Heparan Sulfate Identification and Characterisation: Method II. Enzymatic Depolymerisation and SAX-HPLC Analysis to Determine Disaccharide Composition

Abstract

Heparan sulfate (HS) is purified from complex matrices and often not fully characterised to validate its assignment. The characterisation of heparins and heparan sulfates through enzymatic depolymerisation and subsequent strong anion-exchange high performance liquid chromatography (SAX-HPLC) analysis and quantitation of the resulting disaccharides is a critical tool for assessing the structural composition of this class of compound. This protocol details a methodology to reproducibly determine the disaccharide composition of heparan sulfate by enzymatic depolymerisation and SAX-HPLC analysis. A complementary method for identification and characterisation of heparan sulfate can be found at Carnachan and Hinkley (2017).

Keywords: Chemical characterization; Enzymatic depolymerisation; Glycosaminoglycan’s; HPLC; Heparan sulfate.

Figure 1.. HPLC chromatogram showing resolution of the twelve heparin-derived disaccharides present in the standard mix.

Retention times and species: 2.158 min, Δ-UA-GlcN; 5.092 min, Δ-UA-GlcNAc; 6.567 min, Δ-UA-GlcN(6S); 8.425 min, Δ-UA(2S)-GlcN; 12.175 min, Δ-UA-GlcNS; 14.817 min, Δ-UA-GlcNAc(6S); 16.942 min, Δ-UA(2S)-GlcNAc; 24.092 min, Δ-UA(2S)-GlcN(6S); 26.233 min, Δ-UA-GlcNS(6S); 28.950 min, Δ-UA(2S)-GlcNS; 36.208 min, Δ-UA(2S)-GlcNAc(6S); 44.775 min, Δ-UA(2S)-GlcNS(6S). The elution order of the twelve disaccharide standards was initially determined by running each species separately under the chromatographic conditions described above.

Figure 2.. HPLC chromatogram of a typical enzymatic digest of commercial porcine mucosal heparan sulfate showing separation of the Δ-disaccharide constituents.

Peaks marked with an asterisk represent uncharacterized species that appear reproducibly in chromatographic runs.