Heparins, Low-Molecular-Weight Heparins, and Other Glycosaminoglycans Analyzed by Agarose Gel Electrophoresis and Azure A-Silver Staining

Abstract

A sensitive, nonradioactive azure A-silver staining method combining agarose gel electrophoresis was established and evaluated. Unfractionated heparins (UFHs), low-molecular-weight heparins (LMWHs), heparan sulfate (HS), chondroitin sulfate A (CSA), dermatan sulfate (DS), keratan sulfate (KS), and hyaluronic acid (HA) were analyzed. The detection limit of the method was 0.5 ng for heparin, LMWH, HA, CSA, and DS, 2 ng for KS, and 6 ng for HA in the 2-μl sample volume. Dilution curves demonstrated linear correlation between the logarithm of the concentration of glycosaminoglycans (GAGs) and their optical absorbance at 548 nm. The linear ranges were 1 to 500 ng/μl for heparins, LMWHs, HS, DS, and CSA, 3 to 500 ng/μl for KS, and 8 to 500 ng/μl for HA. GAGs have their characteristic migration patterns and their R _ƒ value decreased from CSA to KS, DS, HS, heparin, and HA. The differences were described for heparins and LMWHs. LMWHs migrated faster and displayed broader bands than unfractionated heparins. It was also observed that some unfractionated heparins contained low sulfated GAGs as contamination, which seemed to be DS as judged by their migration patterns.

Keywords:

Azure A-silver staining - agarose gel electrophoresis - heparin - low-molecular-weight heparin - glycosaminoglycan