Fluorescent Coupled Enzyme Assays for D-alanine: Application to Penicillin-Binding Protein and Vancomycin Activity Assays

Anal Biochem. 2000 Dec 15;287(2):196-202. doi: 10.1006/abio.2000.4835.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alanine / chemistry*
  • Amino Acid Sequence
  • Anti-Bacterial Agents / analysis*
  • Bacterial Proteins*
  • Carrier Proteins / analysis*
  • Fluorescent Dyes / chemistry*
  • Hexosyltransferases*
  • Muramoylpentapeptide Carboxypeptidase / analysis*
  • Penicillin-Binding Proteins
  • Peptidyl Transferases*
  • Reference Standards
  • Sensitivity and Specificity
  • Vancomycin / analysis*

Substances

  • Anti-Bacterial Agents
  • Bacterial Proteins
  • Carrier Proteins
  • Fluorescent Dyes
  • Penicillin-Binding Proteins
  • Vancomycin
  • Peptidyl Transferases
  • Hexosyltransferases
  • Muramoylpentapeptide Carboxypeptidase
  • Alanine