Purification and properties of a lectin from the seeds of Croton tiglium with hemolytic activity toward rabbit red cells

Abstract

A nontoxic lectin from the seeds of Croton tiglium capable of agglutinating erythrocytes of sheep and some other animals, induced hemolysis as well as agglutination of rabbit erythrocytes. The lectin purified by chromatographic and gel filtration procedures was found homogeneous in polyacrylamide gel electrophoresis and immunochemical tests. It gave a single band of 55,000 M r in SDS-gel electrophoresis. In small zone gel filtration studies, it was found to undergo reversible association-dissociation at pH 7, with the dissociated species having a dimeric M r of 100,000 and the associated species approaching a M r value of 400,000. The lectin had 0.75% neutral sugar content and consisted of two hemagglutinating and hemolytic isolectins of p I 5.1 and 5.4. Besides other amino acids, the lectin contained five cysteine and seven methionine residues per 56,000 M r with two of the cysteine residues forming an intrachain disulfide bond. Histidine was the only N-terminal amino acid found. The agglutinating activity of the lectin was not inhibited by simple sugars. However, trypsin released glycopeptides from sheep erythrocytes and their Pronase-digested fragments not only inhibited agglutination of sheep and rabbit erythrocytes but also hemolysis of the latter, indicating the implication of a carbohydrate binding site of the lectin in inducing both hemagglutination and hemolysis. Partial inhibition of lysis of rabbit erythrocytes pretreated with some galactose-specific lectins suggested the involvement of galactose residues on rabbit red cells in binding. While trypsinization increased agglutinability of rabbit red cells without affecting the extent of lysis, at 4 °C onset of lysis was delayed with agglutination remaining almost unaffected, suggesting divergent pathways of hemagglutination and hemolysis subsequent to binding of the lectin to carbohydrate receptors. The variation of extent of lysis as well as agglutination of erythrocytes from individual rabbits indicated the probability of the lectin being blood type-specific among rabbits. In dose-response studies, the departure from linearity at low lectin concentrations with a given red cell population indicated hetero-geneity of the population regarding susceptibility to lysis.