Glycosaminoglycans and proteins: different behaviours in high-performance size-exclusion chromatography

The influence of the conformation of globular proteins and glycosaminoglycans in high-performance size-exclusion chromatography (HPSEC) was studied. Glycosaminoglycans (heparin, chondroitin sulphate and dermatan sulphate) with different primary structures, sulphate-to-carboxyl ratios and physico-chemical properties were extracted and purified. Their physico-chemical properties and purity were evaluated by several analytical techniques. Glycosaminoglycans with different relative molecular masses (M(r)) were prepared by a chemical depolymerization process. These heteropolysaccharides were evaluated by HPSEC and compared with globular proteins of known relative molecular mass. The two third-degree polynomial regression curves for proteins and glycosaminoglycans have different coefficients and the columns present different exclusion limits. In particular, under the experimental conditions, the M(r) exclusion limits for high M(r) are 44,000 for glycosaminoglycans and 240,000 for globular proteins. In contrast, the behaviours of these two classes of macromolecules are similar for lower M(r). In fact, the two third-degree polynomial curves show the same regression below about M(r) = 1000. The behaviour in HPSEC is discussed in relation to the different steric conformations for proteins and glycosaminoglycans with different relative molecular masses.