Correlation between structure and function of heparin

We have fractionated crude porcine heparin to obtain highly active as well as relatively inactive species of molecular weight ≈7000 with specific anticoagulant activities of 360 and 12 units/mg, respectively. Nitrous acid degradation of both of these polymers yielded a tetrasaccharide fraction, 1β, that contained equimolar amounts of iduronic and glucuronic acids, possessed an internal N -acetylated glucosamine, and carried anhydromannitol at the reducing end position. The 1β tetrasaccharide derived from the highly active heparin, 1β a , was recovered in a yield of 1.1 mol/7000 daltons. Our analyses indicate that at least 95% of the 1β a is a single structure that consists of the following unique monosaccharide sequence: L-iduronic acid → N -acetylated D-glucosamine-6-sulfate → D-glucuronic acid → N -sulfate D-glucosamine-6-sulfate. The 1β tetrasaccharide fraction from relatively inactive mucopolysaccharide, 1β i , was recovered in a yield of 0.3 mol/7000 daltons and was a mixture of several components. Only 8.5% of the 1β i tetrasaccharide fraction exhibited the same uronic acid placement and sulfate group position found in 1β a . Thus, 2.6% of relatively inactive mucopolysaccharide molecules contain the unique tetrasaccharide sequence found within each molecule of highly active heparin. Given the correlation between abundance of this unique 1β a tetrasaccharide sequence and biologic potency, we suggest that this structure represents the critical site responsible for anticoagulant activity.