Study On Purification, Characterization And Conformation Of Lycoris Chinensis Agglutinin

A new mannose-binding lectin was isolated from lycoris chinensis Traub by extraction, precipitation with (NH4)2SO4, anion-exchange chromatography on DEAE-Sepharose column followed by gel filtration on a Sephacryl S-200 column. The apparent Mr of lycoris chinensis agglutinin(LCA)was determined to be 48KD by gel filtration on Sephacryl S-200 column. In SDS-PAGE under reducing condition, purified LCA show a single band of Mr 12KD, Therefore, the intact LCA was considered to be a tetrameric glycoprotein with four identical subunits. The lectin was a potent agglutinin for rabbit erythrocytes but was inactive against human red cells and chook erythrocytes. Manne was the most potent inhibitor of LCA hemagglutination of rabbit erythrocytes, followed by mannose.Metal ion Ca2+, Mg2 were helpful to hemagglutination activity. Treated with EDTA-Na2,LAA lost activity as mentioned,fluorescence intensity decrease and λmax of fluorescence emission spectrum red-shifted. Incubate the deionized LAA with Ca2+, Mg2+,the agglutinate activity recovered.The fluorescence spectrum of LCA excited at 280nm and 295nm showed a maximum peak at 337.2nm.The characteristic peak of Tyr did not exist, which showed that the fluorescence energy of Tyr was transformed to Trp and strength the fluorescence of Trp. When LCA was excited at 295nm, the fluorescence spectrum showed a maximum peak at 337.2nm,the λmax of fluorescence emission spectrumblue-shifted more than 10nm compared with the λmax of free Tyr (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.The conformation changes of LCA in the presence of different concentration GuHCl , urea and SDS were investigated by measurements of fluorescence spectra(FR) ,and compared with the activity changes.The λmax and intensity of fluorescence emission spectrum of LCA increased with the concentration of GuHCl increasing. And then the maximum peak blue-shifted and intensity decreased with the concentration of GuHCl increasing, accompanying by hemagglutination activity loss, when the concentration was raised to 6mol/L,the hemagglutining activity was completely lost.Studies on denaturation by urea indicated that the fluorescence intensity of LCA increased obviously as the concentration of urea increased from 0 to 4mol/L ,while the hemagglutination activity of LCA maintained the original level in this stage, And then Fluorescence intensity and λmax of fluorescence emission spectrum of LCA decreased.when the concentration of urea reached 6mol/L , fluorescence intensity come to the lowest and the hemagglutining activity was completely lost.Denaturation by SDS showed that while the activity of LCA was exponentially decreased with the increasing concentrations of SDS, the maximum wavelength and intensity were first decreased, then increased with increasing SDS concentration. When the concentration was raised to 20mM,the hemagglutining activity was completely lost, and fluorescence intensity reached the lowest.The process of denaturation can be seen as two stages. There exists a intermediate state in the presence of low concentration of denaturation, the conformation of the intermediate is different from the native and unfolding state, the maxium wavelength of FR of the intermediate is the maxium, and fluorescence intensity is the highest.The effect of chemical modification of amino acid residues uponhemagglutination was investigated. There is a definite decrease of fluorescence intensity which corresponds to the oxidation of tryptophan. Under native conditions 2.7 tryptophan residues were available for modification by NBS, which had no effect on the hemagglutinating activity.Modification of tyrosine (NAI and NBSF), His(DEPC),thiol groups (PCMB, NEM) and sulfhydryl (DTT) didn't affect the hemagglutination activity. This rules out the possibility of these residues being involved in its binding activities. Modification of serine/threonie had a little effect on haemagglutinating activity.A 50% loss in hemagglutination activity after the modification of Asp/Glu indicated that phenolic hydroxyl groups was essential for the carbohydrate-binding activity of LCA.Fluorescence quenching study on LCA with CsCl, KL acrylamide and marine showed KI,acrylamide and manne quenched the fluorescence of lectin through dynamic quenching mechanism, and 100 % of Trp were quenched by acrylamide, 62.9% of Trp were quenched by KI.83.3% of Trp were quenched by manne. There was no detectable change in the fluorescence intensity of LCA after treated with CsCl.