianolic acid B with human hemoglobin by multispectroscopic techniques[J]. Spectrochim. Acta A,2011,78(4):1295-1301.
[21] Shi J,Wang J,Zhu Y,et al.. Characterization of interaction between isoliquiritigenin and bovine serum albumin:Spectroscopic and molecular docking methods[J]. J. Lumin.,2014,145:643-650.
[22]张红梅,王彦卿,于黎明,等.锌酞菁与牛血红蛋白相互作用的光谱研究[J].化学试剂,2008,30(2):85-88,146.
[23]周秋华,王彦卿,张红梅,等.单宁酸与牛血红蛋白相互作用的光谱研究[J].化学研究与应用,2008(7):816-820.
[24] Temboot P,Usman F,Ul-Haq Z,et al.. Biomolecular interactions of amphotericin B nanomicelles with serum albumins:A combined biophysical and molecular docking approach[J].Spectrochim. Acta A,2018,205:442-456.
[25] Wang C,Liu B,Bian G,et al.. Investigation on the interaction of glipizide with bovine hemoglobin by spectroscopy and molecular docking[J]. Spectrosc. Lett.,2017,50(9):476-481.
[26] Tunc S,Cetinkaya A,Duman O. Spectroscopic investigations of the interactions of tramadol hydrochloride and 5-azacytidine drugs with human serum albumin and human hemoglobin proteins[J]. J. Photoch. Photobio. B,2013,120:59-65.
[27] Yu X,Jiang B, Liao Z, et al.. Study on the interaction between 21-(Ph-NN)-NCTPP and bovine serum albumin by spectroscopic techniques[J]. Spectrochim. Acta A,2015,142:260-265.
[28] Zhou J,Wu X,Gu X,et al.. Spectroscopic studies on the interaction of hypocrellin A and hemoglobin[J]. Spectrochim.Acta A,2009,72:151-155.
[29] Bani-Yaseen A D. Spectrofluorimetric study on the interaction between antimicrobial drug sulfamethazine and bovine serum albumin[J]. J. Lumin.,2011,131(5):1042-1047.
[30] Azimipour S,Ghaedi S,Mehrabi Z,et al.. Heme degradation and iron release of hemoglobin and oxidative stress of lymphocyte cells in the presence of silica nanoparticles[J]. Int.J. Boil. Macromol.,2018,118:800-807.