Hemocyanin from shrimp Litopenaeus vannamei shows hemolytic activity

Hemocyanin is an extracellular copper-containing protein present in the hemolymph of both mollusks and arthropods. The traditionally recognized function of hemocyanin is for oxygen transport. Lately, it was demonstrated that hemocyanin is a multifunctional protein, especially participating in multiple roles of immune defense. For better understanding its actions in immune defense, the hemolytic activity of hemocyanin from shrimp Litopenaeus vannamei and the mechanism were investigated in this study. The results showed that shrimp hemocyanin exhibited hemolytic activity against vertebrate erythrocytes. The hemolysis displayed dependencies on hemocyanin concentration, pH, temperature and divalent cations. The highest activity occurred at a concentration of 0.125 mg ml−1, and pH 6.0, 40 °C in the presence of calcium. Moreover, from the incubation products of erythrocytes with hemocyanin, besides two subunits of hemocyanin, two molecules around 150 and 230 kD were isolated and speculated as oligomers of hemocyanin. Further evidence revealed that the hemolysis could be inhibited to different degrees by osmoprotectants with high molecular masses, suggesting that it follows a colloid-osmotic mechanism. These results indicate that L. vannamei hemocyanin has a novel function with hemolytic activity, partly related to a colloid-osmotic mechanism mediated by its oligomers.