Figure 2.

HBD2 does not act as an alternative substrate for V8. (a,b) Accurate mass measurements of synthetic HBD2 (100 µg/ml) before (a) and after (b) 1-h coincubation with V8 (12.5 µg/ml). Peptides were electrosprayed at a concentration of 50 µg/ml from a solution of 50:49:1 water/methanol/acetic acid (v/v/v). (c,d) FITC-labelled casein substrate was used to measure proteolytic activity of V8 in the presence or absence of either (c) 0.25–2 mg/ml TLCK or (d) 25–100 μg/ml HBD2. Fluorescence was measured at 485/538 nm every 5 min for 1 h and corrected by subtraction of the background fluorescence from the 0-min timepoint. HBD2 data were also normalized for low level HBD2 autofluorescence. 12.5 μg/ml V8 and 5 μg/ml substrate were used throughout. Data represents mean ± SEM for n = 5. Statistical analysis using Mann–Whitney test of final timepoints. ***p < 0.0001.