FIG. 2.

Quantitative measurement of ZapA proteolysis of IgA and IgG by HPLC analysis. In this method, the decrease in the substrate protein peak (rightmost peak in each chromatogram; retention time of ca. 12 min) area and increases in the number and peak areas of product peaks (generally in the range of 2- to 10-min retention time) were used to assess proteolysis as a function of time of incubation and preincubation denaturation of the substrate. (A) Digestion of native IgA over time. The cascade of chromatograms from front to back is IgA without ZapA and in the presence of ZapA at 0, 0.5, 1, 2, 4, 6, and 16 h. (B) Digestion of IgA preincubated in 8 M urea. From front to back, the chromatograms are IgA without ZapA and in the presence of ZapA at 0, 0.5, 1, 2, 4, 6, and 16 h. (C) Digestion of heat-denatured IgA (preincubated at 85°C for 15 min). From front to back, the chromatograms are IgA without ZapA and in the presence of ZapA at 0, 0.5, 1, 2, 4, 6, and 16 h. (D) Digestion of IgG. From front to back, the chromatograms are IgG without ZapA (0 h), IgG plus ZapA (0 h) at 16 h and 37°C, heat-denatured IgG without ZapA, heat-denatured IgG with ZapA, urea-denatured IgG without ZapA, and urea-denatured IgG with ZapA. Reaction mixtures were incubated at 37°C for the specified times in a reaction volume of 100 μl containing 20 nM ZapA and 100 to 125 μM respective protein substrate.