Study On Purification And Characterization Of Ophioglossum Pedunculosum Desv Agglutinin

A novel antifungal lectin was isolated from the roots of Ophioglossum pedunculosum Desv by extraction, precipitation with (NH₄)₂SO₄, anion-exchange chromatography on DEAE-Sepharose column followed by gel filtration on a Sephacryl S-100 column. Ophioglossum pedunculosum Desv agglutinin(OPA) showed one protein band on SDS-PAGE, and its molecular weight was about 19.1KD. The molecular weight was also 19.1KD determined by gel filtration on Sephacryl S-100. Therefore, the intact OPA was considered to be a monomer glycoprotein. OPA was a potent agglutinin for rabbit erythrocyte and the effect concentration is 1.95μg/ml. It also can agglutinin group O of human red cells but was inactiveagainst group A and B. Hemagglutination inhibition assay suggested that its activity could not be inhibited by any of the mono- and disaccharides tested sugars, but thyroglobulin and Mannan showed strong inhibition of OPA hem-agglutination of rabbit erythrocytes.The hemagglutinating activity of the lectin remains stable after exposure to 40℃for 30min and in the pH range of 4-9. Its optimum pH was pH 8.0. Whe n heated at 90℃, The fluorescence spectra weakened a little and its activity fell much. This indicated that OPA was thermal-stable, acid-stable.The fluorescence spectrum of native OPA excited at 295nm showed a maximum peak at 328nm, theλmax of fluorescence emission spectrum blue-shifted 20nm compared with theλmax of free Trp (348nm), which indicated that tryptophan residues were embedded in the protein matrix allowing a modest water accessibility or located near the surface of the molecule. When OPA excited at 280nm, the characteristic peak of Tyr did not exist, and it showed that the fluorescence energy of Tyr was transformed to Trp and strength the fluorescence of Trp.Fluorescence quenching study on OPA with CsCl, KI, CHI and Acrylamide showed Acrylamide and CHI quenched the 100% fluorescence of the agglutinin, and KI quenched about 62.5% fluorescence of agglutinin gradually. However, CsCl almost didn't quench the fluorescence of OPA even when the concentration was 0.5mol/L. It was indicated that the tryptophan residues of OPA were located on the surface of the molecule and most of them were in hydrophobic areas or positively charged areas.Effects of different concentration of GuHCl and SDS on the hemagglutinating activity indicated the activity lost gradually corresponding to the addition of concentration of denaturant, but urea almost didn't have effects on hemagglutinating activity of OPA. Theλ_max and intensity of fluorescence emission spectrum of OPA increased with the concentration of GuHCl increasing.And then the maximum peak red-shifted and intensity decreased with the concentration of GuHCI increasing, accompanying by hemagglutination activity loss, when the concentration was raised to 6mol/L, the hemagglutining activity was completely lost. The intensity of fluorescence emission spectrum of OPA increased with increasing SDS concentration. When the concentration was raised to 20mM, the hemagglutining activity was completely lost.The effect of chemical modification of amino acid residues upon hemagglutination was investigated. There was a definite decrease of fluorescence intensity which corresponds to the oxidation of tryptophan. Under native conditions 1 tryptophan residues were available for modification by NBS, which caused 27% loss of hemagglutinating activity. The result suggested that Try residues might be located in near to the hemagglutinating activity, and probably be involved in the carbohydrate-binding site.Modification of Arg residues led to 70% loss in the hemagglutinating activity of the lectin, indicating the presence of Arg residues in the hemagglutinating activity site. However, modification of His and Tyr residues did not affect the hemagglutinating activities. The results suggested that these amino acid residues did not play any important role in the hemagglutinating activity of the lectin.OPA exerted antifungal activity toward E. neglectum, GibbereUa zeae at the minimum concentration of 20μg/ml and 17.5μg/ml, respectively. However, the lectin did not against Corn Curvalaria Leaf Spot.