Preparation and characterization of tetrasaccharides from beef-lung heparin

Partial N-desulfation of beef-lung heparin prior to degradative deamination with butyl nitrite and reduction with sodium borotritide yielded many large fragments. From these, a tetrasaccharide tetra-O-sulfate (II-4NH; 8% yield from heparin) and a mixture of tetrasaccharide tri-O-sulfates (II-3NHh; 6% yield) were isolated by sequential chromatography on Sephadex G-25 and DEAE-Sephadex. For these and the other tetrasaccharide preparations, the radioactive disaccharides produced by deamination, with and without subsequent relabelling with sodium borotritide, have been quantitatively determined by the methodology described in the preceding paper. In most cases, the results permit a unique reconstruction of the relative proportions of monosaccharide components and of their sequences in the compounds present. Tetrasaccharide II-4NH appeared homogeneous and has the structure (IdoA-SO₄)(GN-O-SO₄)(IdoA-SO₄)(anhMan-SO₄). In tetrasaccharide preparation II-3NHh, the preponderant species (57%) lacks ester sulfate at the terminal l-iduronic residue in the structure just mentioned, and five other species are present. By treatment of the tetra-O-sulfate with mild acid, tetrasaccharide preparations with 3, 2, 1, and no ester sulfate were produced and could be isolated. The isomeric tetrasaccharide tri-O-sulfate species have been partially resolved. Composition and sequence data are given for all of the preparations. The resolution of numerous small fractions suggests minor irregularities in the fine structure of heparin. Ion-exchange electrophoresis was applied to the acidic oligosaccharides and was found to be a useful technique.