Measurement of glyoxalase activities.
Measurement of glyoxalase activities.
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DOI：
10.1042/bst20140010
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发表时间：
2014-04-01
影响因子：
3.9
通讯作者：
Thornalley, Paul J
中科院分区：
文献类型：
--
作者：
Arai, Makoto;Nihonmatsu-Kikuchi, Naomi;Thornalley, Paul J
乙二酸酶I催化甲基乙醛和谷胱甘肽非酶促生成的半硫缩醛异构化为S-D-乳糖基谷胱甘肽。乙草酸酶I的活性通常是用分光光度法测量的，即A240的摩尔吸收系数的变化Deltaepsilon240=2.86 mm⁻·cm⁻。甲基乙二醛和谷胱甘肽在50 mM磷酸二氢钠缓冲液(pH 6.6)中37℃孵育10分钟，原位预制得半硫代缩醛。然后加入细胞提取物，在5分钟内监测A240，并通过校正空白推导出A240的初始增加速率以及乙二醛酶I的活性。乙二酸酶I活性是以每毫克蛋白质或细胞数为单位给出的，其中一个单位是在分析条件下每分钟催化形成1摩尔S-D-乳糖基谷胱甘肽的酶的量。乙二酸酶II催化S-D-乳酸谷胱甘肽的水解制得D-乳酸和谷胱甘肽。乙二酸酶II的活性也是通过跟踪A240的下降而被分光光度测定的，A240的摩尔吸收系数的变化Deltaepsilon240=-3.10 mm⁻·cm⁻。它的单位是每毫克蛋白质或细胞数，其中一个单位是在分析条件下每分钟催化1摩尔S-D-乳糖基谷胱甘肽水解酶的量。乙二酸酶I和乙醛酶II的活性测量已经被修改，用于紫外线透明的微孔板，以获得更高的样品吞吐量。
Glyoxalase I catalyses the isomerization of the hemithioacetal formed non-enzymatically from methylglyoxal and glutathione to S-D-lactoylglutathione. The activity of glyoxalase I is conventionally measured spectrophotometrically by following the increase in A240 for which the change in molar absorption coefficient Deltaepsilon240=2.86 mM⁻·cm⁻. The hemithioacetal is pre-formed in situ by incubation of methylglyoxal and glutathione in 50 mM sodium phosphate buffer (pH 6.6) at 37°C for 10 min. The cell extract is then added, the A240 is monitored over 5 min, and the initial rate of increase in A240 and hence glyoxalase I activity deduced with correction for blank. Glyoxalase I activity is given in units per mg of protein or cell number where one unit is the amount of enzyme that catalyses the formation of 1 mumol of S-D-lactoylglutathione per min under assay conditions. Glyoxalase II catalyses the hydrolysis of S-D-lactoylglutathione to D-lactate and glutathione. Glyoxalase II activity is also measured spectrophotometrically by following the decrease in A240 for which the change in molar absorption coefficient Deltaepsilon240=-3.10 mM⁻·cm⁻. It is given in units per mg of protein or cell number where one unit is the amount of enzyme that catalyses the hydrolysis of 1 mumol of S-D-lactoylglutathione per min under assay conditions. Glyoxalase I and glyoxalase II activity measurements have been modified for use with a UV-transparent microplate for higher sample throughput.