流动注射-分光光度法测定萝卜中过氧化物酶的活性
FI-Spectrophotometric Determination of Peroxidase Activity of Radish
摘 要
采用流动注射-分光光度法测定萝卜中过氧化物酶的活性。优化的试验条件如下:① 反应温度为60 ℃;② 载流为pH 5.5的0.05 mol·L-1磷酸二氢钠-磷酸氢二钠缓冲溶液;③ 采样体积为40 μL;④ 反应盘管长度为200 cm;⑤ 反应试剂为1.5 mmol·L-1过氧化氢,3.5 mmol·L-1愈创木酚的混合液。2-愈创木酚的表观摩尔吸光率为0.012 5 L·μmol-1·cm-1。辣根过氧化物酶的线性范围为454~7 265 U·L-1,检出限(3s/k)为7 U·L-1。方法的加标回收率在94.2%~107%之间。对500,2 500 U·L-1的辣根过氧化物酶标准溶液连续测定11次,测定值的相对标准偏差小于0.62%。
流动注射
过氧化物酶
萝卜
Spectrophotometry
Flow injection
Peroxidase
Radish
Abstract
FI-spectrophotometry was applied to the determination of activity of peroxidase in radish. The optimized conditions found were as follows: ① reaction temperature: 60 ℃; ② carrying solution: 0.05 mol·L-1 NaH2PO4-Na2HPO4 buffer solution (pH 5.5); ③ sampling volume: 40 μL; ④ length of reaction coil: 200 cm; ⑤ reaction reagent: mixture of 1.5 mmol·L-1 H2O2 and 3.5 mmol·L-1 2-guaiacol. Apparent molar absorptivity of 2-guaiacol found was 0.012 5 L·μmol-1·cm-1. Linearity range of horse radish peroxidase was found between 454-7 265 U·L-1 with detection limit (3s/k) of 7 U·L-1. Results of test for recovery of this method were found in the range of 94.2%-107%. Precision of the method was tested at the concentration levels of 500, 2 500 U·L-1 of horse radish peroxidase standard solution for 11 determination, values of RSD′s found were less than 0.62%.
中图分类号 O657.32 DOI 10.11973/lhjy-hx201610008
所属栏目 工作简报
基金项目
收稿日期 2015/10/30
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备注郑巍(1989-),男,河南信阳人,硕士研究生,研究 方向为蛋白质与酶工程研究。
引用该论文: ZHENG Wei,LI Yong-sheng,ZHAO Yang,GAO Xiu-feng. FI-Spectrophotometric Determination of Peroxidase Activity of Radish[J]. Physical Testing and Chemical Analysis part B:Chemical Analysis, 2016, 52(10): 1155～1159
郑巍,李永生,赵炀,高秀峰. 流动注射-分光光度法测定萝卜中过氧化物酶的活性[J]. 理化检验－化学分册, 2016, 52(10): 1155～1159
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参考文献
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【18】张朝,李永生,高秀峰.沙田油皮过氧化物酶的纯化及酶学性质的研究［J］.食品工业科技, 2013,34(12):187-191.
【2】BROWNLEADER M D, AHMED N, TREVAN M, et al. Purification and partial characterization of tomato extension peroxidase［J］. Plant Physiol , 1995,109(3):1115-1123.
【3】AZEVODO A M, MARTINS V C, PRAZERES D M, et al. Horseradish peroxidase: a valuable tool in biotechnology［J］. Biotechnol Annu Rev, 2003,9:199-247.
【4】魏永锋,闫宏涛.催化分光光度法测定辣根过氧化物酶新方法的研究［J］.光谱学与光谱分析, 2001,21(5):704-706.
【5】王琳,王林嵩,马剑敏.萝卜过氧化物酶的稳定性研究［J］.河南师范大学学报(自然科学版), 2004,32(3):81-83.
【6】慈云祥,陈列,魏珊.酪氨酸为底物的过氧化物酶催化荧光反应［J］.高等学校化学学报, 1990,11(1):81-83.
【7】魏永锋,闫宏涛.催化荧光光度法测定辣根过氧化物酶及甲胎蛋白［J］.分析化学, 2000,28(1):99-101.
【8】SHERMAN D H. Increasing sensitivity of luminescent enzyme immunoassays［J］. Trends Biotechnology, 1984,2(1):1-2.
【9】焦奎,徐静,孙伟,等.以变色酸2R为底物测定辣根过氧化物酶的研究［J］.分析化学, 2004,32(5):593-597.
【10】杜鑫,李永生.基于磷钼蓝反应测定钴-钼催化剂浸渍液中钼离子含量［J］.理化检验-化学分册, 2015,51(3):296-299.
【11】RECKTENWALD A, KRONER K H, KULA M R. On-line monitoring of enzymes in downstream processing by flow injection analysis(FIA)［J］. Enzyme and Microbial Technology, 1985,7(12):607-612.
【12】HUANG Hou-ping, CAI Ru-xiu, DU Yu-min, et al. Micelle enhanced spectrofluorimetric assay for laccase activity by a flow-injection stopped-flow technique［J］. Analytica Chimica Acta, 1995,318:63-69.
【13】STADEN J F, MULAUDZI L V. Flow injection spectrophotometric assay of α-amylase activity［J］. Analytica Chimica Acta, 2000,421(1):19-25.
【14】嵇正平,王俊,韩静,等.流动注射化学发光法测定溶菌酶含量［J］.分析化学, 2011,39(7):1100-1103.
【15】HOLM K A. Automated determination of microbial peroxidase activity in fermentation samples using hydrogen peroxide as the substrate and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) as the electron donor in a flow injection system［J］. Analyst, 1995,120(8):2101-2105.
【16】TONAMI H, UYAMA H, NAGAHATA R, et al. Guaiacol oxidation products in the enzyme-activity assay reaction by horseradish peroxidase catalysis［J］. Chemistry Letters, 2004,33(7):796-797.
【17】HWANG S, LEE C H, AHN I S. Product identification of guaiacol oxidation catalyzed by manganese peroxidase［J］. Journal of Industrial and Engineering Chemistry, 2008,14(4):487-492.
【18】张朝,李永生,高秀峰.沙田油皮过氧化物酶的纯化及酶学性质的研究［J］.食品工业科技, 2013,34(12):187-191.
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