Microplate quantification of plant leaf superoxide dismutases
Microplate quantification of plant leaf superoxide dismutases
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DOI：
10.1016/j.ab.2004.06.015
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发表时间：
2004-09-15
影响因子：
2.9
通讯作者：
Stout, R
中科院分区：
文献类型：
--
作者：
Banowetz, GM;Dierksen, KP;Stout, R
超氧化物歧化酶（SOD）催化超氧自由基在包括植物在内的广泛生物体中的歧化。由于存在干扰测定的剂量响应的化合物，因此在粗植物提取物中SOD活性的定量一直是有问题的。虽然存在从植物提取物中部分纯化SOD的策略，但纯化的要求限制了可以进行的测定的快速性和实际数量。在这篇文章中，我们描述了修改的程序，使用邻联茴香胺作为底物，允许相对快速的定量SOD活性在粗叶提取物中的微孔板格式。该方法采用了商业设备的使用，允许一次裂解12个组织样品，并使用Pipes缓冲液，以减少来自粗叶提取物中存在的化合物的干扰。该方法的线性范围为1 ~ 50个SOD单位。使用从一组分类学上不同的植物制备的组织提取物证明了该测定的实用性。与两种草的组织提取物的反应速率在至少60分钟内呈线性。某些物种的组织中含有干扰化合物，其中大部分可以通过超滤去除。植物过氧化氢酶，过氧化物酶，和抗坏血酸在生理量的存在下没有干扰的测定。这种方法提供了一种手段，以量化SOD活性在相对大量的植物样品提供的干扰化合物的存在的可能性被认为是。在某些植物组织中存在干扰化合物，在解释植物胁迫对SOD的影响时必须谨慎。爱思唯尔公司出版
Superoxide dismutases (SODs) catalyze the dismutation of superoxide radicals in a broad range of organisms, including plants. Quantification of SOD activity in crude plant extracts has been problematic due to the presence of compounds that interfere with the dose-response of the assay. Although strategies exist to partially purify SODs from plant extracts, the requirement for purification limits the rapidity and practical number of assays that can be conducted. In this article, we describe modification of a procedure using o-dianisidine as substrate that permits relatively rapid quantification of SOD activity in crude leaf extracts in a microplate format. The method employs the use of a commercial apparatus that permits lysis of 12 tissue samples at once and the use of Pipes buffer to reduce interference from compounds present in crude leaf extracts. The assay provided a linear response from 1 to 50units of SOD. The utility of the assay was demonstrated using tissue extracts prepared from a group of taxonomically diverse plants. Reaction rates with tissue extracts from two grasses were linear for at least 60min. Tissues of certain species contained interfering compounds, most of which could be removed by ultrafiltration. The presence of plant catalases, peroxidases, and ascorbate in physiological quantities did not interfere with the assay. This approach provides a means to quantify SOD activity in relatively large numbers of plant samples provided that the possibility for the presence of interfering compounds is considered. The presence of interfering compounds in certain plant tissues necessitates caution in interpreting the effects of plant stresses on SOD. Published by Elsevier Inc.