Purification And Some Characterization Of A Lectin From Seeds Of Dolichos Purpureus L.Var

A man/glc-specific Dolichos puepureus lectin (DPL) was purified from seeds of Dolichos puepureus L. var. by a procedure including precipitated by ammonium sulphate, DEAE-52 iron-exchange and Sephacryl S-300 HR gel filtration. The purified DPL showed a single band on PAGE and IEF gel, two bands on SDS-PAGE under reducing and non-reducing congditions. The Mr of native DPL was 155kD. The subunits of DPL were DPL- a 39.6kD and DPL- 3 36.8kD, respectively. It may consist two a subunits and two 3 subunits without disulphide bridges. DPL showed no specific agglutination with any type of human erythrocytes. The lectin was a glycoprotein with 1.6% of carbohydrates. The result of IEF showed that the pI of DPL was 6.18.The result of pH on the agglutinating activity of DPL showed that its stability spanned at pH 3,0-10.5. The lectin was stable up to 55℃. Its activity gradually decreased with further increase in temperature. It was completely inactive upon heat treatment at 95℃, in 4moI/L urea and 1mol/L guanidine hydrochloride. The agglutinating activity of DPL vanished after demetalization by dialysis against EDTA. Followed by dialysis against NaCl, full activity was restored by addition of Ca2+, Zn2+, Mn2+andMg2+.The native DPL exhibited two characteristic negative peak centered at 213nm and 226nm. It was estimated DPL contained about 4% a -Helix, 48% 3 -sheet and 48% random coil by the Neural network method in thedicro 2.6 soft.DPL was modified with N-Bromosuccinimide (NBS) in pH 5.0, 0.1mol/L NaAc-HAc buffer containing 8 mol/L urea or not. The results showed 36 Trp residues and 12 of them were modified. Using KI, CsCl and Acr to probe the micro-environments of Trp residues in DPL, the Trp residue fluorescence of DPL was not quenched by KI and CsCl, but 100% quenched by Acr. So, some Trp residues of DPL were located in the inner core and the others located in the hydrophobic pocket.