为了验证邻苯三酚自氧化法的准确性和实用性,对邻苯三酚在Tris-HCl和磷酸盐2种不同反应体系(缓冲液)中的自氧化产物进行UV-Vis吸收光谱扫描, 确定其最适吸收峰; 同时研究了tcv Cu,ZnSOD(简称tcvSOD)和Bovine Cu,Zn-SOD(简称Bovine SOD)分别在2种缓冲液中对邻苯三酚自氧化反应的抑制模式. 波长扫描显示, 终浓度为0.225 mmol/L的邻苯三酚在50 mmol/L Tris-HCl pH 8.2缓冲液中于320 nm处30 s出现明显吸收峰, 30 min吸收峰达到最高值; 30 min 后440 nm附近出现吸收峰; A320处的自氧化速率为0.139/min. 同样浓度邻苯三酚在0.2 mol/L pH8.0磷酸缓冲液中除了A320的自氧化速率稍高(A320 0.152/min)外, 其他性质与50 mmol/L Tris-HCl相同. tcvSOD和Bovine SOD在Tris-HCl缓冲液中对邻苯三酚自氧化反应的抑制作用较磷酸盐缓冲液为强.研究结果确认320 nm可作为检测邻苯三酚自氧化、SOD对其产生抑制及SOD活性间接测定的特定波长.
To verify the accuracy and feasibility of the pyrogallol autoxidation, the UV-Vis absorption spectra of autoxidation products of pyrogallol were scanned systematically in Tris-HCl and phosphate buffers for optimal wavelength determination. The inhibition modes of tcv Cu,Zn-SOD and Bovine Cu,Zn-SOD to pyrogallol autoxidation were characterized in the two buffers. The autoxidation products showed a quickly increased absorption at 320 nm in 30 s, and reached to the maxima in 30 min in a solution of 0.225 mmol/L pyrogallol in 50 mmol/L Tris-HCl, pH8.2; a weak absorption at 440 nm was also observed 30 min after the reaction started; and the rate of autoxidation of pyrogallol was A320 0.139/min.The- DOI:10.3969/j.issn.1000-1565.2018.03.009邻苯三酚自氧化法在SOD活性测定中的应用贾红玉1,王亚森1,田晓辉1,张伟1,杜建芳2,康明1,刘建荣2(1. 河北大学 生命科学学院, 河北省微生物多样性研究与应用实验室,河北 保定 071002;2. 河北大学 生物技术研究中心,河北 保定 071002)摘 要:为了验证邻苯三酚自氧化法的准确性和实用性,对邻苯三酚在Tris-HCl和磷酸盐2种不同反应体系(缓冲液)中的自氧化产物进行UV-Vis吸收光谱扫描, 确定其最适吸收峰; 同时研究了tcv Cu,ZnSOD(简称tcvSOD)和Bovine Cu,Zn-SOD(简称Bovine SOD)分别在2种缓冲液中对邻苯三酚自氧化反应的抑制模式. 波长扫描显示, 终浓度为0.225 mmol/L的邻苯三酚在50 mmol/L Tris-HCl pH 8.2缓冲液中于320 nm处30 s出现明显吸收峰, 30 min吸收峰达到最高值; 30 min 后440 nm附近出现吸收峰; A320处的自氧化速率为0.139/min. 同样浓度邻苯三酚在0.2 mol/L pH8.0磷酸缓冲液中除了A320的自氧化速率稍高(A320 0.152/min)外, 其他性质与50 mmol/L Tris-HCl相同. tcvSOD和Bovine SOD在Tris-HCl缓冲液中对邻苯三酚自氧化反应的抑制作用较磷酸盐缓冲液为强.研究结果确认320 nm可作为检测邻苯三酚自氧化、SOD对其产生抑制及SOD活性间接测定的特定波长. 关键词:邻苯三酚; 自氧化; 铜锌超氧化物歧化酶(Cu,Zn-SOD); 波长扫描中图分类号:Q554 文献标志码:A 文章编号:1000-1565(2018)03-0284-07Pyrogallol autoxidation and its application to SOD activity assay JIA Hongyu1, WANG Yasen1, TIAN Xiaohui1, ZHANG Wei1, DU Jianfang2, KANG Ming1, LIU Jianrong2(1. Key Laboratory of Microbial Diversity Research and Application of Hebei Province, College of Life Sciences, Hebei University, Baoding 071002, China;2. Biotechnology Center,Hebei University, Baoding 071002, China)Abstract:To verify the accuracy and feasibility of the pyrogallol autoxidation, the UV-Vis absorption spectra of autoxidation products of pyrogallol were scanned systematically in Tris-HCl and phosphate buffers for optimal wavelength determination. The inhibition modes of tcv Cu,Zn-SOD and Bovine Cu,Zn-SOD to pyrogallol autoxidation were characterized in the two buffers. The autoxidation products showed a quickly increased absorption at 320 nm in 30 s, and reached to the maxima in 30 min in a solution of 0.225 mmol/L pyrogallol in 50 mmol/L Tris-HCl, pH8.2; a weak absorption at 440 nm was also observed 30 min after the reaction started; and the rate of autoxidation of pyrogallol was A320 0.139/min.The- 收稿日期:2017-10-25 基金项目:国家自然科学基金资助项目(31470192);河北省自然科学基金资助项目(C2016201180) 第一作者:贾红玉(1991—), 女, 河北邢台人, 河北大学在读硕士研究生. E-mail:jhy121818298@sina.com 通信作者:刘建荣(1963—), 女, 河北保定人,河北大学副研究员, 主要从事生化与分子生物学研究. E-mail:bioinfo5@hbu.edu.cn康明(1962—), 男, 河北昌黎人,河北大学教授,博士, 主要从事微生物代谢工程以及病毒宿主相互作用研究. E-mail:mingkang@hbu.edu.cn第3期贾红玉等:邻苯三酚自氧化法在SOD活性测定中的应用autoxidation mode of 0.225 mmol/L pyrogallol in 0.2 mol/L phosphate, pH8.0 was the same as that in 50 mmol/L Tris-HCl except the rate of autoxidation associated with phosphate buffer was a little higher(A320 0.152/min). Both the tcvSOD and Bovine SOD showed slightly stronger inhibition to pyrogallol autoxidation in Tris-HCl than that in phosphate buffer. It was validated that 320 nm is the optimal wavelength for measuring the rate of pyrogallol autoxidation, the inhibition of pyrogallol autoxidation, and the indirectly determination of SOD activity.
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