Recombinant human glyoxalase 1 glo1

Recombinant human glyoxalase I (GLO1) was purchased from R&D Systems (catalog #4959-GL). Assays were carried out in 0.1 M sodium phosphate, pH 7.0 buffer, utilizing 96-well clear UV plates (Corning UV Transparent Microplates, catalog #3635). A fresh solution of GSH (100 mM) and methylglyoxal (MG) (100 mM) was prepared in Millipore grade water. The substrate for the assay was prepared by adding 0.43 mL of GSH and 0.43 mL of MG to 15.14 mL of buffer. The substrate mixture was vortexed vigorously for 45 s and then allowed to sit at room temperature for 15 min. The initial well volume was 50 μL containing GLO1 (40 ng) and inhibitor. This protein and inhibitor mixture was incubated for 15–20 min prior to addition of the substrate. The substrate (150 μL) was then added to the wells yielding a maximum amount of 5% DMSO per well. The enzyme activity was measured utilizing a Biotek Synergy HT plate reader by measuring absorbance at 240 nm every 1 min for a duration of 8 min. The rate of absorbance increase was compared for samples versus controls containing no inhibitor (set at 100% activity). The absorbance reading for background wells containing DMSO, buffer, and substrate (no enzyme or inhibitor) was subtracted from the experimental wells. A positive control (Chugai-3d inhibitor, 50 μM final concentration) showed complete inhibition under the assay conditions described above.⁶¹

Recombinant Human Glyoxalase I (GLO1) was purchased from R&D Systems (Catalog #4959-GL). Assays were carried out in 100 mM Sodium Phosphate, pH 7.0 buffer utilizing 96-well Clear UV Plate (Corning UV Transparent Microplates Catalog #3635). A fresh solution of glutathione (Pre-Substrate 1, 100 mM) as well as methylglyoxal (Pre-substrate 2, 100 mM) was prepared in deionized water. The substrate was prepared by adding 14.5 mL of buffer and 0.99 mL of each pre-substrate components. The substrate mixture was vortexed vigorously for 15 sec, then allowed to sit at room temperature for 20 min. Initial well volume was 50 μL containing GLO1 (40 ng) and inhibitor. This protein and inhibitor mixture was incubated for 15–20 min prior to addition of substrate. To this was then added substrate (150 μL) yielding a maximum amount of 5% DMSO per well. The enzyme activity was measured utilizing a Biotek Synergy H4 plate reader by measuring absorbance at 240 nm every 1 min for 8 min. The rate of absorbance increase was compared for samples versus controls containing no inhibitor (100% activity). Absorbance for background wells containing DMSO, buffer and substrate (no enzyme or inhibitor) were subtracted from the rest of the wells.