PURIFICATION AND PARTIAL CHARACTERIZATION OF CHLORELLA PYRENOIDOSA LECTIN

The freeze-drying powder of a fresh microalga, Chlorella pyrenoidosa, was extracted overnight by PBS at 4℃ and a tow-step precipitate with solid ammonium sulfate(25% and 75%) was performed for the supernatant after centrifugation. The crude protein obtained was purified by DEAE-Sepharosa ion exchange, followed by Sephdex G-100 filtration. The activity of the lectin was tested wih fresh rabbit erythrocyte in each step of above procedure and protein concentration was determined using Coomassie Brilliant G250. This purified lectin(CPL) dose not contain neutral saccharide but is a monomeric protein with a relative molecular weight of 14,000-15,000, estimated by SDS-PAGE and Sephdex G-100 filtration. In its amino acid composition, Phe has the highest content, followed by Asp and Glu, but there is a lack of His. Assays for sugar or glycoproteiin inhibition and erythrocyte agglutination were done in microtiter plates. The result indicated that CPL could agglutinated erythrocytes from rabbit, sheep and pigeon but not from duck, chicken and human A, B or O blood cell groups and the highest activity was found in the agglutination with rabbit erythrocytes at 6.88μg/mL. The agglutinic activity of CPL was inhibited by seven types of monosaccharides when its concentration was at 13.75μg/mL and by ovalbumin at a range of 6.88-432μg/mL. CPL was tolerant of high temperature and its activity could be maintained even when the lectin solution was heated to 90℃ for 10min.