Evidence for the presence of a calcium-ion-binding site within fibrinogen fragment D [proceedings].

HCI in lOml of methanol and diluting to IOOml with butan-1-o1;just before use, 0.1 g of sodium dithionite was added. The dried electrophoresis sheets were sprayed with the reagent and developed at 120°C for 1 5min. With 4and 6-sulphated disaccharides a strong colour-positive reaction occurred with p-anisidine at the highest amount examined (20pg) and a detectable coloured product was observed at the lowest amount (156ng). The unsulphated disaccharide produced a weaker reaction, but was detected at a concentration of 1 pg. The p-anisidine reagent provided more than 50 times the sensitivity of alkaline AgN03. The best of several alkaline AgNO, reagents examined was that described by Trevelyan et al. (1950). Cartilage extracts of glycosaminoglycans, enzymes and buffers did not cause interference with the disaccharide-anisidine reaction. In an examination of the monosaccharide components of glycosaminoglycans, the amino sugars, glucosamine and galactosamine did not react withp-anisidine. Glucose, galactose and uronic acids gave a positive (brown) reaction confirming previous reports that this reagent reacts with reducing monosaccharides (Merck, 1966). Under the assay conditions described monosaccharides were not produced from the chondroitin sulphate. The electrophoresis conditions described produced migration distances of 9.5 cm for unsulphated disaccharide with the 4-sulphated disaccharide located between 12.5 and 14.5cm and the 6-sulphated disaccharide between 13.0 and 15.0cm. The desulphation of the 4-sulphated disaccharide by chondro-4-sulphatase readily permitted separation and identification of mixtures containing the two disaccharides. The depolymerization of chondroitin sulphate from articular cartilage extracts with chondroitin ABC lyase produced p-anisidine-detectable sulphated disaccharides after 45min at 37°C. The nature of the reaction between p-anisidine and the chondroitin sulphate disaccharides was not resolved. For the quantitative determination of microgram quantities of 4and 6-sulphated disaccharides the periodate/thiobarbituric acid and borate,$-dimethylaminobenzaldehyde assays were used (Elliott & Gardner, 1977).