Analytical and Preparative Strong Anion-Exchange HPLC of Heparan Sulfate and Heparin Saccharides

Abstract

Studies on the structure-function relationships of the complex linear polysaccha-rides, known as glycosaminoglycans (GAGs), are becoming increasingly important as biological functions are established for them. However, structural analysis of GAGs presents a difficult technical problem, particularly in the case of the N-sulfated GAGs heparan sulfate (HS) and heparin, which display remarkable structural diversity (1). A widely used and effective approach is to degrade the chains into smaller saccharide units that can then be separated and analyzed. In this regard, strong anion-exchange (SAX) HPLC techniques have proved particularly useful for both the analysis of disac-charide composition (2,3) and the separation of complex mixtures of larger saccharides (3–6). However, in many methods the columns used have been silica-based and suffer from drawbacks related to poor stability of the support (e.g., inconsistency of run times, peak broadening, and short column life). There is clearly a need for improvements in column performance, especially for the purification of larger saccharides to homogeneity for sequencing and bioactivity testing. This chapter describes how a single type of polymer-based SAX column, ProPac PA1, can be used to provide high-resolution separations of both disaccharides and larger oligosaccharides derived from HS and heparin, with consistent elution times and excellent column performance characteristics (see Notes 1 and 2). Disaccharides from chondroitin sulfate and dermatan sulfate can also be separated (see Note 3). The improved resolution of saccharides compared to other SAX-HPLC methods, combined with the versatility and longevity of the columns, makes them a valuable tool for purification and structural analysis of HS/heparin and other GAG saccharides.