muramidase and 8-amino-1-3-6-naphthalenetrisulfonic-acid

The muropeptide composition of bacterial peptidoglycan is currently most efficiently determined by reverse-phase high-pressure liquid chromatography (HPLC). Though sensitive, the HPLC procedure is technically demanding and has been applied to a relatively small number of bacterial strains and species. We have found that fluorescence-assisted carbohydrate electrophoresis (FACE) is a simple, rapid method by which reducing muropeptides from multiple peptidoglycan samples can be visualized. Individual reducing muropeptides were covalently labeled with the fluorescent molecule 8-aminonaphthalene-1,3,6-trisulfonic acid, after which they were separated by electrophoresis through a 35% polyacrylamide gel and visualized by exposure to UV light. FACE detected the appropriate numbers of reducing muropeptides in the proper proportions for four bacteria: Escherichia coli, Pseudomonas aeruginosa, Enterobacter cloacae, and Yersinia enterocolitica. As little as 2 to 5 pmol per muropeptide was detected when the intensity of the fluorescent signal was measured with a charge-coupled device camera, at a level of sensitivity between 50 and 250 times higher than that of the classic HPLC technique. Thus, FACE may be used to identify interesting peptidoglycan samples prior to more-extensive analysis by HPLC, or FACE may eventually replace HPLC for some applications.

Topics: Amino Acid Sequence; Chromatography, High Pressure Liquid; Disaccharides; Electrophoresis; Enterobacter cloacae; Escherichia coli; Fluorescent Dyes; Molecular Sequence Data; Muramidase; Mutation; Naphthalenes; Peptidoglycan; Pseudomonas aeruginosa; Sensitivity and Specificity; Yersinia enterocolitica