BOCILLIN FL, a sensitive and commercially available reagent for detection of penicillin-binding proteins - PubMed (original) (raw)

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

G Zhao et al. Antimicrob Agents Chemother. 1999 May.

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 beta-lactam antibiotics with the aid of fluorescence polarization technology and to monitor a PBP2x protein during purification.

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Figures

FIG. 1

FIG. 1

Structure of BOCILLIN FL.

FIG. 2

FIG. 2

Detection and quantitation of the penicillin-sensitive PBP2x protein of S. pneumoniae by BOCILLIN FL-binding assays. The penicillin-sensitive PBP2x protein of S. pneumoniae was purified (33), labeled with BOCILLIN FL (10 μM) for 30 min, separated by SDS-PAGE, and visualized by using a FluorImager. (A) Detection of the PBP2x protein; lanes 1 to 8: 2, 4, 8, 12, 16, 24, 36, and 48 ng of the purified PBP2x protein, respectively. (B) Quantitation of the PBP2x protein; fluorescence intensities of each band from panel A were quantified and plotted.

FIG. 3

FIG. 3

Detection of PBPs of S. pneumoniae, E. coli, and P. aeruginosa. The membrane fractions of S. pneumoniae, E. coli, and P. aeruginosa were prepared and labeled with BOCILLIN FL (25 μM). The labeled membrane preparations (≈7.5 μg of protein each) were separated by SDS-PAGE and visualized by using a FluorImager. Lanes 1 to 3: BOCILLIN FL-labeled membrane preparations of S. pneumoniae, E. coli, and P. aeruginosa, respectively.

FIG. 4

FIG. 4

Minimal amount of BOCILLIN FL required for the detection of E. coli PBPs. The E. coli membrane preparation (≈15 μg of protein each) was labeled with BOCILLIN FL (final concentration, 1.6 to 25 μM). The labeled PBPs are separated by SDS-PAGE and detected by using a FluorImager. Lanes 1 to 5: membrane preparation labeled with 1.6, 3.2, 6.4, 12.5, and 25 μM BOCILLIN FL, respectively.

FIG. 5

FIG. 5

Determination of IC50s of the penicillin-sensitive and -resistant PBP2x proteins of S. pneumoniae (hex) R6 and 328, respectively, for penicillin G (Pen G). Both PBP2x proteins were purified (33), labeled with BOCILLIN FL in the presence of various amounts of penicillin G, separated by SDS-PAGE, and visualized by using a FluorImager. (A) Penicillin-resistant PBP2x protein of S. pneumoniae 328. (B) Penicillin-sensitive PBP2x protein of S. pneumoniae (hex) R6.

FIG. 6

FIG. 6

Determination of the IC50 of the penicillin-sensitive PBP2x protein of S. pneumoniae (hex) R6 for penicillin G (Pen G). The fluorescence polarization for the penicillin-sensitive PBP2x protein (1.3 nM) in a competitive interaction with BOCILLIN FL (2 nM) and increasing concentrations of unlabeled penicillin G (0.01 to 10,000 nM) were measured as described previously. Data points represent the average of four replicates (± standard deviations), and the curve is the predicted nonlinear regression result.

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