Measurement of glyoxalase activities

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 A 240 for which the change in molar absorption coefficient Δe 240 =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 A 240 is monitored over 5 min, and the initial rate of increase in A 240 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 A 240 for which the change in molar absorption coefficient Δe 240 =−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.