chondroitin-sulfates and 8-amino-1-3-6-naphthalenetrisulfonic-acid

Fluorophore-assisted carbohydrate electrophoresis (FACE) was applied to determine the molecular mass (M) values of various chondroitin sulfate (CS) samples. After labeling with 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS), FACE was able to resolve each CS sample as a discrete band depending on the M value. After densitometric acquisition, the migration distance of each CS standard was acquired and the third grade polynomial calibration standard curve was determined by plotting the logarithms of the M values as a function of migration ratio. Purified CS samples of different origin and the European Pharmacopeia CS standard were analyzed by both FACE and conventional high-performance size-exclusion liquid chromatography (HPSEC) methods. The molecular weight value on the top of the chromatographic peak (M(p)), the number-average M(n), weight-average M(w), and polydispersity (M(w)/M(n)) were examined by both techniques and found to be quite similar. This study demonstrates that FACE analysis is a suitable, sensitive and simple method for the determination of the M values of CS macromolecules with possible utilization in virtually any kind of research and development such as quality control laboratories.

Topics: Animals; Calibration; Cattle; Chickens; Chondroitin Sulfates; Chromatography, Gel; Chromatography, High Pressure Liquid; Electrophoresis; Fluorescent Dyes; Molecular Weight; Naphthalenes; Reference Standards; Reproducibility of Results; Swine

Various combinations of fluorescent dyes, polyacrylamide gels, and electrophoresis buffers were tested by fluorophore-assisted carbohydrate electrophoresis (FACE) for the purpose of analyzing sulfated and nonsulfated glycosaminoglycan (GAG) oligosaccharides in which disaccharides and low-molecular weight oligosaccharides were included. A nonionic fluorescent dye was found to be suitable for analyzing sulfated disaccharides derived from sulfated GAGs (e.g., chondroitin sulfate, dermatan sulfate) because sulfated disaccharides themselves had enough anionic potential for electrophoresis. The migration rates of chondroitin sulfate (CS) disaccharides in polyacrylamide gels were affected by the number of sulfate residues and the conformation of each disaccharide. When an anionic fluorescent dye, 8-aminonaphthalene-1,3,6-trisulfonic acid disodium salt (ANTS), was coupled with sulfated GAG oligosaccharides, nearly all of the conjugates migrated at the electrophoretic front due to the added anionic potential. Nonsulfated hyaluronan (HA) oligosaccharides (2-16 saccharides) were subjected to electrophoresis by coupling with a nonionic fluorescent dye, 2-aminoacridone (AMAC), but did not migrate in the order of their molecular size. Especially di-, tetra-, hexa-, and octasaccharides of HA migrated in the reverse order of their molecular size. HA/CS oligosaccharides were able to migrate in the order of their chain lengths by coupling with an anionic fluorescent dye in a nonborate condition.

Topics: Aminoacridines; Animals; Borates; Chondroitin Sulfates; Decapodiformes; Disaccharides; Electrophoresis, Polyacrylamide Gel; Fluorescent Dyes; Glycosaminoglycans; Hyaluronic Acid; Molecular Structure; Molecular Weight; Naphthalenes; Oligosaccharides; Whales