Isolation and characterization of earthworm hemolysins and agglutinins

Hemolytic activity in coelomic fluid of Eisenia fetida (ECF) is due to three proteins H1, H2, H3 with molecular weights of 46, 43 and 40 kD, respectively. These proteins were isolated by preparative PAGE. H1 and H2 were shown to be stable in SDS and alpha-2-ME whereas H3 splits into two fragments with molecular weights of 18 and 21 kD after SDS treatment. IEF indicates that each protein consists of different isoforms with pIs between 5.1 and 6.2 H3 was demonstrated to be a bifunctional protein that can lyse and agglutinate erythrocytes. At 56 degrees C hemolytic activity of all three proteins was inactivated, but the agglutination activity of H3 was stable. Intracoelomic injection of erythrocytes reduced the number of hemolysins from three to two. Monospecific antisera were raised against the isolated hemolysins H1,2 and 3. The use of these antibodies and of carbohydrates as inhibitors of the biological activity of the molecules demonstrates the close structural relationship of agglutinins and hemolysins in the CF of E. fetida.