Figure 2. VP4 possesses hemolytic activity.

(A) Percent hemolysis of bovine RBCs as a function of GST-VP4 concentration. RBCs (0.5%, v/v) were incubated with various concentrations of the protein for 30 min at 37°C. (B) Kinetics of hemolysis of bovine RBCs as a function of temperature as indicated. RBCs were incubated at different temperatures with GST-VP4 (10 µg/ml) for 30 min. (C) Hemolysis as a function of pH. Hemolysis reactions were carried out at varying pH. Data are normalized with respect to samples containing only buffer and RBCs (0%), and containing RBCs with 50% water to mediate complete lysis (100%). (D) Hemolysis in presence of different metal ions. Purified GST-VP4 was mixed with bovine RBCs. Reactions were incubated in 10 mM Tris (pH 7.5)/150 mM NaCl containing Cs⁺, K⁺, Mg²⁺, Ca²⁺ ions. (E) Calcium concentrations were varied for the hemolytic assay over a wider range. The error bars represent the standard deviation from three independent experiments.