Characterization of heparins with different relative molecular masses (from 11,600 to 1600) by various analytical techniques

Abstract

Heparin was extracted and purified from beef intestinal mucosa, and its structure and physico-chemical properties, e.g. disaccharide pattern (by specific enzymatic cleavage), relative molecular mass and sulfate-to-carboxyl ratio, were evaluated by different techniques. Heparin fractions with different relative molecular mass (from M(r) = 7560 to M(r) = 1600) were prepared by chemical degradation and gel-permeation chromatography. The fractions were characterized with respect to relative molecular mass, disaccharide pattern, sulfate-to-carboxyl ratio and percentage of slow moving and fast moving components by agarose-gel electrophoresis. The percentage of the two heparin species was calculated by densitometric analysis and specific calibration curves. The amount of the slow moving component decreases with relative molecular mass. The disaccharide pattern is different for the two heparin fractions. The percentage of trisulfated disaccharide decreases and the amount of mono- and disulfated disaccharides increases with a decrease of the relative molecular mass. The charge density, evaluated as the sulfate-to-carboxyl ratio, also decreases with the molecular mass of the fractions. This study confirms the heterogeneity of the structure, as evaluated by the constituent disaccharides, of the physico-chemical properties, such as relative molecular mass and charge density, and of the relative amount of the two heparin components also for low molecular mass heparins particularly produced by chemical depolymerization in the presence of free radicals.