Studies On The Properties Of Dolichos Purpureus Lectin

It was determined that each DPL molecule possesses 4 Tryptophan residues by means of pronase degradation with DAB as visualization reagent. Only one Trp residue was detected through NBS chemical modification in the pH 4.0, 0.1 mol/L acetic acid buffer. The activity of DPL in sample buffer without urea remained after being NBS chemical modified and full dialysis, while DPL in buffer with 8 mol/L urea underwent a total loss of activity after the same treatment. This indicated that the Trp residues on molecular surface are not related to the hemagglutination activity of DPL, while the Trp residues in the inner part of molecules are located in the centre of activity, or although not in the centre of activity can result in the variation of conformation of activity center after being modified by NBS.The variation of DPL conformation in different solution conditions and the conformational characteristics of Trp local environment were followed by intrinsic fluorescence, with the corresponding activity determined. Studies indicated that the λ_max of natural DPL was at 328 run when the excitation wavelength was 295 nm, which suggested that the Trp residues were located in hydrophobic environment. Further, the λ_max remained at 328 nm when the excitation wavelength was changed to280 nm, and the typical emission spectra of Tyr was observed, and that suggested that the fluorescence energy of Tyr was transferred to Trp residues; the change of ironic strength from 0.01 mol/L to 1 mol/L did not lead to variation of Trp local environment; Temperature, pH value, denaturant urea and reducing agent DTT could all reduce the fluorescence strength of DPL, but could not lead to changes in the profile of fluorescence spectra and the position of emission peak, which hinted that there was no obvious variation in the Trp local environment; the maintenance or minor change of hemagglutination activity suggested that the structure of DPL is stable. The relatively high temperature (above 85 °C) or high concentration of urea resulted in the obvious red shift of emission peak and the significant loss of hemagglutination activity, which further demonstrated that the Trp local environment is closely related to the activity of DPL. Finally, we studied the enzymolysis of DPL by trypsin and found that DPL was apparently degraded, and the degree of degradation rised as duration increased. On the other hand, trypsin did not hydrolyze the two subunits of DPL at the same levels, with the higher level of degradation in smaller subunit. The hemagglutination activity of DPL remained after enzymolysis for a certain time, which demonstrated the stability of DPL.Specific anti-serum was raised against New Zealand rabbits using purified DPL, and underwent double immunodiffusion, through which the titer was determined as 1:32. The three species of Phaseolus L, Phaseolus albiflora L. var, Phaseolus purpurea L. var and Phaseolus coccineus L.var have been purified by extraction with Tris-HCl buffer, ammonium fractionation, ion-exchange column, gel filtration chromatography. Only one band was observed on the gel after SDS-PAGE. The three species of Phaseolus L could cross react with the rabbit anti serum against DPL, which indicated that their content can be obtained conveniently by immunoprecipitation for their applications in clinical events and determination of protein expression in transgenic plants.