Tritiation of commercial heparins by reaction with NaB 3H 4: Chemical analysis and biological properties of the product

Abstract

A simple method to tritiate commercial samples of heparin is described. Heparin is reacted with NaB 3H 4 at pH 8.0 for 3 h at room temperature, after which the 3H-labeled mucopolysaccharide is isolated by gel filtration. Using NaB 3H 4 of low specific radioactivity (227–555 mCi/mmol), [ 3H]heparins containing 3.7–8.0 × 10 5 dpm/mg are made whereas with NaB 3H 4 of higher specific activity (5–10 Ci/mmol), preparations of 1.2–1.8 × 10 7 dpm/mg are obtained. From analysis of [ 3H]heparin using several techniques (periodate oxidation; mild acid hydrolysis accompanied by ion-exchange chromatography and high-voltage electrophoresis), the labeling procedure largely relies on a small proportion (approximately 4–6%) of heparin molecules possessing a terminal monosaccharide which, on reaction with NaB 3H 4, is reduced to yield an alditol. Consequently, 3H is incorporated on C 1 of this modified terminus. The product, [ 3H]heparin as the Na salt, is very stable under normal conditions of treatment and storage. The behavior of [ 3H]heparin when chromatographed on Sepharose-antithrombin III and on Sepharose-thrombin compares closely with that of unlabeled heparin. However, gel filtration on Sephadex G-200 reveals that [ 3H]heparin ( V e V 0 = 2.19 ) chromatographs more slowly than native heparin ( V e V 0 = 2.08 ), a feature which may reflect the true nature of the constituent molecules present in the heparin sample.