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

Determination of superoxide dismutase activity in methanobactin-copper complexes by pyrogallol autoxidation

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-Na2 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-Na2 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 O2^-., and the optimal reaction system suitable for the analysis conditions are established in this article.