A simple and rapid method for detecting penicillin-bindingproteins using ¹²⁵I-labeled penicillin V

Abstract

A simple and rapid method to detect target enzymes of β-lactams the penicillin-binding proteins (PBPs), using ¹²⁵ I-labeled penicillin V ([¹²⁵I] PCV) was constructed.[¹²⁵ I] PCV was easily synthesized by incubation of p-trimethylstannyl PCV with [¹²⁵ I] Na.[¹²⁵ I] PCV was detected by radioluminography (RLG) using an imaging plate (IP) which is more sensitive than an X-ray film. Use of [¹²⁵I] based RLG in combination with a preparation of a membrane fraction withoutultracentrifugation, and use of a 96-well microtiter plate and a mini-gel electrophoresis system made it possible to determine the affinity of β-lactams for PBPs easily in a short time (less than 3 days). Electrophoretic patterns of PBPs detected by simil PCV were similar to those detected by [¹⁴C] penicillin G ([¹⁴C] PCG). Binding affinities of cefoselis (FK037) and imipenem/cilastatin for PBPs from MRSA and Escherichia coli, determined with [¹²⁵I] PCV were similar to those determilled with [¹²⁵ I] PCG. In conclusion, using [¹²⁵ I] PCG instead of [¹⁴C] PCG provides a useful alternative for assay of binding affinity of β-lactams for PBPs.