The latent ampholytic nature of glycosaminoglycan (GAG) oligosaccharides facilitates their separation by isoelectric focusing

Abstract

Glycosaminoglycans are medically and biologically important polysaccharides. Glycosaminoglycan oligosaccharides are of increasing pharmaceutical relevance but, oligosaccharides from two in particular, heparan sulfate and heparin, are difficult to separate owing to their structural complexity. This hinders further progress because it is difficult to obtain pure saccharides in sufficient quantities. The ampholytic nature of heparin and heparan sulfate is realised by reversible de-N-sulfation or de-N-acetylation to reveal free amine groups, providing an opportunity to separate them using isoelectric focusing. Ampholytic heparan sulfate and heparin saccharides are characterised by low pI values, and isoelectric focusing required bespoke polyacrylamide gels capable of supporting pH gradients below pH 2. Representative oligosaccharide pools from heparin and chemically modified heparin, which had been fractionated previously to the best current levels by high performance anion exchange chromatography and gel permeation chromatography, were further separated and focused. The isoelectric focusing method provides an additional separation dimension for glycosaminoglycan oligosaccharides and has implications for the future development of related semi-preparative techniques.