Purification of a lectin from M. rubra leaves using immobilized metal ion affinity chromatography and its characterization.
Lectins represent a heterogeneous group of proteins/glycoproteins with unique carbohydrate specificity, with wide range of biomedical applications. The multi-step purification protocols generally used for purification of lectin result in a significant reduction in the final yield and activity. In the present study, Morus rubra lectin (MRL) was purified to homogeneity from the leaves using a single-step immobilized metal ion affinity chromatography (IMAC) procedure. The approximate molecular weight of purified MRL resolved as a single band on SDS-PAGE was 52 kDa. Final percentage yield of purified lectin by IMAC was calculated as 74.7 %. Purified MRL was specific to three sugars, galactose, D-galactosamine and N-acetyl-D-galactosamine, and rendered haemagglutination (HA) activity towards different human blood group RBCs. MRL showed stability over a wide range of temperature (up to 80 °C) and pH (4-11). Chelation of the lectin with EDTA did not alter HA which indicates that metal ion is not required for activity. In the presence of Fe(2+), Ca(2+), Zn(2+), Ni(2+), Mn(2+), Na(+) and K(+), HA activity was reduced to 50 %, whereas the presence of trivalent metal ions (Fe3(+) and Al(3+)) and Cu(2+) did not affect the activity. In the presence of Mg(2+) and Hg(2+), only 25 % of HA activity remained.
Centre for Bio-Separation Technology (CBST), VIT University, Vellore, Tamil Nadu, 632 014, India.
Thavamani
T
Sureshkumar
Sulochana
S
Priya
D002241:Carbohydrates / Q000032:analysis; D005591:Chemical Fractionation / Q000379:methods*; D002846:Chromatography, Affinity / Q000379:methods*; D006384:Hemagglutination / Q000187:drug effects; D006801:Humans; D008670:Metals / Q000737:chemistry* / Q000494:pharmacology; D031625:Morus / Q000737:chemistry* / Q000166:cytology; D010503:Periodic Acid-Schiff Reaction; D018515:Plant Leaves / Q000737:chemistry* / Q000166:cytology; D037121:Plant Lectins / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology
D002241:Carbohydrates; D008670:Metals; D037121:Plant Lectins