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.
Viola
Manuela
M
Department of Experimental and Clinical Biomedical Sciences, University of Insubria, Varese, Italy.
Vigetti
Davide
D
Karousou
Evgenia
E
Bartolini
Barbara
B
Genasetti
Anna
A
Rizzi
Manuela
M
Clerici
Moira
M
Pallotti
Francesco
F
De Luca
Giancarlo
G
Passi
Alberto
A
D000107:Acetylation; D000609:Aminoacridines / Q000032:analysis; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis; D006025:Glycosaminoglycans / Q000032:analysis; D006493:Heparin / Q000032:analysis* / Q000737:chemistry; D006497:Heparan Sulfate / Q000032:analysis* / Q000737:chemistry; D051381:Rats; D012680:Sensitivity and Specificity; D013552:Swine
D000609:Aminoacridines; D005456:Fluorescent Dyes; D006025:Glycosaminoglycans; D006493:Heparin; D006497:Heparan Sulfate; C013786:A73025; C072515:2-aminoacridone