High-resolution polyacrylamide gel electrophoresis of carbohydrates derivatized with a visible dye.
A technique for carbohydrate analysis that is both inexpensive and easily performed is currently unavailable. In this communication we address the problem and have outlined a method for labeling saccharides with a visible dye, 4-amino-1,1'-azobenzene-3, 4'-disulfonic acid, which has an absorption maximum of 489 nm and an extinction coefficient of 37,615, to facilitate visible detection at low levels. The visible dye was coupled by a reductive amination to different sugars. The sugar-dye adducts were then separated by electrophoresis on alkaline polyacrylamide gels. The gels were scanned with a densitometer, and visible sugar-dye adducts were qualitatively analyzed by identifying them according to their mobilities. The sugar-dye adducts were quantified by determining their densitometric volume. The kinetics of the reductive amination reactions, performed at 37 degrees C, were different for each of three saccharides tested. The rate constants for glucose and fucose were 1.31 times greater and 1.8 times greater, respectively, than that of maltotriose. The reductive amination reactions were essentially complete after approximately 16 h under the given experimental conditions. A linear dose-response relationship was observed between the amount of sugar (monosaccharide, trisaccharide, or heptasaccharide) in the reductive amination reaction. The quantity of saccharide-dye adduct that could be visually detected for glucose, maltotriose, and maltoheptaose, was 25, 25, and 50 nmol, respectively. Sugar-dye adducts were separated from one another by varying the acrylamide concentration in the polyacrylamide gels. Sugar-dye adducts of monosaccharides, disaccharides, trisaccharides, and heptasaccharides were separated on alkaline 30% polyacrylamide gels with mobilities of 0.778, 0.667, 0.639, and 0.375. Adducts of glucose, fucose, galactose, and mannose were separated with mobilities of 0.844, 0.833, 0.820, and 0.810, respectively, on a 30 to 40% gradient polyacrylamide gel. Adducts of glucose and glucose derivatives were separated on a 35% polyacrylamide gel. This technique provides an inexpensive and easily performed method of carbohydrate analysis to laboratories that do not have the highly trained personnel nor the expensive equipment needed for other methods of carbohydrate analysis. The method is most applicable to research problems where sensitivity (20 pmol) is not a problem. The simplicity of the method also makes it easily incorporated into teaching laboratories.
Westfall
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Division of Life Sciences, The University of Texas at San Antonio, 6900 North Loop 1604 West, San Antonio, Texas, 78249-0662, USA.
Flores
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Negrete
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Martinez
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Haro
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D002241:Carbohydrates / Q000032:analysis* / Q000737:chemistry; D004396:Coloring Agents / Q000737:chemistry; D003720:Densitometry; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D007700:Kinetics
D002241:Carbohydrates; D004396:Coloring Agents