New electrophoretic and chromatographic techniques for analysis of heparin and heparan sulfate |
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Authors: | Viola Manuela Vigetti Davide Karousou Evgenia Bartolini Barbara Genasetti Anna Rizzi Manuela Clerici Moira Pallotti Francesco De Luca Giancarlo Passi Alberto |
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Affiliation: | Department of Experimental and Clinical Biomedical Sciences, University of Insubria, Varese, Italy. |
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Abstract: | 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. |
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Keywords: | 2‐Aminoacridone derivatization Fluorescence Glycosaminoglycan Heparin Polyacrylamide gel electrophoresis of fluorophore‐labeled saccharides |
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