Interaction of monolayers of concanavalin A with mono- and polysaccharides. A study by means of infrared-attenuated total reflectance spectroscopy.
The effects of pH, Mn2+ and Ca2+ and urea denaturation on the interaction of monolayers of concanavalin A on saline with the polysaccharide dextran B-1355 and the monosaccharides methyl alpha-D-mannopyranoside and D-galactose have been investigated. Infrared absorption spectra of compressed monolayers of the protein and the protein-dextran complex coated on a germanium plate have been obtained by means of attenuated total reflectance spectroscopy. Except in one case of denaturation, the amide I absorption of concanavalin A peaked around 1631 cm-1, indicating a predominance of the beta-pleated sheet conformation, in agreement with its secondary structure in the solution and crystalline phases. The contribution to the absorbance of the concanavalin A-dextran films at 3300 cm-1 due to absorption by the O-H stretching modes of the polysaccharide is a measure of its binding. Increasing the pH from 6.1 to 7.5 appreciably reduced the dextran binding, at pH 9.3 the binding was zero. Adding 1 mM Mn2+ and Ca2+ to the subphase at pH 7.5 restored both the dextran binding and the affinity of concanavalin A for methyl alpha-D-mannopyranoside to that of the native protein at pH 6.1. At this latter pH, the weak binding of dextran to monolayers of demetallized concanavalin A (apo-concanavalin A) was also restored to that for the native molecule by the addition of these divalents. This indicates the requirement of concanavalin A for these ions to maintain the integrity of the saccharide-binding site. The loss of dextran binding with urea denaturation was also observed. These results parallel those for solutions of the protein, indicating the validity of the monolayer system for the study of these interactions.
D002118:Calcium; D003208:Concanavalin A; D003911:Dextrans; D006863:Hydrogen-Ion Concentration; D008345:Manganese; D009005:Monosaccharides; D011134:Polysaccharides; D011489:Protein Denaturation; D013053:Spectrophotometry; D013055:Spectrophotometry, Infrared; D014508:Urea
D003911:Dextrans; D009005:Monosaccharides; D011134:Polysaccharides; D003208:Concanavalin A; D008345:Manganese; D014508:Urea; D002118:Calcium