A new, efficient method to study glutathione-independent glyoxalases: a continuous spectrophotometric assay

by Aizat

ABSTRACT

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We developed a new enzymatic assay for type III glyoxalases. Type III, glutathione-independent glyoxalases catalyze the conversion of methylglyoxal, a toxic byproduct of glycolysis, to lactic acid. This continuous assay utilizes the reversible reaction between methylglyoxal and dithiothreitol to form hemithioacetal that absorbs UV light at 286 nm. When a glyoxalase is present, free methylglyoxal is converted to lactic acid via enzyme catalysis, thus decreasing its concentration, which leads to a decrease in hemithioacetal concentration according to Le Chatelier’s principle. Hemithioacetal decomposition can be measured spectrophotometrically at 286 nm and the rate of decomposition can be used to calculate the rate of methylglyoxal conversion by glyoxalase enzyme. We determined equilibrium constant of hemithioacetal formation and its molar absorptivity coefficient at 286 nm to be able to obtain concentration from absorbance data. The method has a broad dynamic range due to its indirect measurement of analyte concentration. By mixing different ratios of methylglyoxal to dithiothreitol, free methylglyoxal concentrations between 20 μM to 2 mM can be reached and the rate of its conversion can be calculated. This enables determinations of Michaelis-Menten parameters for different glyoxalases from various organisms. The method is continuous; we compared it with one of the existing discontinuous assays to find that the new method is simpler and less error prone. And it uses a common laboratory reagent dithiothreitol, rendering itself cost-efficient. Overall, the new method is easy to use, reproducible and effective over a broad range of analyte concentrations.