Uv transparent microplates

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

A 1 : 1 mixture of ^HAlgMA and ^LAlgMA (15 mg mL⁻¹), GelMA (10% w/v), or PVAMA (67 kDa or 31 kDa; 10, 15, 20, or 40% w/v) was dissolved in buffer A and mixed with LPA (0.1% w/v in water) and the required amount of MBP-TaHAL and HdeA (if applicable). These mixtures (50 μL) were pipetted into the wells of a 24-well plate and crosslinked by UV irradiation (Eurolite LED IP FL-50 COB UV, 395 nm, 50 W) for 30 s. The crosslinked disks were washed with 1 mL HEPES buffer, and 100 μL of this HEPES buffer as well as the disks themselves were transferred to a 96-well plate (Corning® UV-Transparent Microplates), where 100 μL HEPES buffer was added followed by the addition of histidine (0.5 or 25 mM final concentration). Observation of the histidine conversion reaction was performed as outlined above. Leakage of the enzyme from the hydrogels was measured by incubating the hydrogel disks at room temperature in HEPES buffer for 2 h, removing the wash buffer, and determining the enzyme activity in this washing solution after 2 h. The pH titration and the measurements of the stabilities of the encapsulated MBP-TaHAL in hydrogels when exposed to simulated gastrointestinal fluids were conducted as previously outlined. Specifically, the disks were incubated with 1 mL of the required solution for 2 h at 37 °C, washed, and transferred to a 96-well plate, and activity was measured.