FIGURE 1.

Antibacterial activities and protease effects of peptides. A, antimicrobial activity as assessed by RDA against S. aureus of peptides with either N-terminal (left panel) or C-terminal (right panel) hydrophobic modifications. For determination of activity, bacteria (4 × 10⁶ CFU/ml) were inoculated in 0.1% TSB-agarose gel, and 4-mm wells were punched. In each well, 6 μl of peptide (at 100 μm) was loaded. The zones of clearance correspond to the inhibitory effect of each peptide after incubation at 37 °C for 18–24 h (mean values, n = 3). The activity is plotted versus peptide hydrophobicity (Kyte and Doolittle scale). (The correlation coefficient (R) and the coefficient of determination (R²) in Fig. 1A are 0.583 and 0.340, respectively, for the N-terminal modifications, and 0.881 and 0.776, respectively, for the C-terminal modifications.) B, dose-dependent activity of the selected peptides GKH17 and HKH17 and WWW-tagged variants against S. aureus (4 × 10⁶ CFU) in RDA. (The difference between the original peptides GKH17, HKH17, and their tagged variants is significant (p < 0.05, two-way ANOVA).) C, effect of tag length on GKH17 peptide activity (at 50 μm) against S. aureus ATCC 29213 as assessed by RDA in the presence and absence of 0.15 m NaCl. * denotes no detectable clearance zone. (In absence of salt the difference between all tagged peptides, and no tag is significant. In presence of 0.15 m NaCl, the difference between the WWW and WWWWW peptides versus no tag is significant (p < 0.05, one-way ANOVA).) D, MIC of indicated peptides against different S. aureus isolates. E, protease sensitivity of peptides. The indicated peptides were incubated with (+) or without (−) the S. aureus enzymes aureolysin (Aur), V8 proteinase (V8), or human leukocyte elastase (HLE) and analyzed by SDS-PAGE (16.5% Tris-Tricine gels).