Affiliation:
1. Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285-0438,1and
2. Molecular Probes, Inc., Eugene, Oregon 974022
Abstract
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
3
H-,
14
C-, or
125
I-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.
Publisher
American Society for Microbiology
Subject
Infectious Diseases,Pharmacology (medical),Pharmacology