Improved method for analysis of glycosaminoglycans in glycosaminoglycan/protein mixtures: Application in Cohn–Oncley fractions of human plasma

BackgroundGlycosaminoglycans 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.MethodsIn 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.ResultsChondroitin 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.ConclusionsSince 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.