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The importance of 3-O- and 6-O-sulfated glucosamine residues within the heparin octasaccharide iduronic acid(1)----N-acetylglucosamine 6-O-sulfate(2)----glucuronic acid(3)----N-sulfated glucosamine 3,6-di-O-sulfate(4)----iduronic acid 2-O-sulfate(5)----N-sulfated glucosamine 6-O-sulfate(6)----iduronic acid 2-O-sulfate(7)----anhydromannitol 6-O-sulfate(8) was determined by comparing with synthetic tetra- and penta-saccharides its ability to bind human antithrombin. The octasaccharide had an affinity for antithrombin of 1 X 10(-8) M (10.2 kcal/mol) measured by intrinsic fluorescence enhancement at 6 degrees C. The synthetic pentasaccharide, consisting of residues 2-6, had an affinity of 3 X 10(-8) M (9.6 kcal/mol). The same pentasaccharide, except lacking the 3-O-sulfate on residue 4, had an affinity of 5 X 10(-4) M (4.5 kcal/mol) measured by equilibrium dialysis. The tetrasaccharide, consisting of residues 2-5, bound antithrombin with an affinity of 5 X 10(-6) M (6.8 kcal/mol). The tetrasaccharide, consisting of residues 3-6, had an affinity of 5 X 10(-5) M (5.5 kcal/mol). Since the loss of either the 6-O-sulfated residue 2 or the 3-O-sulfate of residue 4 results in a 4-5 kcal/mol or a 40-50% loss in binding energy of the pentasaccharide, these two residues must be the major contributors to the binding and must be linked to the biologic activity of the octasaccharide.

The chemical composition and the 13C n.m.r. spectra of heparin oligosaccharides (essentially octasaccharides), having high affinity for antithrombin III and high anti-(Factor Xa) activity, prepared by three independent approaches (extraction, partial deaminative cleavage with HNO2 and partial depolymerization with bacterial heparinase), leading to different terminal residues, have been studied and compared with those of the corresponding inactive species. Combined with chemical data, the spectra of the active oligosaccharides and of their fragmentation products afforded information on composition and sequence. The three types of active oligosaccharides were shown to have the common hexasaccharide core

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The serum thymic factor (FTS) utilized in its synthetic or natural form loses its biological activity in a rosette assay after treatment with a metal ion-chelating agent, Chelex 100. This activity is restored by the addition of Zn salts and, to a lesser extent, certain other metal salts. FTS activation is secondary to the binding of the metal to the peptide. The metal-to-peptide molar ratio of 1:1 provides the best activation. These data indicate the existence of two forms of FTS. The first one lacks Zn and is biologically inactive; the second one contains Zn and is biologically active, for which we propose the name of "thymulin" (FTS-Zn). The presence of Zn in synthetic FTS was confirmed by atomic absorption spectrometry. The interaction between Zn and FTS was fiurther suggested by microanalysis demonstrating the presence of this metal in thymic reticuloepithelial cells.

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