Electrophoresis Separation of Glycosaminoglycans on Nitrocellulose Membranes

Abstract

A rapid and simple electrophoretic separation method on nitrocellulose membranes by a single run of glycosaminoglycans, mainly slow-moving and fast-moving heparins, dermatan sulfate, and chondroitin sulfate, is reported. Different dyes, azure A, toluidine blue, and Alcian blue, were used to stain the nitrocellulose strips, and azure A showed the highest sensitivity. The mobility for slow-moving heparin was 0.00 cm/A/min, for fast-moving species about 2.87 × 10−3cm/A/min, for dermatan sulfate 4.00 × 10−3cm/A/min, and for chondroitin sulfate about 5.75 × 10−3cm/A/min. Calibration curves were determined by the increasing amounts (from 0.1 to 2.5 μg) of single glycosaminoglycan species. The curves demonstrated a linear relationship between the amount of glycosaminoglycans (slow-moving and fast-moving heparin, dermatan sulfate, and chondroitin sulfate) and the optical density at 600 nm calculated by densitometric scanning. The limit of detection for fast-moving heparin, dermatan sulfate and chondroitin sulfate was about 0.2–0.3 μg, while that for slow-moving heparin was 0.1 μg. Hyaluronic acid, keratan sulfate, and highly sulfated chondroitin sulfate from shark cartilage were also separated electrophoretically on nitrocellulose. No difference in mobility was detected for chondroitin sulfates with different structure and physicochemical properties. Hyaluronic acid has about the same mobility as that of dermatan sulfate, while keratan sulfate has a mobility intermediate between those of dermatan sulfate and chondroitin sulfate (about 4.75 × 10−3cm/A/min). Glycosaminoglycans extracted and purified from bovine aorta and bovine lung were submitted to electrophoresis on nitrocellulose and the amount of each polysaccharide species was calculated by densitometric scanning. By this analysis bovine aorta glycosaminoglycans consist of about 11% fast-moving heparin (heparan sulfate), 23% dermatan sulfate, and 65% chondroitin sulfate. Bovine lung polysaccharides are composed of about 16% slow-moving and 46% fast-moving species (62% heparin), 15% dermatan sulfate, and 22% chondroitin sulfate.