| Assay Method Information | |
| | Biochemical Assays |
| Description: | Stock solutions for all assays were prepared in DMSO (10 mM) and stored at −20 °C. Assays were performed in 96 well plates (Greiner) at a total reaction volume of 100 μL. First, enzyme (final concentration: 50 nM) was incubated with inhibitor (25 μM) or positive control (DMSO, 0.25%) in a reaction buffer (finalconcentration: 100 μM CoCl2 6H2O, 100 mM NaCl, 0.075% bovine serum albumine, 50 mM Tris pH 7.5) for 20 min at 37 °C. Then, the substrate Met-Gly-Met-Met was added to a final concentration of 400 μM. After an incubation period of 15 min (HsMetAP1), 20 min (EcMetAP), or 60 min (HsMetAP2) at 37 °C, the enzyme reactionwas stopped by adding 10 μL 4% trifluoroacetic acid (TFA), and the plate was centrifuged at 1000g for 10 min. Detection of the product Gly-Met-Met was performed by HPLC: C18 column (Dr. Maisch, ReproSil-Pur 120 ODS-3, 3 μm, 50 × 2 mm). An 80 μL injection volume was eluted with mobile phases A (H2O and 0.1% TFA, filtered, degasse |
| Affinity data for this assay | |
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