Fig. 4. Redox state-dependent spectral properties of rxYFP¹⁴⁹₂₀₂ at 30°C. (A) Absorption spectra were recorded after equilibration of the protein in redox buffers with [GSH]²/[GSSG] ratios of 0.004 M (open circles), 0.55 M (open squares), 2.17 M (open diamonds) and 8.13 M (open triangles) (see Figure 3A). Reduced protein (open inverted triangles) was obtained by incubation with 10 mM DTT for 2 h. (B) The relative fluorescence (open symbols) and relative absorbance (closed symbols) of reduced (circles) and oxidized (squares) rxYFP¹⁴⁹₂₀₂ as a function of pH. Data were fitted as described in Materials and methods. The estimated values of the chromophore pK_a and the Hill coefficient (n) are given in the text.