Affinity purification and characterization of a seed lectin from Crotalaria medicaginea.
Purification of lectin from the seeds of Crotalaria medicaginea Lamk by affinity chromatography on asialofetuin-linked amino activated silica, yielded a single band on non-denatured PAGE at pH 4.5 and 8.3 and, a single peak on HPLC size exclusion and cation exchange columns. The molecular mass of the native C. medicaginea lectin was determined to be 125 kDa by gel filtration. In SDS-PAGE, the lectin migrated as a single band of M(r) 31.6 kDa under reducing and nonreducing conditions, indicating that it is a tetramer of apparently identical subunits. It agglutinated red blood cells (RBCs) from rabbit and human ABO blood groups. It also reacted with RBCs from rat, sheep, goat and guinea pig but after desialylation with neuraminidase. The hemagglutination activity of the lectin was inhibited by D-galactose and its derivatives. Amino acid analysis showed that lectin was rich in aspartic and glutamic acid and, did not contain sulphur containing amino acids. The lectin is a glycoprotein having 1.41% of neutral sugars. It is labile at temperature above 60 degrees C. It needs divalent cations for its activity, as a loss of activity was observed on removal of Ca2+ and Mn2+. Denaturing agents like urea, thiourea and guanidine-HCl have no effect on its activity.
Department of Molecular Biology and Biochemistry, Guru Nanak Dev University, Amritsar 143 005, India.
Navjot
N
Kaur
Jatinder
J
Singh
Sukhdev Singh
SS
Kamboj
D000017:ABO Blood-Group System; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001212:Asialoglycoproteins / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D002118:Calcium / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods; D029864:Crotalaria / Q000378:metabolism*; D060748:Fetuins / Q000737:chemistry; D018698:Glutamic Acid / Q000737:chemistry; D006388:Hemagglutinins / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007477:Ions; D037102:Lectins / Q000737:chemistry* / Q000302:isolation & purification*; D008345:Manganese / Q000737:chemistry; D009439:Neuraminidase / Q000737:chemistry; D011817:Rabbits; D051381:Rats; D012639:Seeds / Q000378:metabolism*; D013696:Temperature
D000017:ABO Blood-Group System; D000596:Amino Acids; D001212:Asialoglycoproteins; D060748:Fetuins; D006388:Hemagglutinins; D007477:Ions; D037102:Lectins; C017029:asialofetuin; D001224:Aspartic Acid; D018698:Glutamic Acid; D008345:Manganese; D009439:Neuraminidase; D002118:Calcium