Fluorescent Coupled Enzyme Assays for d-Alanine: Application to Penicillin-Binding Protein and Vancomycin Activity Assays
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- 作者：Gutheil ; William G. ; Stefanova ; Miglena E. ; Nicholas ; Robert A.
- 刊名：Analytical Biochemistry
- 出版年：2000
- 出版时间：December 15, 2000
- 年：2000
- 卷：287
- 期：2
- 页码：196-202
- 全文大小：95.7 K
文摘
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/ET 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.