(2008). Briefly, 0.2 mM of DPPH in methanol was mixed with 100 μL of twofold increasing concentrations of sample (1.95–250 μg mL−1) and 50 mM Tris-HCl buffer, pH 7.4. The mixture was shaken vigorously and left at room temperature for 30 min in the dark. The A517 nm was then measured. l-Ascorbic acid was used as a positive control. The free-radical-scavenging activity was then calculated as the Selleckchem Tacrolimus percentage of inhibition according to the following equation: The S07-2 compound was purified to homogeneity and its activity was tested against P. aeruginosa. Data of the purification steps
are summarized in Table 2. Extraction with methanol increased the specific activity to 400 AU mL−1 and led to 100% recovery. The active compound was eluted from a Sep-Pak C18 cartridge at 40% acetonitrile (F40) with a specific activity
of 3200 AU mL−1 and a 64% recovery. F40 was further loaded onto a DEAE-Sepharose column. The S07-2 compound eluted with 10 mM ammonium acetate, pH 3, showed a specific activity of 4000 AU mL−1 and a 40% recovery. To achieve purification, the active fraction was further loaded onto a C18 RP-HPLC column. One major peak was separated from contaminants and subjected to a second HPLC run on a discovery HS PEG column. A single peak was purified to homogeneity (Fig. 1). Its specific activity was increased 3500 times, reaching a value of 7000 AU mL−1. The purity of the S07-2 compound was controlled by TLC as reported in Fig. 1 (see inset). A single spot with Rf 0.7 exhibiting antibacterial INNO-406 mw activity against P. aeruginosa (Fig. 1, inset a) was detected by both UV light at 254 nm and exposure to iodine reagent (Fig. 1, insets b and c, respectively). The optimal temperature and pH values of the
S07-2 compound were also investigated. The compound conserved its antibacterial activity until 90 °C and Pregnenolone lost 50% of its initial activity after autoclaving at 121 °C for 20 min. It was stable in the pH range from 3 to 10 and was resistant to proteases. The S07-2 compound showed a positive reaction with TDM reagent (Fig. 1, inset d), but was negative to ninhydrin. Data indicate the absence of free N-terminal amino group and the presence of peptide bonds. Therefore, the antibacterial compound could be a cyclic peptide antibiotic. A cyclic structure should increase the rigidity of the peptide, reducing its proteolytic degradation by hampering enzyme access to the cleavage sites. Various cyclic peptides containing N- and/or C-terminal blocked residues as well as unusual amino acids have already been described, such as maltacine complex (Hagelin, 2005), subtilosin A lantibiotic (Kawulka et al., 2004), and surfactin, iturin and fengycin lipopeptides (Tamehiro et al., 2002). The molecular mass of the S07-2 compound was determined by matrix-assisted laser desorption/ionization-time-of-flight MS (Fig. 2).