BOCILLIN FL, a sensitive and commercially available reagent for detection of penicillin-binding proteins

We describe a new, sensitive, rapid, and nonradioactive method involving th e use of the commercially available BOCILLIN FL, a fluorescent penicillin, as a labeling reagent for the detection and study of penicillin-binding pro teins (PBPs). This method allowed rapid detection of 30 ng of a purified PB P protein under UV light and of 2 to 4 ng of the protein with the aid of a FluorImager. This method also allowed rapid determination of the PBP profil es of Escherichia coli, Pseudomonas aeruginosa, and Streptococcus pneumonia e. The PBP profiles obtained are virtually identical to those reported prev iously with H-3-, C-14-, or I-125-labeled penicillin. Using this method ena bled us to determine the 50% inhibitory-concentrations of the penicillin-se nsitive and -resistant PBP2x proteins of S. pneumoniae for penicillin G, th ereby allowing a direct evaluation of their relative affinities for penicil lin G. Finally, this method also allowed us to compare relative affinities of a PBP2x protein for different beta-lactam antibiotics with the aid of fl uorescence polarization technology and to monitor a PBP2x protein during pu rification.