Determination of IC50 and Ki values of beta-lactamase inhibition by BLI with nitrocefin or imipenem as a substrate.

Enzymes were mixed with BLIs at concentrations varying from 160 to 0.0027 μM in 50 mM sodium phosphate (pH 7.0)-0.1 mg/ml bovine serum albumin (BSA) (buffer A; 20 μM ZnCl₂ was also added for all metallo enzymes) and incubated for 10 min at 37°C. A 50 μM concentration of nitrocefin (10 μM for SHV-12) or 100 μM imipenem (prewarmed at 37°C for 10 min) was added, and substrate cleavage profiles were recorded at 37°C at 490 nm every 10 s for 10 min or at 294 nm every 30 s for 1 h for nitrocefin and imipenem, respectively. Initial rates of reaction were calculated and exported to Prism software to calculate IC₅₀ values using the “dose-response—inhibition, variable slope (four parameters)” equation. K_i values were calculated by the method of Waley (31). This method was previously used to calculate K_i values for boronic fast-on–fast-off BLIs.