| Assay Method Information | |
| | Biochemical Assay |
| Description: | Nitrocefin (Cayman, CAS 41906-86-9) was used as the chromogenic substrate for all biochemical assays. The enzymatic activity of purified VIM-2 and NDM-1 were determined spectrophotometrically (spectramax-M5-reader) at room temperature in 50 mM potassium phosphate buffer at pH 7.0. The rate of product formation was monitored based on the λmax=486 nm absorbance taken at 10 s intervals for 30 mins. The Km and kcat values were determined from 10 different concentrations of nitrocefin ranging from 0.001 to 100 μM with at least four independent initial-velocity measurements. Velocity versus substrate concentration curves were fitted by nonlinear regression using Michaelis-Menten Enzyme kinetics with Graphpad Prism 6. |
| Affinity data for this assay | |
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