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AND HYGSTEDT gel filtration is presented here as a fast, gentle method for the isolation of sialyloligosaccharides. With this method, monosialyllactose and disialyllactose have been isolated from cow colostrum as their stable sodium salts.
Methods
Colostrum. Fresh cow colostrum from the first two days after delivery was stored deep frozen.
Chemicals and Solvents. All chemicals and solvents were of analytical grade. Furthermore, solvents used for chromatography and analysis were redistilled before use. Sephadex G-25 fine (Pharmacia Fine Chemicals, Uppsala, Sweden) was used for gel filtration.
Ion-Exchange Resins. Dowex l-X8, 200400 mesh, was heated in a steam bath for 30 min in 2 N hydrochloric acid and allowed to settle for 10 min, after which the supernatant was discarded. The resin was then thoroughly washed with distilled water, transferred to a glass column, and converted to acetate form by passing 2 N aqueous sodium acetate through the column until the effluent was negative for chloride ions tested with a HNO,-AgNO, reagent. After washing with distilled water, the moist resin was stored at 4°C. Immediately before use it wa$ washed on a Biichner funnel with 0.1 N acetic acid and distilled water. Dowex 5OW-X8, lo&200 mesh, was twice slurried in 2 N sodium hydroxide and left for 1 hr. The resin was then washed several times on a Biichner funnel with 2 N hydrochloric acid and after that with distilled water, and finally stored at 4°C. Before use it was again washed with hydrochloric acid and distilled water.
Sialic Acids. Crystalline samples of N-acetyl-, N,O-diacetyl-, and Nglycolylnemaminic acids were obtained from Dr. L. Svennerholm. Neuraminidase.
The enayme was obtained from Behringwerke AG, Marburg Lahn, Germany (Vibrio cholerae Op 20261 II, 100 IU/ml)! essentially free from aldolase activity. Hydrolysis with neuraminidase was performed in 0.05 M acetate buffer, pH 5.5, 26 mM for NaCl and 1.5 mM for CaC12. Substrate was incubated with 10 IU enzyme in 0.5 ml buffer at 37°C for 12 hr.
Quantitative Determination. Sialic acid, calculated as N-acetylneuraminic acid, was determined by the resorcinol method (20). The amount of neutral sugars could be estimated approximately by comparing the absorptions at 580 and 450 mp. Free sialic acid was assayed according to Warren (21). The analytical determination of hexose was made with an orcinol method (22) using lactose as standard substance. Hexosamine was determined according to Elson and Morgan (23) with glucosamine as standard substance. Formaldehyde was assayed with the chromotropic