Collect. Czech. Chem. Commun. 1992, 57, 2151-2156

Preparation and Characterization of the High Molecular Weight [3H]Hyaluronic Acid

Peter Chabrečeka, Ladislav Šoltésb, Hynek Hradecc, Jiří Filipc and Eduard Orviskýd

a Institute of Biotechnology, Slovak Technical University, 812 37 Bratislava
b Institute of Experimental Pharmacology, Slovak Academy of Sciences, 842 16 Bratislava
c Institute for Research, Production and Application of Radioisotopes, 102 27 Prague 10
d Research Institute of Rheumatic Diseases, 921 01 Piestany


Two methods for the preparation of high molecular weight [3H]hyaluronic acid were investigated. In the first one, hydrogen atoms in the molecule were replaced by tritium. This isotopic substitution was performed in aqueous solution using Pd/CaCO3 as the catalyst. In the second method, the high molecular weight hyaluronic acid was alkylated with [3H]methyl bromide in liquid ammonia at a temperature of -33.5 °C. High-performance gel permeation chromatographic separation method was used for the isolation and characterization of the high molecular weight [3H]hyaluronic acid. Molecular weight parameters for the labelled biopolymers were Mw = 128 kDa, Mw/Mn = 1.88 (first method) and Mw = 268 kDa, Mw/Mn = 1.55 (second method). The high molecular weight [3H]hyaluronic acid having Mw = 268 kDa was degraded further by specific hyaluronidase. Products of the enzymatic depolymerization were observed to be identical for both, labelled and cold biopolymer. This finding indicates that the described labelling procedure using [3H]methyl bromide does not induce any major structural rearrangements in the molecule.