Fig 1.
Activity of bacterial proteases with antimicrobials in SDS-PAGE zymography.
(A) A mixture of bacterial proteases was separated on polyacrylamide gels with gelatin and incubated overnight in developing buffer that contained decreasing amounts of povidone-iodine (PVP-I), silver lactate (Ag), chlorhexidine digluconate (Chl), and octenidine hydrochloride (Oct). The values are expressed as relative protease activity related to control (Ctrl) with no added antimicrobial. (B) The activity of eukaryotic proteases similarly incubated with varying concentrations of antimicrobials. In panels A and B, bars represent standard deviation, * denotes p < 0.05 (Student's one sample t-test to control), n = 4.
Fig 2.
Activity of bacterial proteases with antimicrobials in fluorogenic assay.
Pure proteases (MMP-2 or trypsin) or single-source proteases from bacterial or eukaryotic media were evaluated in fluorogenic assay with quenched gelatin as a substrate. Reactions were in the presence or absence of different concentrations of silver lactate (A), chlorhexidine digluconate (B), and octenidine hydrochloride (C). Differences in fluorescence increase were related to control without antiseptics. Measurements were repeated two to three times. X-axis is in log10 scale. PA–P. aeruginosa, SL–S. liquefaciens, SM—S. marcescens.
Fig 3.
Activity of porcine skin endogenous proteases with antimicrobials.
Whole skin was incubated with the tested compounds in a tube, homogenized, and assessed by means of gelatin zymography (A) or incubated in Franz diffusion cells (dermal side up) and analysed using in situ zymography, as illustrated with representative images (B). Color-inverted images, darker shades correspond to increased proteolysis. Each sample was developed with fluorogenic substrate either at 37°C (the upper sample section of each treatment) or at -20°C (assay control, the lower sample section). Squares show areas of fluorescence intensity measurement, which were evaluated in replicates (n = 5) with image analysis (C). Bars in (A) and (C) show mean protease activity related to PBS control ± SD. * denotes p < 0.05 (Student's one sample t-test to control). The tested compounds were povidone-iodine (PVP-I), silver lactate (Ag), chlorhexidine digluconate (Chl), octenidine hydrochloride (Oct), commercial protease inhibitors (P. Inh.).