Hemolytic Activity

The hemolytic activity of peptides was determined by measuring the amount of hemoglobin released by human red blood cells (hRBCs) as described in a previous study (Luna-Ramírez et al., 2014). The hRBCs were washed and resuspended in 10 mM PBS (pH 7.4) to attain a dilution of ∼2% heamatocrit. Then, 50 μL of the hRBCs solution was incubated with 50 μL of a serial dilution of peptides dissolved in PBS for 1 h at 37°C. The mixtures were centrifuged, and the absorbance of the supernatant was measured using a microplate reader (Bio-Rad, ıHercules, CA, United States) at 570 nm. The hRBCs treated with PBS (0% hemolysis, A₀) and 0.01% Triton X–100 (100% hemolysis, A_t) acted as negative and positive controls, respectively. Melittin was used as a control peptide. The percentage of hemolysis was calculated using the following equation: Hemolysis = [(A × A₀)/(A_t × A₀)] × 100%.