FIG. 4.

HPLC analysis of the PL50 cleavage products. BoNT/A (10 ng/ml) was incubated for 5 h at 37°C with PL50 (15 μM) in standard assay buffer (HEPES, 20 mM, pH 7.4; ZnSO₄, 200 μM; DTT, 5 mM; BSA, 1 mg/ml) in a final reaction volume of 100 μl. The reaction products were then separated by HPLC on a Kromasil C₁₈ column using a gradient of 10 to 90% CH₃CN (0.1% TFA) in 30 min, and the resulting chromatograms are shown. (A) Peptide 12 metabolite in assay buffer (1 μM), with an Rt of 13.03 min; (B) PL50 (15 μM) incubated in assay buffer without toxin (Rt = 15.96 min); (C) result of the hydrolysis of PL50 (15 μM) by BoNT/A. At 343 nm, the wavelength at which Pya emits, the only observed peaks are those corresponding to PL50 and to peptide 12.