Isolation And Partial Characterization Of The Lectin From Crotalaria Mucronata

Crotalaria mucronata lectin (CML) was purified from seeds of Crotalaria mucronata by extraction,fraction with (NHi) 2864,hog gastric mucin-Sepharose 4B affinity chromatography and followed by gel filtration of Sephacryl S-200 HR. CML agglutinated type A human red blood cells specially. The purified CML gave one band pel e'ectronhoresis and on SDS nolvacrvlamide gelelectrophoresis. CML's pi is 5.7.The purified CML modified by N-ethymaleimide in the physiological saline containing 8mol/L urea or not didn't lose its agglutinate activity. About 3.1 sulfhydryl groups in CML were modified in 8mol/L urea and 3.2 sulfhydryl groups in CML were modified in the physiological saline without urea. There were 5.9 Tip residues in CML. The purified CML was modified with N-Bromosuccinimide (NBS) in HAc-NaAc buffer containing 8mol/L urea or not. The result indicated 1.8 Tip residues were modified in the buffer without urea and they were not essential groups to the agglutinate activity of CML. In the buffer with urea,5.0 Tip residues were modified and 2.8 of them were essential groups to the agglutinate activity of CML. These trials also indicated that the agglutinate activity of denatured CML by urea was revertible. The trials of NR/R single-dimensional SDS-PAGE and NR/R two-dimensional SDS-PAGE proved that CML had no disulfide bond.EDTA inhibited the agglutinate activity of CML and a small part of CML recovered its agglutinate activity after removing the EDTA. Incubated the deionized CML with Ca2 Mg2+ Mn2 Zn2+ respectively,CML's agglutinate activity recoveredto the level of the natural CML. The series of trials indicated some ions were helpful to stabilize the configuration of CML and the inhibition of CML's activity by EDTA was dual. EDTA inhibited the agglutinate activity of CML directly and also made CML lose its agglutinate activity through chelating the ions in CML.Circular dichroism(CD) of natural CML indicated the P -fold dominated the second structure of CML. The changes of CD and agglutinate activity of CML in different pH,temperature,concentration of guanidiniumhydrochloride indicated that CML had a comparatively stable structure despite of its lack of disulfide bond and having so much 3 -fold. The denaturation was a two-steps' process. First,CML didn't lose activity despite of its structure changes because the changes were out of the active center. Second,the active center was destroyed and CML lost its agglutinate activity completely. The fluorescence spectra (FLS) of CML exited at 280nm and 295 nm showed a maximum peak at 335 nm. FLS of CML with different temperature also showed there were two steps in the process of denaturing. Time-scanning FLS indicated the process of denaturing by urea was faster than that of recovering its activity.The trials of inhibiting bacteria indicated CML didn't inhibit the growth of Bacillus Subtilis,Escherichia coli. Bacillus licheniformos,Pseudomonas psudoalcaligenes and Staphylococcus aureus.