Location of specific oligosaccharides in heparin in terms of their distance from the protein linkage region in the native proteoglycan.
Studies were conducted to define the location of components and sequences in heparin with respect to their distance from the peptide linkage in the native proteoglycan. A purified heparin-oligopeptide was linked via its amino terminus to a matrix containing an azo bond and an activated carboxyl group. The polysaccharide chain was maximally degraded, either with heparinase or nitrous acid, and the soluble products were removed. The heparin-oligopeptide fragments that remained on the matrix were released by reductive cleavage of the azo linkage and characterized. The fragments, as well as heparin released without prior degradation, contained serine and glycine as the principal amino acids; the ratio of galactose to xylose was 2:1. The ratio of glucosamine to serine of 33:1 in the undegraded heparin was reduced to 6:1 and 1:1 in the heparinase-treated and nitrous acid-treated products, respectively. The undegraded sample and the fragments contained phosphate in equivalent amounts, demonstrating its presence in the heparin-protein linkage region. The heparin-oligopeptide preparation was also fractionated by gel filtration and high and low molecular weight fractions thus obtained were each linked to the insoluble matrix. The products that were subsequently released were subfractionated on a molecular weight-calibrated column of Sephadex G-200, and eluates were assayed for activity in promoting the neutralization of thrombin and factor Xa by antithrombin. The results revealed a sharp decrease in specific activity in heparin-oligopeptide fractions below Mr = 15,000 indicating that the anticoagulant-conferring segment is located at about 20 disaccharide units away from the peptide linkage region.
Rosenfeld
L
L
Department of Biochemistry, New York Medical College, Valhalla 10595.
Danishefsky
I
I
D000596:Amino Acids / Q000032:analysis; D002241:Carbohydrates / Q000032:analysis; D006493:Heparin; D008970:Molecular Weight; D009842:Oligopeptides / Q000032:analysis; D009844:Oligosaccharides / Q000302:isolation & purification; D011509:Proteoglycans
D000596:Amino Acids; D002241:Carbohydrates; D009842:Oligopeptides; D009844:Oligosaccharides; D011509:Proteoglycans; D006493:Heparin