Determination of Oxidoreductase Activity Using a High-Throughput Microplate Respiratory Measurement

详细信息    查看全文

• 作者：Gregor Hommes ; Christoph A. Gasser ; Erik M. Ammann ; Philippe F.-X. Corvini
• 刊名：Analytical Chemistry
• 出版年：2013
• 出版时间：January 2, 2013
• 年：2013
• 卷：85
• 期：1
• 页码：283-291
• 全文大小：414K
• 年卷期：v.85,no.1(January 2, 2013)
• ISSN：1520-6882

文摘

High-throughput multiparallel activity profiling for oxygen consuming cell layers has been recently developed for extracellular flux analysis. This technology has great potential for determining the enzymatic activity of oxidoreductases (i.e., laccase) both in vivo and in vitro, which is usually measured using photometrical tests monitoring the colored oxidation products. Improvements in terms of sample throughput, comparability, and gain of information (i.e., stoichiometry, electron transfer rate) can be achieved by means of a multiwell plate-based fluorimetric oxygen sensor. In the present study, various laccases have been applied to develop protocols that allow the multiparallel measurement of O₂-consumption by enzymatic reactions. The developed and validated method enables the comparative quantitation of laccase characteristics (i.e., profiles of activity at various pH values) and minimizes the time it usually takes to collect respiratory data of oxygen-consuming enzymes. Furthermore, the possibility to assess differences between single and multisubstrate kinetics of laccases has been demonstrated.