ristocetin and alanylalanine

We aimed to develop and validate sensitive fluorescence techniques to assess the binding of magnetic microbeads derivatized with ristocetin from Amycolatopsis lurida to carboxyfluorescein-labeled D-Ala-D-Ala-D-Ala and in competition with Staphylococcus aureus bacteria. Glycopeptide antibiotics have been widely used to treat bacterial infections. However, new antibiotics are needed because of growing bacterial resistance and serious side effects. To screen potential candidates for new antibiotics, there is a great demand for sensitive, fast and inexpensive techniques to analyze the interactions between these molecules and bacterial cells.. Fluorometry, an in-house fluorescent instrument and fluorescence microscopy were used to determine binding constants of 2.75 × 10(4), 2.21 × 10(4) and 0.81 × 10(4) M(-1), respectively, for the interaction between ristocetin and the labeled peptide.. The methods detailed herein have been successfully applied to assess the binding of carboxyfluorescein- labeled D-Ala-D-Ala-D-Ala to ristocetin on microspheres. The magnetic bead-based immunoassay could be used to detect bacteria at low concentrations.

Topics: Anti-Bacterial Agents; Bacteria; Dipeptides; Drug Evaluation, Preclinical; Fluoresceins; Fluorometry; Immunoassay; Limit of Detection; Microspheres; Peptides; Ristocetin; Spectrometry, Fluorescence; Staphylococcus aureus

Binding constants between the glycopeptides teicoplanin (Teic) and ristocetin (Rist) and their derivatives to D-Ala-D-Ala terminus peptides were determined by on-column receptor synthesis coupled to partial-filling affinity capillary electrophoresis (PFACE) or affinity capillary electrophoresis (ACE). In these techniques, the column is first partially filled with increasing concentrations of D-Ala-D-Ala terminus peptides. This is followed by plugs of buffer, antibiotic and two noninteracting standards, and acetic and/or succinic anhydride (and buffer in the case of ACE). The order of the reagent plugs containing the antibiotic and anhydride varies with the charge of the glycopeptide. Upon electrophoresis, the antibiotic reacts with the anhydride yielding a derivative of Teic or Rist. Continued electrophoresis results in the overlap of the derivatized antibiotic and the plug of D-Ala-D-Ala peptide. Analysis of the change in the relative migration time ratio (RMTR) of the new glycopeptide relative to the standards, as a function of the concentration of the D-Ala-D-Ala ligand yields a value for the binding constant K(b). The techniques described here can be used to assess how the derivatization of drugs alters their affinities for target molecules.

Topics: Acetic Anhydrides; Anti-Bacterial Agents; Buffers; Dipeptides; Electrophoresis, Capillary; Mathematics; Molecular Structure; Pharmaceutical Preparations; Ristocetin; Succinic Anhydrides; Teicoplanin