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

✍ Scribed by M.W.C. Hatton; L.R. Berry; R. Machovich; E. Regoeczi

• Publisher: Elsevier Science
• Year: 1980
• Tongue: English
• Weight: 834 KB
• Volume: 106
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(80)90542-4

✦ Synopsis

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 NaB3& of low specific radioactivity (227-555 mCi/mmol), [3H]heparins containing 3.7-8.0 x 105 dpm/mg are made whereas with NaB3H, of higher specific activity (5-10 Wmmol), preparations of 1.2-1.8 X IO' 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 C, 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 t3H]heparin (VJV, = 2.19) chromatographs more slowly than native heparin (VJV,, = 2.08), a feature which may reflect the true nature of the constituent molecules present in the heparin sample.