A new reagent for the detection of penicillin-binding proteins (PBPs) was developed. An N-hydroxysuccinimide ester of biotin was used to tag f8-lactam antibiotics with free side chain amino groups such as ampicillin (BIO-AMP), 6-aminopenicillanic acid (BIO-APA), and 7-aminocephalosporanic acid (BIO-ACA). Bacterial PBPs from cells or isolated cytoplasmic membranes of Escherichia coli, Haemophilus influenzae, Staphylococcus aureus, and Streptococcus pneumoniae were labeled with BIO-AMP, subjected to electrophoresis on sodium dodecyl sulfate (SDS)-polyacrylamide gels, and transferred onto nitrocellulose membranes. Electrophoretic PBP profiles were detected on blots, using colorimetric or chemiluminescence systems, on the basis of the interaction of BIO-AMP-PBP complexes and a streptavidin-peroxidase conjugate. The chemiluminescent reaction permitted a high sensitivity of detection, and PBP profiles could be determined within seconds. All PBP profiles were similar to those obtained with a traditional PBP labeling technique with '251-labeled penicillin V, except that an additional unidentified PBP (approximately 55,000 Da) was labeled with BIO-AMP in E. coli and H. influenzae. Differences in the intensities of labeling for specific PBPs were observed between the chemiluminescent and radioactive labeling agents and were attributed to the differences in their affinities for PBPs. Similarly, BIO-AMP, BIO-APA, and BIO-ACA produced different PBP profiles. We also investigated the use of BIO-AMP in PBP purification. BIO-AMP-PBP complexes from a mixture of H. influenzae proteins were allowed to bind to avidin immobilized on an agarose support in a microcentrifuge tube. After several washes in the presence of salts, PBPs were eluted by boiling and treatment with SDS. The eluted proteins were separated by electrophoresis on SDS-polyacrylamide gels, and biotinylated proteins were identified on blots by a chemiluminescence reaction. Biotinylation of P-lactams is rapid, safe, and inexpensive. Our results demonstrate the feasibility of using biotinylated P-lactams as nonradioactive reagents for the study of PBPs and for the purification of these proteins.,B-Lactam drugs must bind to specific targets located in the cytoplasmic membrane of bacteria to exert their inhibitory effects. These target proteins can be identified by their ability to covalently bind isotope-labeled penicillin and are termed penicillin-binding proteins (PBPs). The enzymatic functions of higher-molecular-weight PBPs are essential in the cross-linking of the bacterial cell wall. P-Lactam antibiotics kill bacteria through inhibition of these high-molecular-weight PBPs as substrate analogs of the acyl-D-alanyl-D-alanine component of peptidoglycan (11,28 428-3550. reus, and Streptococcus pneumoniae), and the chemiluminescence detection system permitted the rapid and sensitive detection of these PBPs. Furthermore, we showed that biotin-,-lactam-PBP complexes could be purified by affinity chromatography by using the ligand avidin immobilized on beaded agarose...