High-performance gel permeation chromatography of glycosaminoglycans. Column calibration by gel electrophoresis.

High-performance gel permeation chromatography and sensitivity-enhanced polyacrylamide gel electrophoresis (SE-PAGE) have been combined to investigate the chromatographic properties of structurally related glycosaminoglycans. Enzymatically digested samples of hyaluronate, chondroitin 4-sulfate and dermatan sulfate were fractionated on a column series of TSK G4000SW and TSK G2000SW, eluted with 0.15 M sodium chloride. Isolated fractions were subjected to sensitivity-enhanced polyacrylamide gel electrophoresis for determination of molecular weight. These procedures allowed the rapid development of column calibration profiles for each glycosaminoglycan, under a given set of chromatographic conditions. The profiles obtained in 0.15 M sodium chloride yielded the following data: (1) at equal degrees of polymerization, hyaluronic acid chains have an apparently smaller hydrodynamic radius than the sulfated polymers, and (2) at equal molecular weights, all three glycosaminoglycans have approximately equal hydrodynamic radii.

Polyacrylamide-gel electrophoresis and Alcian Blue staining of sulphated glycosaminoglycan oligosaccharides.

Oligosaccharide fragments of glycosaminoglycans may be separated for rapid analysis by electrophoresis through a 10% polyacrylamide matrix. An extensive ladder-like set of bands is observed for partial testicular hyaluronidase digests of chondroitin 4- or 6-sulphate, and for dermatan sulphate. Co-electrophoresis of purified oligosaccharides has established that the major bands of these patterns represent fragments differing in chain length by one disaccharide unit, with the smallest fragments having the greatest mobility. Additional minor bands, representing heterogeneity in the repeating unit structure, are also observed. There are slight differences in the mobilities of oligosaccharides derived from the three major types of sulphated glycosaminoglycans. Alcian Blue is employed for visualization of the digest fragments. Sample loads of 5-10 micrograms per band appear optimum. The smallest oligosaccharide which may be stained by this method is the hexasaccharide. After consideration of this effect, a good correlation is found to exist between densitometric scans of the gel-electrophoretic patterns and gel-filtration chromatographic profiles based on uronic acid concentration.