Isolation and characterization of two lectins from the seeds of lablab bean (Lablab purpureus Linn cv. Highworth)

Two mitogenic lectins were isolated from the seeds of lablab bean (Lablab purpureus Linn cv Highworth) by protein extraction using phosphate buffered saline (PBS) pH 7.2, ammonium sulfate fractionation and gel permeation chromatography using superfine sephadex G-200. Both lectins were non-blood type specific but blood group specific because they agglutinated and treated blood types A, B, O, and AB but not trypsinized and untrypsinized blood from calf and goat. Hemagglutination by lectin 1 was inhibited by the addition of D-fructose, D-glucose, maltose, sucrose and methyl-alpha-D-mannopyranoside using blood type O and by D-glucose and D-mannose using blood type B. On the other hand, lectin 2 was inhibited by the presence of D-fructose, D-mannose, G-glucose and methyl-alpha-D-mannopyranoside regardless of blood type. Among the haptenic sugars, strongest inhibition was elicited by methyl-alpha-D-mannopyranoside. Agglutination of blood types A and AB by lectin 1 was not inhibited by any of the sugars used. Precipitation of lectins 1 and 2 occurred at pH 4-5 and 8-9, respectively, indicating that their isoelectric point lie in these ranges and that the former is an acidic protein while the latter is basic. Activity of both lectins were stable from pH 2-12. Extreme temperature destroyed the activity of both lectins. Optimum temperature for lectin 1 activity was 40-50 deg C using blood types B and O, and 50 deg C lectin 2 using blood type O. Using gel permeation chromatography, the molecular weights of lectins 1 and 2 were estimated to be 267 and 57 KD, respectively. From SDS-PAGE, lectin 1 was found to consist of six sub-units with apparent molecular weights of 32, 35.5, 39, 51.5, 55.5 and 59.5 KD. Lectin 2 was made up of sub-units weighing 15 and 17.5 KD, presumably of the alpha 2 beta 2 type. Lectins 1 and 2 were found to be glycoproteins containing 2.30 percent and 4.23 percent total sugars, respectively. The reducing sugar residues were identified to be glucose and galactose by high performance liquid chromatography (HPLC). Both lectins contained high amounts of aspartate and glutamate, a large proportion of which exist in the amide form