During the purification procedure, the antimicrobial activities of the samples were monitored by a liquid growth inhibition assay using the Gram-negative bacteria Escherichia

coli SBS363, Gram-positive bacteria Micrococcus luteus A270 that were cultured in poor broth nutrient medium (PB: 1.0 g peptone in 100 ml of water containing 86 mM NaCl at pH 7.4; 217 mOsM), whereas yeast strain Candida albicans MDM8 was cultured in poor dextrose broth (1/2 PDB:1.2 g potato dextrose in 100 ml of H2O at pH 5.0; 79 mOsM). Determination of antimicrobial peptide was performed using 5-fold microtiter broth dilution assay in 96-well sterile plates at a final volume of 100 μL. Mid-log 17-AAG phase culture were diluted to a final concentration of 1×105 colony forming units/mL. Dried fractions were dissolved in 200 μL of water ultrapure and 20 μL applied into each well and added to 80 μL of the bacterium/yeast dilution. The fractions were tested in triplicate. The microtiter plates were incubated for 18 h at 30 °C; growth inhibition was determined by measuring CDK inhibitor absorbance at 595 nm. For purification of antimicrobial peptides, the plasma was homogenised and then trifluoracetic acid was added to a final concentration of 0.05%. The sample was agitated on ice for 30 min and centrifuged at 16,000g at 4 °C.

The acidic supernatant was loaded onto classic Sep-Pak C18 cartridges equilibrated in acidified water (TFA 0.05%). After washing with acidified water, three stepwise elutions were successively performed with 5%, 40% and 80% acetonitrile (ACN/TFA 0.05%). The 40% Sep-Pak fraction was concentrated in a vacuum centrifuge and reconstituted in Milli-Q water (Millipore™) and directly subjected to C18 reverse-phase on a semi-preparative Jupiter C18 column equilibrated at room temperature with 0.05% trifluoracetic acid in water. The sample was purified using

acetonitrile/water/0.05% trifluoracetic acid gradients of 2–60% acetonitrile in 60 min at a flow rate 1.5 mL/min. Ultraviolet absorbance was monitored at 225 nm. The eluted peaks fractions were collected by hand find more and were vacuum dried (Speed-Vac Savant) and used for assay of antimicrobial activity and determination of amino acid sequence. Briefly, 0.35 μL of sample in Milli-Q water was mixed with 0.35 μL of saturated matrix α-cyano-4-hydroxycinnamic acid solution deposited onto the sample slide and dried on the bench. The analysis was performed with the spectrometer operating in positive mode, which detects positively charged ions. To determine the amino acid sequence of rondonin, the doubly charged ions were subjected to “de novo” sequencing in a Q-TOF Ultima API (Micromass) spectrometer operating in positive ionisation mode. The spectrum was analysed, and the “y” and “b” fragments were used to elucidate the primary structure of the molecule. Synthetic rondonin was synthesised by solid phase peptide synthesis using the Fmoc procedure [1].