2.8. Measurement of Glo1 Activity

The activity of Glo1 was determined spectrophotometrically by measuring the initial rate of increase in absorbance at 235 nm following addition of total protein extract (ca. 10 µg protein) to hemithioacetal, prepared by pre-incubating 2 mM methylglyoxal and 2 mM GSH in 50 mM sodium phosphate buffer (pH 6.6). For the conversion of the hemithioacetal to S-D-lactoylglutathione, the change in molar extinction coefficient Δε235 = 1.07 mM⁻¹cm⁻¹ was used. The activity of Glo1 was expressed in units (U), where 1 U is the amount of Glo1 which catalyzes the formation of 1µmol of S-D-lactoylglutathione per minute at 37 °C [33].