Investigation of the characteristics of recombinant buforin I peptide

Antimicrobial effect of recombinant buforin I peptide for S. aurous (ATCC 25923), E. coli O157: H7 (ATCC 35150), Methicillin-resistant S. aureus (ATCC 33591), L. monocytogenes (PTCC 1297), P. aeruginosa(PTCC 1707), K. pneumonia (ATCC 13882), C. perfringens (ATCC 13124), S. Typhi(PTCC 1609), A. niger (PTCC 5010), and C. albican (PTCC 5027) were examined. Microbial strains were incubated for 18 to 24 h at optimum strain temperature until reaching half McFarland concentration. The minimum concentration of growth inhibitor (MIC) was determined using the standard sequential dilution method. 90 μl of the prepared concentrations with 10 μl of microbial suspension was incubated in a 96-well plate for 24 h at 37 °C for bacterial strains and 48 h at 25 °C for fungal strains for MIC determination. To determine the MIC, the absorbance at 630 nm (OD₆₃₀) was measured by an ELISA reader. The lowest concentration at which no microorganism growth was observed was reported as the minimum concentration of MIC growth inhibitor. No inoculated growth medium was used as a negative control³⁵.

To prepare the red blood cells, 4 ml of fresh human blood (from volunteer donors) was first centrifuged in 50 μl of EDTA for 10 min at 2422 g. The precipitate was dissolved in 4 ml of PBS buffer and the resulting solution was centrifuged at 1024 g for 10 min. 190 μl of the prepared blood suspension was poured into 7 μl of 1.5 ml and 10 μl of concentrations of 25, 50, 100, 200, 400 μg/ml peptide was added to 5 μL. 10 μl of PBS buffer was added to a microtube as a negative control and 10 μl of Triton X100 added to another tube as a positive control. All microtubes were placed in an incubator at 37 °C. After 30 min, the samples were centrifuged at 1919 g for 5 min. 100 μL of the supernatant from the centrifuge was taken, and its volume was reduced to a final volume of 1 ml by PBS buffer, and then the absorbance of the samples was measured using a spectrophotometer at 570 nm. The uptake of all blood cells treated with serial concentrations of peptide was measured by spectrophotometer and compared with the uptake of a sample containing Triton, and the percentage of hemolysis was calculated.

As sample adsorption, Ac is negative control sample adsorption and A100 is also positive control sample adsorption. A minimum hemolytic concentration (MHC) was defined as the highest concentration of peptide that does not cause hemolysis³⁶.Cytotoxicity evaluated by using HSF (Human Splenic Fibroblasts) cell line DMEM medium containing 1% penicillin and streptomycin and 10% FBS by ATTC culture were maintained. The cells were incubated at 37 °C at 95% humidity and 5% carbon dioxide³⁷. The cells were treated in 96-well platelets at concentrations of 0, 250, 500, 1000, and 1500 μg/ml peptide and incubated for another 72 h. Cell viability was assessed by resazurin dye reduction method and by adsorption at 640 nm. Cell viability was calculated using the following formula:

As adsorption of the sample, Ac adsorption of the control sample (DMEM medium, dye and cell) and A₀ also adsorption of blank (dye alone)³⁸.