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. Assa ys for D-Ala can be used to investigate several aspects of cell wall biosyn thesis and the effects of antibiotics on this process. High-sensitivity flu orescent 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-ph enylenediamine (OPD) was also adapted to microtiter plates. OPD gave a lowe r limit of sensitivity of 2 nmol and was linear up to 60 nmol. Two commerci ally available fluorogenic HRP substrates were then tested in this assay. A mplex Red (AR) gave a lower Limit of sensitivity of 2 pmol and was Linear u p to 400 pmol D-Ala. QuantaBlu (QB) based assays exhibited a lag in their r esponse to D-Ala corresponding to 50 pmol D-Ala. This lag complicated calib ration, 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 characteriza tion of the DD-carboxypeptidase activity of a soluble form of Escherichia c oli 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 pro files, whereas OPD based assays gave slightly (10%) higher activity. This i s consistent with the loss of a small amount of E. coli PBP 5 activity duri ng the dilution necessary prior to its use in the highly sensitive fluoresc ent assays. These assays were then demonstrated for characterization of van comycin binding to a D-Ala-D-Ala-based substrate. (C) 2000 Academic Press.