Separation of 8-aminonaphthalene-1,3,6-trisulfonate (ANTS)-labeled oligomers containing galacturonic acid by capillary electrophoresis: application to determining the substrate specificity of endopolygalacturonases.
Slight modifications of published procedures (Stefansson, M. and Novotny, M., Carbohydr. Res. 1994, 258 1-9) allow separation by relative size of 8-amino-naphthalene-1,3,6-trisulfonate (ANTS)-labeled neutral and acidic oligosaccharides by electrophoresis in uncoated capillaries. The separations are performed at pH 2.5 to suppress both any charge from carboxylic acid groups on the oligosaccharides and electroosmotic flow. Utility of the procedure is demonstrated by its application to characterization of substrate specificity of endopolygalacturonases. The results show that both a fungal and a bacterial endopolygalacturonase need four adjacent nonesterified galacturonic acid residues in a pectin to be able to act.
Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater 74078-3035, USA.
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D019075:Electrophoresis, Capillary / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D005690:Galactose / Q000378:metabolism; D006603:Hexuronic Acids / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D009281:Naphthalenes / Q000737:chemistry*; D009844:Oligosaccharides / Q000032:analysis*; D011096:Polygalacturonase / Q000378:metabolism*; D013379:Substrate Specificity
D005456:Fluorescent Dyes; D006603:Hexuronic Acids; D009281:Naphthalenes; D009844:Oligosaccharides; C007819:galacturonic acid; C046465:8-amino-1,3,6-naphthalenetrisulfonic acid; D011096:Polygalacturonase; D005690:Galactose