邻苯三酚自氧化法测定甲烷氧化菌素-铜配合物的超氧化物歧化酶活性
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摘要
目的建立甲烷氧化菌素(methanobactin,Mb)-铜配合物(Mb-Cu)模拟超氧化物歧化酶(superoxide dismutase,SOD)的邻苯三酚自氧化体系分析方法。方法利用HP 20大孔树脂从甲基弯菌IMV301l发酵液中分离纯化得到Mb,制备Mb-Cu测定其具有的SOD活性,利用紫外光谱分析对SOD邻苯三酚测活法进行系统研究。考察缓冲液浓度、邻苯三酚浓度、Mb-Cu浓度、EDTA-Na_2浓度、pH值和温度等对Mb-Cu模拟SOD活性的影响。结果在Mb-Cu浓度为0.8 mg/L,Tris-HAC缓冲溶液为0.05 mol/L(其中缓冲溶液的pH值为8.2、EDTA-Na_2的浓度为2 mmol/L),邻苯三酚浓度为0.363 mmol/L,温度为20℃时,邻苯三酚的自氧化速率相对较低,此时Mb-Cu对超氧阴离子自由基(O_2~-.)的抑制率较高,以此建立Mb-Cu模拟SOD活性的邻苯三酚自氧化分析方法。发现Mb-Cu符合影响生物催化剂条件的一般规律,但比生物酶具有更高的热稳定性。结论Mb-Cu可作为清除O_2~-.的SOD模拟酶,本试验建立的分析条件是最佳反应体系。
Objective To establish a method for determination of superoxide dismutase(SOD) activity in methanobactin(Mb)-copper complex(Mb-Cu) by pyrogallol autoxidation system. Methods Mb was isolated and purified by macroporous resin HP20 from Methylosinus trichosporium IMV3011 fermented fluid. Mb-Cu was prepared and its SOD activity was determined with the pyrogallol autoxidation system by ultraviolet spectrum analysis, systematically. The effects of buffer concentration, pyrogallol concentration, Mb-Cu concentration, EDTA-Na_2 concentration, pH value and temperature on the mimic of SOD were investigated. Results When the concentration of Mb-Cu was 0.8 mg/L, concentration of Tris-HAC buffer was 0.05 mol/L(the buffer pH value was 8.2 and the concentration of EDTA-Na_2 was 2 mmol/L), pyrogallol concentration was 0.363 mmol/L under temperature of 20 ℃, the autoxidation rate of pyrogallol was relatively low and the inhibition rate on superoxide anion radical was higher. The method for determination of mimic of SOD of Mb-Cu based on the pyrogallol autoxidation system was established. It was found that Mb-Cu was in accordance with the general rule of the biocatalyst conditions influenced, but had higher thermal stability than other biological enzymes. Conclusion Mb-Cu can be used as mimic of SOD for scavenging O_2~-., and the optimal reaction system suitable for the analysis conditions are established in this article.
Objective To establish a method for determination of superoxide dismutase(SOD) activity in methanobactin(Mb)-copper complex(Mb-Cu) by pyrogallol autoxidation system. Methods Mb was isolated and purified by macroporous resin HP20 from Methylosinus trichosporium IMV3011 fermented fluid. Mb-Cu was prepared and its SOD activity was determined with the pyrogallol autoxidation system by ultraviolet spectrum analysis, systematically. The effects of buffer concentration, pyrogallol concentration, Mb-Cu concentration, EDTA-Na_2 concentration, pH value and temperature on the mimic of SOD were investigated. Results When the concentration of Mb-Cu was 0.8 mg/L, concentration of Tris-HAC buffer was 0.05 mol/L(the buffer pH value was 8.2 and the concentration of EDTA-Na_2 was 2 mmol/L), pyrogallol concentration was 0.363 mmol/L under temperature of 20 ℃, the autoxidation rate of pyrogallol was relatively low and the inhibition rate on superoxide anion radical was higher. The method for determination of mimic of SOD of Mb-Cu based on the pyrogallol autoxidation system was established. It was found that Mb-Cu was in accordance with the general rule of the biocatalyst conditions influenced, but had higher thermal stability than other biological enzymes. Conclusion Mb-Cu can be used as mimic of SOD for scavenging O_2~-., and the optimal reaction system suitable for the analysis conditions are established in this article.
引文
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[16]张锁慧,王改香,唐晓军,等.邻苯三酚自氧化法测定超氧化物歧化酶活性研究[C].第十届中国化妆品学术研讨会论文集,2014:260-264.Zhang SH,Wang GX,Tang XJ,et al.Study of pyrogallol autoxidation method to determine SOD activity[C].Proceedings of the Tenth Symposium on Chinese Cosmetics,2014:260-264.
[17]王炳娟,李玲霞,邹洪.邻苯三酚自氧化法测定SOD活性的电化学研究[J].现代仪器,2008,2:29-30,38.Wang BJ,Li LX,Zou H.Determination the activity of superoxide dismutase enzyme by electrochemical method based on the reaction of pyrogallol autoxidation[J].Mod Instrum,2008,2:29-30,38.
[18]杨磊.厚朴提取物抗氧化药物动力学研究[D].保定:河北农业大学,2008.Yang L.The pharmacokinetic study of antioxidation of extracts of magnolia offieinalis[J].Baoding:Agricultural University of Hebei,2008.
[19]周叶峰.甲烷氧化菌的分离及氧化活性的影响因子研究[D].长沙:湖南农业大学,2008.Zhou YF.Studies on the influential factors of oxidation aetivities and isolation of methane oxidizing bacteria[J].Changsha:Agricultural University of Hunan,2008.
[2]陈佳乐,高飞,易喻,等.超氧化物歧化酶化学修饰的研究进展及展望[J].浙江化工,2008,39(7):22-26,32.Chen JL,Gao F,Yi Y,et al.The Development of superoxide dismutaseschemical modification[J].Zhejiang Chem Ind,2008,39(7):22-26,32.
[3]殷晓春,李刚,王荣民,等.金属卟啉白蛋白结合体模拟SOD酶性能研究[J].中国科学(化学),2013,43(2):171-177.Duan XC,Li G,Wang RM,et al.Antioxidant activity of conjugation of metalloporphyrin combining with bovine serum albumin[J].Sci Sin(Chim),2013,43(2):171-177.
[4]李晨,杨征,厍梦尧,等.超氧化物歧化酶化学模拟的新进展[J].高等学校化学学报,2011,32(9):2046-2061.Li C,Yang Z,Ku MY,et al.Recent progress in superoxide dismutase mimics[J].Chem J Chin Univ,2011,32(9):2046-2061.
[5]袁泽利,吴庆,杨兴变,等.新型水溶性大环席夫碱锰(Ⅱ)配合物的合成及其模拟超氧化物歧化酶的活性研究[J].合成化学,2011,19(4):446-449.Yuan ZL,Wu Q,Yang XB,et al.Synthesis and activity study of sod novel water-soluble schiff base macrocycic Mn(Ⅱ)compounds[J].Chin J Synth Chem,2011,19(4):446-449.
[6]李唤民,高楠,刘磊,等.新型兼具GPX,SOD,CAT活性的水杨醛Schiff碱衍生物的合成[J].高等学校化学学报,2009,30(7):1353-1356.Li HM,Gao N,Liu L,et al.Novel trifunctional salicylaldehyde schiff base derivative with GPX,SOD and CAT activities[J].Chem J Chin Univ,2009,30(7):1353-1356.
[7]Choi DW,Zea CJ,Do YS,et al.Spectral,kinetic and thermodynamic properties of Cu(Ⅰ)and Cu(II)binding by methanobactin from methylosinus trichosporium OB3b[J].Biochemistry,2006,45(5):1442-1453.
[8]Kim HJ,Graham DW,Di Spir Uo AA,et al.Mehanobactin,a copper-acquisition compound from mettiane-oxidizing bacteria[J].Science,2004,305(5690):1612-1615.
[9]Choi DW,Semrau JD,Antholine WE,et al,Oxidase,superoxide dismutase,and hydrogen peroxide reductase activities of methanobactin from types I and II methanotrophs[J].J Inorg Biochem,2008,102(8):1571-1580.
[10]Xin JY,Zhang YX,Dong J,et al,Production,purification and SOD-like activity of methanobactin from M.trichosporium 3011[J].Ad Mater Res,2011,160-162:19-23.
[11]Vincent CHY,Kenkel I,Delsuc N,et al.Bioinspired superoxide-dismutase mimics:The effects of functionalization with cationic polyarginine peptides[J].J Inorg Biochem,2016,160:172-179.
[12]Shearer J.Dioxygen and superoxide stability of metallopeptide based mimics of vs.bis-amidate ligation nickel containing superoxide dismutase:The influence of amine/amidate[J].J Inorg Biochem,2013,129:145-149.
[13]邢海丽.甲烧氧化菌素抗菌性的研究[D].哈尔滨:哈尔滨商业大学,2016.Xing HL.Antibacterial activity of methanobactin from Methylosinus trichosporium 3011[D].Harbin:Harbin University of Commerce,2016.
[14]闫超泽,张帅,辛嘉英,等.铬天青S分光光度法测定甲烷氧化菌素的研究[J].分析测试技术与仪器,2011,17(2):69-73.Yan CZ,Zhang S,Xin JY,et al.Study on determination of methanobactin by spectrophotometry using chrome azure S[J].Anal Test Technol Instrum,2011,17(2):69-73.
[15]程德竹,杜爱玲,李成帅,等.生姜提取物对邻苯三酚自氧化生成超氧自由基的清除[J].中国调味品,2014,39(11):35-39.Cheng DZ,Du AL,Li CS,et al.Antioxidant capacity of ginger active ingredients by pyrogallol auto-oxidation action[J].China Condiment,2014,39(11):35-39.
[16]张锁慧,王改香,唐晓军,等.邻苯三酚自氧化法测定超氧化物歧化酶活性研究[C].第十届中国化妆品学术研讨会论文集,2014:260-264.Zhang SH,Wang GX,Tang XJ,et al.Study of pyrogallol autoxidation method to determine SOD activity[C].Proceedings of the Tenth Symposium on Chinese Cosmetics,2014:260-264.
[17]王炳娟,李玲霞,邹洪.邻苯三酚自氧化法测定SOD活性的电化学研究[J].现代仪器,2008,2:29-30,38.Wang BJ,Li LX,Zou H.Determination the activity of superoxide dismutase enzyme by electrochemical method based on the reaction of pyrogallol autoxidation[J].Mod Instrum,2008,2:29-30,38.
[18]杨磊.厚朴提取物抗氧化药物动力学研究[D].保定:河北农业大学,2008.Yang L.The pharmacokinetic study of antioxidation of extracts of magnolia offieinalis[J].Baoding:Agricultural University of Hebei,2008.
[19]周叶峰.甲烷氧化菌的分离及氧化活性的影响因子研究[D].长沙:湖南农业大学,2008.Zhou YF.Studies on the influential factors of oxidation aetivities and isolation of methane oxidizing bacteria[J].Changsha:Agricultural University of Hunan,2008.