New electrophoretic and chromatographic techniques for analysis of heparin and heparan sulfate

Heparin (HE) and heparan sulfated glycosaminoglycans are well‐known mediators of tissue development, maintenance and functions; the activities of these polysaccharides are depending mainly on their sulfate substitutions. The HE structure is also a very important feature in antithrombotic drug development, since the antithrombin binding site is composed by sequences of a specific sulfation pattern. The analysis of disaccharide composition is then a fundamental point of all the studies regarding HE/heparan sulfate glycosaminoglycan (and thereby proteoglycan) functions. The present work describes two analytical methods to quantify the disaccharides constituting HE and heparan sulfate chains. The use of PAGE of fluorophore‐labeled saccharides and HPLC coupled with a fluorescence detector allowed in one run the identification of 90–95% of HE disaccharides and 74–100% of rat kidney purified heparan sulfate. Moreover, the protocol here reported avoid the N ‐sulfation disaccharides degradation, which may affect N ‐sulfated/ N ‐acetylated disaccharides ratio evaluation. These methods could be also very important in clinical treatments since they are useful for monitoring the availability kinetics of antithrombotic drugs, such as low‐molecular‐weight HEs.