Isolation of N-Acetyllactosamine and Galactosyl-β-d-(1 → 4)-N-acetyllactosamine from Beef Brain Glycopeptides

Abstract

Abstract Forty-eight beef brains were dehydrated with acetone, extracted with chloroform-methanol solutions to remove lipid, and digested with Pronase. The glycopeptides were quantitatively recovered after gel filtration on P-4 resin (yield, 98 g with a carbohydrate content of 55%). The glycopeptides were further purified by chromatography on DEAE-cellulose and P-30 resin to give three fractions, obtained in a total yield of 75%, based on the fucose content of the Pronase digest. The three fractions were analyzed for pentoses, hexoses, hexosamines, hexuronic acid, sialic acid, sulfate, phosphorus, nitrogen, fatty acid, and peptide. Two of the fractions, which were free of fatty acid and hexuronic acid, were pooled (total weight, 5.6 g), desialized, and subjected to partial acid hydrolysis (pH 1.1, 100°, 90 min). The partial hydrolysate was fractionated by gel filtration on P-2 resin to give four peaks. Peak 3 was further purified by DEAE-cellulose and charcoal-Celite chromatography, and yielded 26 mg of a substance that was identified as N-acetyllactosamine by its composition, reduction with NaBH4 to give galactose and N-acetylglucosaminitol, complete cleavage with β-galactosidase, optical rotation, resistance to heating under alkaline conditions, and cochromatography with authentic N-acetyllactosamine in two solvent systems and with borate electrophoresis. Its identity was confirmed by mass spectroscopy of the per(trimethylsilyl)ated derivative. Peak 2 of the partial acid hydrolysate was further purified by deionization and preparative paper chromatography (yield, 3 mg). The substance contained 2 moles of galactose and 1 mole of N-acetylglucosamine. Reduction with NaBH4 established that the N-acetylglucosamine occupied the reducing end. Extended hydrolysis with β-galactosidase quantitatively cleaved the trisaccharide to monosaccharides. Partial hydrolysis with β-galactosidase yielded galactose and N-acetyllactosamine. Smith degradation gave erythritol in an 85% yield. These results indicated that the trisaccharide was galactosyl-β-d-(1 → 4)-N-acetyllactosamine.