Study Of The Purification And Characterization Of Aspidistra Elatior Lectin

Aspidistra elatior lectin, namely AEL was isolated from the rhizomes of Aspidistra elatior Bulme by extraction, precipitation with (NH₄)₂SO₄, ion-exchange chromatography on DEAE-Sepharose, CM-Sepharose and followed by gel filtration on Sephacryl S-100. AEL was a heterotetramer with molecular mass of 56 kDa and composed of two different homodimers consisting of two different polypeptides of 13.5 and 14.5 kDa that are held together by noncovalent interactions. Hapten inhibition assay indicated that hemagglutinating activity of AEL toward rabbit erythrocytes could be exclusively inhibited by D-mannose,mannan, thyroglobulin and ovomucoid. The lectin was stable up to 70℃and showed maximum activity in a narrow pH range of 7-8.The far-UV CD spectrum of native AEL showed a broad negative minimum peak at 215 nm which indicated that AEL might be mainly composed ofβ-sheets. The fluorescence spectrum of AEL excited at 280 nm and 295 nm showed a maximum peak at 310 nm and 328 nm, respectively which suggest that the fluorescence energy of Trp was transfered to Tyr. Compared with the fluorescence spectrum of free Trp residues, theλ_max of fluorescence emission spectrum excited at 295 nm blue-shifted about 20 nm. These results indicated that the Trp residues of AEL might located in the hydrophobic core of lectin.Treating purified AEL with NBS, an affinity-modifying reagent specific for Trp residues in restricted condition, completely inactivated the lectin with total modification of four Trp residues in native conditions. Of these residues only the third or fourth modified residue seemed to play a crucial role in hemagglutinating activity of AEL. The presence of the haptenic sugar mannose in the assay medium did not provide protection for AEL against NBS, which indicated that Trp residues might not be present in mannose-binding site of the lectin. The loss in hemagglutinating activities of AEL might be due to the secondary structure change, since the CD spectrum of the protein in the far-UV region (200-250 nm) experienced a significant change. Modifications of other residues such as carboxyl groups, Arg, Cys residues also resulted in significant loss of the hemagglutinating activity. Of these residues, only carboxyl groups might located in the sugar-binding sites, the other residues might be also play a important role like Trp residues in maintaining the active conformation of the protein to exhibit hemagglutinating activity. This conclusion was further supported by the significant change of the CD spectrum.Fluorescence quenching was carried out with four quenchers. Maximum quenching was observed with acrylamide, followed by succinimide and iodide, and was significantly lower with cesium ion. Increase in the extent of quenching upon denaturation of AEL was maximum with I" and minimum with Cs⁺, suggesting the presence of positively charged residue(s), near at least one tryptophan residue. The fluorescence quenching parameters experienced a obvious increase in the presence of mannose. These results might be result from the conformational changes that was induced by ligand binding.In-vitro anti-proliferative activity of AEL was evaluated against five human cancer cell lines representing different organs and tissues namely Pro-01 (prostate), Lu-04 (lung), Bre-04 (breast), HepG2 (liver) and Hela (cervix). Of the five cancer cell lines, AEL possesses the most deleterious effect towards Bre-04 (66%), relatively milder toxicity towards Lu-04 (60%), and only minor inhibitory towards HepG2 (56%), while the effect on the viability of Hela cell lines was undetectable. AEL also showed significant anti-virus activity toward different virus. All the IC50 of AEL toward these virus were 4μg/ml.