Improved method for analysis of glycosaminoglycans in glycosaminoglycan/protein mistures: application in Cohn-Oncley fractions of human plasma

Abstract: BACKGROUND: Glycosaminoglycans are found in human tissues including plasma. They encompass chondroitin sulphates, heparan sulphate/heparin, hyaluronic acid, and keratan sulphate. Glycosaminoglycans, in particular heparan sulphate and heparin, are strongly associated with plasma proteins, so that their purification results quite difficult. METHODS: In order to study the distribution of glycosaminoglycans in plasma subfractions, we developed a novel method that allows their identification even if they were still associated with proteins or peptides. Plasma was fractionated following the procedure of Cohn-Oncley, and each fraction was treated with proteases. After centrifugation, glycosaminoglycan/protein complexes in the supernatant were analysed using a modified cellulose acetate electrophoresis which allowed identification of glycosaminoglycans in mixtures of glycosaminoglycans/proteins. RESULTS: Chondroitin sulphate was recovered in cryoprecipitate and in all Cohn-Oncley fractions. Glycosaminoglycans belonging to the class of heparan sulphate/heparin, however, were recovered in the cryoprecipitate and in fractions I and IV-1, and, in smaller amount, in fraction II+III. CONCLUSIONS: Since the largest amount of plasma proteins is partitioned in Factions II+III and V, these results demonstrate that heparan sulphate/heparin are not randomly distributed in Cohn-Oncley fractions and are associated with certain plasma proteins. This association might play a role in the physiological function of heparan sulphate/heparin, regulating hemostasis and atherogenesis.