Purification and characterization of anti-N lectin from Vicia unijuga leaves

1. An anti-N lectin was extracted from Vicia unijuga leaves with phosphate-buffered saline (PBS). Purification of the lectin was achieved, after pretreatment of the PBS extract by ammonium sulfate fractionation and absorption with human M erythrocytes, by using a combination of conventional chromatographic techniques with asialoglycophorin AN-Sepharose CL-4B affinity chromatography. Purification steps were followed by increase of specific activity. 2. Homogeneity of the purified lectin was demonstrated by HPLC and SDS-PAGE. The purified lectin was a glycoprotein with 11.4% carbohydrate and relatively high percentages of serine, threonine and aspartic acid residues and had a Mw of 120,000 Da. 3. This lectin agglutinated human N and MN erythrocytes, but did not agglutinate M erythrocytes. Hemagglutination of the lectin was inhibited by glycophorin AN and N-active sialoglycopeptide released from human N erythrocytes by treatment with Pronase or trypsin. However, it was not inhibited by any of mono- and di-saccharides, ABH-active glycoproteins, glycophorin AM and M-active sialoglycopeptide liberated from human M erythrocytes by treatment with Pronase or trypsin.