Study On Purification,Characterization,Structure And Bioactivity Of Sophora Flavescens Lectin

The study on Sophora Flavescens Lectin (SFL) includes three parts: purification and characterization of SFL; study on the relationship between structure and bioactivity of SFL and preliminary study on the cytotoxic activity of SFL.SFL was isolated from the roots of Sophora Flavescens Ait by extraction,precipitation with (NH4)2SO4, ion-exchange chromatography on DEAE-Sepharose and followed by gel filtration on Sephacryl S-200 HR. The purified lectin showed a single protein band on SDS-PAGE. The molecular weight and the subunit molecular weight were all 32kD, determined by gel filtration on Sephadex G-100 and SDS-PAGE respectively. The results implied that there was only one subunit in a SFL molecule. SFL contained 3.5% neutral saccharide. There were 3 Trp residues in a SFL molecule, determined by colorimetry. The result of isoelectrofocusing showed that the isoelectric point of SFL was 5.56. The lectin agglutinated rabbit erythrocytes at 0.97?g/mL and showed no specific agglutination with any type of human erythrocytes. The result of carbohydrate inhibition assay showed that D-Manose was a specific inhibitory carbohydrate of SFL.SFL exhibited a characteristic circular dichroic (CD) spectrum, a negative band centered at 217 nm. It was estimated that SFL contained about 15.8% ?-helix, 48.3% P -sheet and 35.9% random coil. Theeffect of different temperature, various pH and different concentration of urea on CD spectra and hemagglutinating activity of SFL was studied. The results showed that SFL was inactive or only had a little hemagglutinating activity at pH4.0 , a temperature above 80癈 and concentration of urea higher than 2 mol/L. Under these conditions, the CD spectra and the secondary structure of SFL changed greatly.The fluorescence spectra of SFL were studied. The results showed that the Trp residues contributed most to the fluorescence intensity of SFL. Native SFL had 2 exposed Trp residues . Modifiying them with N-bromosuccinimide (NBS) led to thorough loss of the hemagglutinating activity of SFL. Treating SFL with Mannose could protest the Trp residues from oxidation and a fairly high hemagglutinating activity could be retained. These results indicated that Trp residues were essential to the hemagglutinating activity and were involved in carbohydrate-binding site. Modification of Trp residues with NBS induced changes in the fluorescence spectra of SFL. The changes implied that the 2 Trp residues in the surface of the SFL molecule were located in a hydrophobic pocket of the glycoprotein and the other Trp residue was in the interior of the SFL molecule. The study on fluorescene quenching showed that the fluorescence from Trp residues were not quenched by KI, but 87% were quenched by acrylamide.Modification of thiol groups had no influence on the hemagglutinating activity of SFL. No sulfhydryl group has been detected when using N-ethymaleimide (NEM) as a modificatory reagent. It was speculated there was no cysteine in SFL molecules. The pattern of NR/R two-dimensional diagonal SDS-PAGE showed that SFL had no disulfide bond. The result was an additional proof for confirming above speculation.Modification of tyrosine in SFL with N-acetylimidazole (NAI) and p-nitrobenzene sulfonyl fluoride (NBSF) had no effect on the hemagglutinating activity, indicating that Tyr residues were not essential for the activity.MTT colormetric assay was performed to determine the inhibitory rate of SFL on HeLa cells. The result showed that SFL had apparent inhibitory effect on the growth of HeLa cells. The inhibitory rate increased with the increase of the concentration of SFL. In order to investigate whether SFL could induce apoptosis in HeLa cells, the methods of Hoechst33342 and PI double nucleic staining, DNA Ladder Assay and flow cytometry were used. That SFL could induce apoptosis in HeLa cells was confirmed. But the apoptosis was not induced intensively and only occurred after treating for a certain time. When the treating time was longer than 60 hours, many necrosis cells began to emerge, indic...