Forschungs- & Entwicklungsinformationsdienst der Gemeinschaft - CORDIS

Set up of a mass spectrometric method for quantitative evaluation of products generated by heparanase digestion of heparin/HS oligosaccharides

With the aim of establishing a simple, reliable method for studying the cleavability by heparanase of heparin/heparan sulfate oligosaccharides in the absence and in the presence of heparanase inhibitors, an on-line liquid chromatography/mass spectrometric (ESI-MS) method was set up. The method works in the nanomolar range concentration of substrate and does not need derivatization (radioactive nor fluorofore) of the substrate or the products.

The method is based on mass identification of oligosaccharide fragments generated by heparanase and their quantification with reference to an internal stardard (a heparin disaccharide). In its most practical version, cleavage of oligosaccharidic substrates by the enzyme was monitored from the area of HPLC/mass signals of the substrate. Substrates used to set up the method were two pentasaccharides (AGAIA and AGAIA) and two octasaccharides containing the AGAIA sequence in different locations along the chain. (AGAIA is the typical sequence of the active site of heparin/HS for antithrombin. When part of larger oligosaccharides, previous studies had shown that heparanase cleaves this sequence between the G (glucuronic acid) and A (3-O-sulfated glucosamine residues.).

The present study showed that AGAIA is cleaved by the enzyme even when it is not part of larger oligosaccharides. The commercial availability of AGAIA makes it an ideal substrate to determine the specific activity of heparanase preparations in substitution of the currently used complex and difficult to reproduce heparan sulfates or HS proteoglycans extracted from animal organs. The present method is also suitable for rapid screening of potential heparanase inhibitors.

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