Digoxigenin-ampicillin conjugate for detection of penicillin-binding proteins by chemiluminescence.
This paper describes a highly sensitive new method for the identification of penicillin-binding proteins (PBPs) that is based on the use of an ampicillin-digoxigenin conjugate (DIG-AMP conjugate) which is detected by immunoblotting and chemiluminescence. The sensitivity of chemiluminescence permitted X-ray film exposure times to be decreased to minutes, as opposed to the days or weeks which are requisite when conventionally radiolabeled beta-lactams are used. Coupling of ampicillin to digoxigenin yielded a product containing digoxigenin (detected by chemiluminescence) which also was inhibitory for Staphylococcus aureus and Escherichia coli. Unconjugated digoxigenin at concentrations of up to 100 micrograms/ml was not inhibitory for either organism. For S. aureus the MICs of DIG-AMP (0.7 microgram of conjugated ampicillin per ml) and of free ampicillin (0.5 microgram/ml) were comparable, indicating that ampicillin retained its bioactivity when coupled to digoxigenin. However, for E. coli the MICs of DIG-AMP (70 micrograms of conjugated ampicillin per ml) and of free ampicillin (8 micrograms/ml) were widely disparate, suggesting that the DIG-AMP conjugate was too large and/or hydrophobic to traverse the E. coli outer membrane via porins. DIG-AMP binding assays with E. coli and S. aureus cell envelopes revealed profiles of PBPs similar to those detected with 125I-ampicillin or [3H]penicillin. DIG-AMP binding to PBPs was completely inhibited in competition experiments with free ampicillin or penicillin, supporting the specificity of the DIG-AMP conjugate for PBPs. DIG-AMP thus represents an advantageous alternative to radioactive beta-lactams for the identification and analysis of PBPs.
Weigel
L M
LM
Department of Microbiology, University of Texas Southwestern Medical Center, Dallas 75235.
Belisle
J T
JT
Radolf
J D
JD
Norgard
M V
MV
D000667:Ampicillin / Q000138:chemical synthesis / Q000494:pharmacology; D001426:Bacterial Proteins / Q000032:analysis*; D002352:Carrier Proteins / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D004076:Digoxigenin / Q000138:chemical synthesis / Q000494:pharmacology; D004926:Escherichia coli / Q000737:chemistry; D006602:Hexosyltransferases; D015151:Immunoblotting; D008163:Luminescent Measurements; D002267:Muramoylpentapeptide Carboxypeptidase / Q000032:analysis*; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases; D012680:Sensitivity and Specificity
D001426:Bacterial Proteins; D002352:Carrier Proteins; D046915:Penicillin-Binding Proteins; D000667:Ampicillin; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase; D004076:Digoxigenin