BOCILLIN FL, a Sensitive and Commercially Available Reagent for Detection of Penicillin-Binding Proteins

Abstract

We describe a new, sensitive, rapid, and nonradioactive method involving the use of the commercially available BOCILLIN FL, a fluorescent penicillin, as a labeling reagent for the detection and study of penicillin-binding proteins (PBPs). This method allowed rapid detection of 30 ng of a purified PBP 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 profiles of Escherichia coli, Pseudomonas aeruginosa, and Streptococcus pneumoniae. The PBP profiles obtained are virtually identical to those reported previously with 3H-,14C-, or 125I-labeled penicillin. Using this method enabled us to determine the 50% inhibitory concentrations of the penicillin-sensitive and -resistant PBP2x proteins of S. pneumoniae for penicillin G, thereby allowing a direct evaluation of their relative affinities for penicillin G. Finally, this method also allowed us to compare relative affinities of a PBP2x protein for different β-lactam antibiotics with the aid of fluorescence polarization technology and to monitor a PBP2x protein during purification.

Fluorescent Bocillins: Synthesis and application in the detection of penicillin-binding proteins

“…Figure 2 shows that the IC 50 s of the penicillin-sensitive and -resistant PBP2x proteins for penicillin G were 10 and 90 mM, respectively. This result is in general agreement consistent with our previous finding that the affinity of penicillin-sensitve PBP2x protein for penicillin G as determined with Bocillin FL is 10-15-fold higher than that of penicillinresistant PBP2x protein [11], and with the finding that the K d values of the penicillin-sensitive and -resistant PBP2x proteins for penicillin V and 125 I-labeled penicillin V are about 12-fold different [18]. …”

“…Cells were grown and membrane preparations obtained as previously described for E. coli and P. aeruginosa [11]. Purified penicillin-sensitive PBP2x protein was obtained from S. pneumoniae as previously described [18].…”

“…Purified penicillin-sensitive PBP2x protein was obtained from S. pneumoniae as previously described [18]. To determine the sensitivity of Bocillin FL and Bocillin 650/665 for the detection of PBP2x, binding assays were performed as previously described [11]. Briefly, reaction mixtures contained 4-48 ng of the purified penicillin-sensitive PBP2x protein of S. pneumoniae, 10 mM fluorescent Bocillin, 20 mM potassium phosphate (pH 7.5), and 140 mM NaCl.…”

“…Determination of the IC 50 s of penicillin-sensitive and resistant PBP2x proteins of S. pneumoniae for penicillin G were performed as described previously [11]. Briefly, reaction mixtures (20 mL each) contained 25 mM potassium phosphate (pH 7.5), 3 mg of each PBP2x protein, 10 mM Bocillin 650/665, and various amounts of penicillin G. The reaction mixtures were incubated at 357C for 15 min, denatured with 20 mL of Tris-glycineSDS sample buffer (Novex), and subjected to SDS-PAGE (10% polyacrylamide; 5-10 mL of reaction mixture per lane; BioRad Laboratories).…”

“…We describe the synthesis of these new probes, and their electrophoretic and chromatographic behavior with PBPs from purified protein and bacterial membrane preparations. A preliminary account of this work has appeared previously [11].…”