Fluorescent coupled enzyme assays for D-alanine: application to penicillin-binding protein and vancomycin activity assays.
D-Alanine (D-Ala) is a ubiquitous constituent of bacterial cell walls. Assays for D-Ala can be used to investigate several aspects of cell wall biosynthesis and the effects of antibiotics on this process. High-sensitivity fluorescent assays for D-Ala were developed in a microtiter plate format based on d-aminoacid oxidase/horseradish peroxidase (DAO/HRP)-coupled reactions. For comparative purposes the classic chromogenic (UV-vis) assay using o-phenylenediamine (OPD) was also adapted to microtiter plates. OPD gave a lower limit of sensitivity of 2 nmol and was linear up to 60 nmol. Two commercially available fluorogenic HRP substrates were then tested in this assay. Amplex Red (AR) gave a lower limit of sensitivity of 2 pmol and was linear up to 400 pmol d-Ala. QuantaBlu (QB) based assays exhibited a lag in their response to D-Ala corresponding to 50 pmol D-Ala. This lag complicated calibration, but could be eliminated by addition of 150 pmol D-Ala to all assays. The QB assays were linear up to 3000 pmol D-Ala and gave a lower limit of sensitivity of 10 pmol. These assays are demonstrated for the characterization of the dd-carboxypeptidase activity of a soluble form of Escherichia coli penicillin-binding protein 5 (PBP 5) against the classic PBP substrate diacetyl-L-Lys-D-Ala-D-Ala. AR and QB based assays gave identical v/E(T) profiles, whereas OPD based assays gave slightly (10%) higher activity. This is consistent with the loss of a small amount of E. coli PBP 5 activity during the dilution necessary prior to its use in the highly sensitive fluorescent assays. These assays were then demonstrated for characterization of vancomycin binding to a D-Ala-D-Ala-based substrate.
Gutheil
W G
WG
Division of Pharmaceutical Sciences, University of Missouri-Kansas City, 5005 Rockhill Road, Kansas City, Missouri 64110, USA.
Stefanova
M E
ME
Nicholas
R A
RA
D000409:Alanine / Q000737:chemistry*; D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000032:analysis*; D001426:Bacterial Proteins; D002352:Carrier Proteins / Q000032:analysis*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase / Q000032:analysis*; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases; D012015:Reference Standards; D012680:Sensitivity and Specificity; D014640:Vancomycin / Q000032:analysis*
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D002352:Carrier Proteins; D005456:Fluorescent Dyes; D046915:Penicillin-Binding Proteins; D014640:Vancomycin; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase; D000409:Alanine