FIGURE 1.

Spectroscopic pH titration to determine pK_a of Asp-75^ppR, the counterion of the protonated Schiff base. The ordinate represents the ratio of the 541-nm absorbance at the respective pH (see text). The protein (∼10 μM) was suspended in 400 mM NaCl supplemented with six-mixed buffer (see Materials and Methods), and the pH was adjusted with H₂SO₄ or NaOH. The titration data were analyzed with the scheme shown in the inset and pK_a values were estimated with a nonlinear regression. Temperature was 20°C.