The alkali labile linkage between keratan sulphate and protein

Keratan sulphate was isolated from adult intervertebral disc in 90% yield by sequential digestion of the whole tissue with papain, Pronase and Proteus vulgaris chondroitin sulphate lyase. Treatment of this preparation with alkali cleaved a glycosidic bond between N acetylgalactosamine and threonine and produced, by an alkali catalyzed 'peeling' reaction, an unsaturated derivative of N acetylgalactosamine which reacted as a chromogen in the Morgan Elson reaction, but remained covalently bonded to the keratan sulphate chain. This derivative was reduced and labelled by alkaline NaB³H₄. The substituent at position 3 of N acetylgalactosamine in the keratan sulphate/protein linkage was identified as a disaccharide, N acetylneuraminylgalactose, which was isolated from the reaction mixture after alkali treatment.