At longer incubation times, the activity of the proline-rich peptide seemed further MLN2238 mouse inhibited, especially by murine serum (Figure 1). We also assayed the effects of serum albumin, the most abundant protein in blood, on the peptide activity.
In contrast to what observed for other AMPs , the bactericidal activity of Bac7(1-35) did not change upon addition of 40 mg/mL BSA, a concentration corresponding BI 2536 nmr to that present in the blood (data not shown). Figure 1 Antimicrobial activity of Bac7(1-35) in the presence of biological fluids. Kinetics of the bactericidal activity of 10 μM Bac7(1-35) against S. enterica ATCC 14028 in the absence (filled squares) or in the presence of 66% murine serum (filled circles), or 66% murine plasma (filled triangles). Bacterial growth without peptide is indicated by empty symbols. Results represent the EX 527 mean ± SD of three independent determinations performed in triplicate. Stability of Bac7(1-35) in serum and plasma Inhibition of the peptide due to enzymatic degradation by blood proteases was taken into account to explain the reduced activity of Bac7(1-35) in biological fluids. The stability of Bac7(1-35) was therefore evaluated by incubating the peptide up to 24 h with murine plasma or serum followed by Western blot analysis. Immunodetection indicated a slow and progressive reduction of the band corresponding to intact
Bac7(1-35), which disappeared after 24 h-incubation in serum (Figure 2A). The degradation of Bac7(1-35) in plasma Interleukin-2 receptor was slower (Figure 2A), suggesting that the activation of proteases of the coagulation cascade in serum may contribute to the faster peptide degradation in this medium. LC-MS analysis indicated that the amount of intact Bac7(1-35) in murine serum decreases by 10% after 1 h of incubation and that the peptide was almost completely degraded after 8 h (Figure 2B and 2C). The degradation process is slower in plasma than in serum, (Figure 2B and 2C), confirming the result observed in the Western blot analysis, while in PBS alone, no peptide degradation was observed even after several
days of incubation at 37°C. Figure 2 Bac7(1-35) stability in blood fractions. (A) Western blot analysis of Bac7(1-35) incubated for different times at 37°C in 25% murine serum or plasma. Lane 1: 0.5 μg Bac7(1-35); lanes 2-6: Bac 7(1-35) after incubation with murine serum or plasma for respectively 0, 1, 4, 8, 24 hrs; lane 7: serum or plasma alone. (B) MC-LC chromatograms of Bac7(1-35) incubated at 37°C in 25% murine serum or plasma. (C) The percentages of Bac7(1-35) with respect to the t0 control were calculated following LC-MS analysis (see section Methods for further details) after incubation of the peptide with murine serum (filled squares) or plasma (filled triangles) for different times. No fragments of Bac7(1-35) were detected by LC-MS analysis.