Materials and Methods

Preparation of Octasaccharide. Porcine mucosal heparin (7.5 g of batch no. 41681 at 169 USP units/mg, Diosynth Inc., Chicago, IL) was dissolved in 150 mL of cold 0.2 M citric acid and the pH adjusted to 1.5 with concentrated sulfuric acid. Sodium nitrite was added to a final concentration of 0.05 M. The reaction proceeded for 9 min at 0 C and was quenched with excess ammonium sulfamate (0.075 M). The reaction mixture was precipitated with a final concentration of 80%(v/v) cold ethanol, centrifuged at lOOOOg for 15 min, dissolved in a minimum volume, and gel filtered at a flow rate of 270 mL/h on a polyacrylamide P-10 (Bio-Rad) column (4.7 X 200 cm) in 0.5 M ammoniumbicarbonate. The octasaccharide fraction was selected from well-resolved peaks ranging from disaccharide to dodecasaccharide as monitored by absorbance at 254 nm and colorimetric assay of uronicacid (Bitter & Muir, 1962) and rechromatographed on the same column to remove traces of residual hexasaccharide and decasaccharide (Oosta et al., 1981). The octasaccharides from four prepa-rations as described above were combined for a total yield of

~ 1.5 g.

Affinity Fractionation. Bovine antithrombin (1.5 g) was added to the octasaccharide (1.3 g) at final concentrations of 450 jiM and 10 mM, respectively, in 0.01 Mtris (hydroxy-methyl) aminomethane hydrochloride (Tris-HCl) containing 0.15 M NaCl, pH 7.5. The solution, in four 13-mL additions, was applied to polyacrylamide P-100 columns (2.5 X 100 cm), equilibrated in the same buffer, and gel filtered at30 mL/h. The column effluent was monitored by absorbance at 280 nm and colorimetric assay of uronic acid (Bitter & Muir, 1962). The antithrombin-octasaccharide complex, which completely