Holt Andrew, Palcic Monica M
Department of Pharmacology, 9-70 Medical Sciences Building, University of Alberta, Edmonton, Alberta, Canada, T6G 2H7.
Nat Protoc. 2006;1(5):2498-505. doi: 10.1038/nprot.2006.402.
This absorbance plate-reader-based assay is suitable for the examination of monoamine oxidase and copper amine oxidase activities versus numerous substrates. The assay is robust, continuous, rapid, highly quantitative, reasonably sensitive, inexpensive and suitable for automation. In the presence of a suitable amine substrate, amine oxidase enzymes generate hydrogen peroxide, which then drives the peroxidase-dependent oxidation of 4-aminoantipyrine. A subsequent interaction with vanillic acid generates stoichiometric amounts of a red quinoneimine dye, the appearance of which is monitored at 498 nm. An alternative procedure in which vanillic acid is replaced by 2,4-dichlorophenol enhances sensitivity but precludes the measurement of monoamine oxidases due to inhibition of these enzymes by dichlorophenol. Some substrates with low redox potentials, such as catecholamines, are not suitable for inclusion in this assay. A researcher familiar with the procedure can manually generate data for 30 full kinetic curves, composed of ten triplicate points, in 8 h.
这种基于酶标仪的吸光度测定法适用于检测单胺氧化酶和铜胺氧化酶针对多种底物的活性。该测定法稳健、连续、快速、高度定量、灵敏度适中、成本低廉且适合自动化操作。在合适的胺类底物存在的情况下，胺氧化酶会产生过氧化氢，然后过氧化氢驱动4-氨基安替比林的过氧化物酶依赖性氧化反应。随后与香草酸相互作用会生成化学计量的红色醌亚胺染料，通过在498nm处监测其吸光度来进行检测。另一种方法是用2,4-二氯苯酚替代香草酸，这种方法可提高灵敏度，但由于二氯苯酚会抑制单胺氧化酶，因此无法用于检测单胺氧化酶。一些氧化还原电位较低的底物，如儿茶酚胺，不适合用于该测定法。熟悉该操作流程的研究人员在8小时内可手动生成30条完整的动力学曲线数据，每条曲线包含10个重复三次的测量点。
