Tritiation of commercial heparins by reaction with NaB3H4: Chemical analysis and biological properties of the product

Abstract

A simple method to tritiate commercial samples of heparin is described. Heparin is reacted with NaB3H4 at pH 8.0 for 3 h at room temperature, after which the 3H-labeled mucopolysaccharide is isolated by gel filtration. Using NaB3H4 of low specific radioactivity (227–555 mCi/mmol), [3H]heparins containing 3.7–8.0 × 105 dpm/mg are made whereas with NaB3H4 of higher specific activity (5–10 Ci/mmol), preparations of 1.2–1.8 × 107 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 NaB3H4, is reduced to yield an alditol. Consequently, 3H is incorporated on C1 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 (Ve/V0 = 2.19) chromatographs more slowly than native heparin (Ve/V0 = 2.08), a feature which may reflect the true nature of the constituent molecules present in the heparin sample. © 1980, All rights reserved.