Magnetic microsphere-based methods to study the interaction of teicoplanin with peptides and bacteria.
Teicoplanin (teic) from Actinoplanes teichomyceticus is a glycopeptide antibiotic used to treat many gram-positive bacterial infections. Glycopeptide antibiotics inhibit bacterial growth by binding to carboxy-terminal D-Ala-D-Ala intermediates in the peptidoglycan of the cell wall of gram-positive bacteria. In this paper we report the derivatization of magnetic microspheres with teic (teic-microspheres). Fluorescence-based techniques have been developed to analyze the binding properties of the microspheres to two D-Ala-D-Ala terminus peptides. The dissociation constant for the binding of carboxyfluorescein-labeled D-Ala-D-Ala-D-Ala to teic on microspheres was established via fluorimetry and flow cytometry and was determined to be 0.5 x 10(-6) and 3.0 x 10(-6) mol L(-1), respectively. The feasibility of utilizing microparticles with fluorescence methods to detect low levels (the limit of bacterial detection was determined to be 30 colon-forming units; cfu) of gram-positive bacteria has been demonstrated. A simple microfluidic experiment is reported to demonstrate the possibility of developing microsphere-based affinity assays to study peptide-antibiotic interaction.
Piyasena
Menake E
ME
Department of Chemistry and Biochemistry, California State University, Los Angeles, 5151 State University Drive, Los Angeles, CA 90032-8202, USA.
Real
Lilian J
LJ
Diamond
Rochelle A
RA
Xu
H Howard
HH
Gomez
Frank A
FA
D000192:Actinomycetales / Q000737:chemistry*; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000378:metabolism*; D001419:Bacteria / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D005434:Flow Cytometry / Q000379:methods; D005452:Fluoresceins / Q000737:chemistry; D005453:Fluorescence; D008280:Magnetics; D044085:Microfluidics / Q000379:methods; D008855:Microscopy, Electron, Scanning / Q000379:methods; D008863:Microspheres; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D013194:Staining and Labeling; D017334:Teicoplanin / Q000737:chemistry / Q000378:metabolism*; D013997:Time Factors
D000900:Anti-Bacterial Agents; D005452:Fluoresceins; D010455:Peptides; C024098:6-carboxyfluorescein; D017334:Teicoplanin