FIGURE 3. Azido-Q uptake of RegB and the HPLC profile of digested RegB.

A, effect of illumination time on azido-Q uptake and inactivation of the sensor kinase RegB. Purified RegB in 20 mm Tris-Cl, pH 8.0, containing 150 mm NaCl and 30% glycerol was incubated with azido-Q in ethanol (open circles and open squares) or ethanol only (solid circles) for 1 h at 4 °C in the dark. The samples were then illuminated with long wavelength UV light for the indicated times at 0 °C. The determination of activity (open circles and solid circles) and radioactivity (open squares) were performed as described under “Experimental Procedures.” B, ³H radioactivity distribution in an HPLC chromatogram of a protease K-digest of [³H]azido-Q-labeled RegB. The labeled protein was digested, fractioned, and assayed for radioactivity as described under “Experimental Procedures.”