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.