Arai Makoto, Nihonmatsu-Kikuchi Naomi, Itokawa Masanari, Rabbani Naila, Thornalley Paul J
†Department of Psychiatry and Behavioral Sciences, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506, Japan.
*Clinical Sciences Research Laboratories, Warwick Medical School, University of Warwick, University Hospital, Coventry CV2 2DX, U.K.
Biochem Soc Trans. 2014 Apr;42(2):491-4. doi: 10.1042/BST20140010.
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 Δε240=2.86 mM⁻¹·cm⁻¹. 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 μmol 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 Δε240=-3.10 mM⁻¹·cm⁻¹. 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 μmol 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.
乙二醛酶I催化由甲基乙二醛和谷胱甘肽非酶促形成的半硫代乙缩醛异构化为S-D-乳酰谷胱甘肽。传统上，通过跟踪240nm吸光度（A240）的增加来分光光度法测定乙二醛酶I的活性，其摩尔吸收系数变化Δε240 = 2.86 mM⁻¹·cm⁻¹。通过在37°C下于50 mM磷酸钠缓冲液（pH 6.6）中孵育甲基乙二醛和谷胱甘肽10分钟，原位预先形成半硫代乙缩醛。然后加入细胞提取物，在5分钟内监测A240，并扣除空白校正后推导出A240的初始增加速率，从而得出乙二醛酶I的活性。乙二醛酶I的活性以每毫克蛋白质或细胞数的单位给出，其中一个单位是在测定条件下每分钟催化形成1μmol S-D-乳酰谷胱甘肽所需的酶量。乙二醛酶II催化S-D-乳酰谷胱甘肽水解为D-乳酸和谷胱甘肽。乙二醛酶II的活性也通过跟踪240nm吸光度（A240）的降低来分光光度法测定，其摩尔吸收系数变化Δε240 = -3.10 mM⁻¹·cm⁻¹。它以每毫克蛋白质或细胞数的单位给出，其中一个单位是在测定条件下每分钟催化水解1μmol S-D-乳酰谷胱甘肽所需的酶量。乙二醛酶I和乙二醛酶II活性测量方法已进行修改，以便与紫外线透明微孔板一起使用，以实现更高的样品通量。
