Purification and characterization of two types of Cytisus multiflorus hemagglutinin by affinity chromatography.
Two hemagglutinins were separated from extracts of Cytisus multiflorus seeds by successive affinity chromatographies on columns of galactose- and di- N-acetylchitobiose-Sepharose 4B. One was found to be inhibited by di- N-acetylchitobiose or tri- N-acetylchitotriose and shown to possess anti-H(O) activity [Cytisus-type anti-H(O) hemagglutinin designated as Cytisus multiflorus hemagglutinin I]. The other, which was not a blood group-specific hemagglutinin, was inhibited by galactose or lactose (hemagglutinin II). Hemagglutinins I and II were further purified by gel filtration on Sephacryl S-300. These preparations were homogeneous as judged by polyacrylamide gel electrophoresis and gel filtration. The molecular weights of the purified hemagglutinins I and II were found to be 86000 by sedimentation equilibrium analysis and 80000 by gel filtration. On disc gel electrophoresis in the presence of sodium dodecyl sulfate and dithiothreitol, both hemagglutinins gave a single component of a molecular weight of 42000 +/- 2000, suggesting that these hemagglutinins are dimeric proteins of two identical subunits. Hemagglutinins I and II contain 2.7% and 1.5% carbohydrate, respectively, and only very small amounts of cystine and methionine were detected, but they are rich in aspartic acid and serine. Treatment of human O erythrocytes with a purified H-decomposing enzyme (alpha-L-fucosidase from Bacillus fulminans abolished the agglutinability of the cells with hemagglutinin I. This indicates that the L-fucosyl residue is important even for the H-specificity detected by this di-N-acetylchitobiose-specific hemagglutinin I.
D000596:Amino Acids / Q000032:analysis; D000606:Amino Sugars / Q000032:analysis; D001407:Bacillus / Q000201:enzymology; D055598:Chemical Phenomena; D002621:Chemistry; D002846:Chromatography, Affinity / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004912:Erythrocytes / Q000187:drug effects; D006801:Humans; D066298:In Vitro Techniques; D037102:Lectins / Q000302:isolation & purification*; D008970:Molecular Weight
D000596:Amino Acids; D000606:Amino Sugars; D037102:Lectins