An application of ellipsometry. Assessment of polysaccharide and glycoprotein interaction with lectin at a liquid/solid interface.
Lectins were specifically adsorbed from solution onto metallized glass slides coated with polysaccharide, glycopeptide and glycoprotein films. The degree of interaction was determined by measuring the thickness of the bound lectin layer with an ellipsometer after washing and drying the slide. The binding of concanavalin A (tetrameric) and succinyl concanavalin A (dimeric) to a yeast mannan film was studied as a function of lectin concentration, temperature, rinsing time and the extent of stirring of the slide. The maximum thickness of bound concanavalin A and succinyl concanavalin A was 11 amd 3.8 nm, respectively. The method permitted the measurement of the association constants for both lectins (1.0 x 10(7) M-1 for concanavalin A, 2 x 10(6) M-1 for succinyl concanavalin A) and the detection of 0.6 pmol concanavalin A. The same sensitivity was observed with anti-mannan antibodies. The binding of both lectins was shown to be specific using sugar haptens. When compared with methyl alpha-D-mannoside, the affinity of concanavalin A for D-mannose and D-glucose was 14 and 3%, respectively. A film of mucin glycopeptide (universal adsorbent) interacted similarly with concanavalin A, Ricinus communis I, soya bean and wheat germ lectins. However, films of glycoproteins such as fetuin, ceruloplasmin and Aspergillus niger beta-D-galactosidase interacted to different degrees with those lectins. The relative affinity of wheat germ agglutinin for N-acetyl-D-glucosamine and for chitin-derived oligosaccharides was also determined. When films of sialoglycoproteins were treated wih neuraminidase, the thickness of the bound peanut agglutinin layer increased. Although this method cannot determine quantitatively the sugar composition of the film, it permits rapid estimation of the interaction of lectins with polysaccharides and glycoproteins, using little material.
D000042:Absorption; D000906:Antibodies; D003208:Concanavalin A / Q000031:analogs & derivatives; D006020:Glycopeptides; D006023:Glycoproteins; D037102:Lectins; D008351:Mannans; D011134:Polysaccharides; D013499:Surface Properties
D000906:Antibodies; D006020:Glycopeptides; D006023:Glycoproteins; D037102:Lectins; D008351:Mannans; D011134:Polysaccharides; C026887:biotinylconcanavalin A; D003208:Concanavalin A; C019558:succinylconcanavalin A