Figure 1. Comparison of v_dark, v_light, k_d and k_th of wild-type visual pigments.

(a) Schematic procedure of the sample preparation for the measurement of k_th. (b,c) Absolute spectra of bRh-n and bRh-7mr in DM with 100 mM NH₂OH. Dark, light-irradiated, and heat-denatured samples are shown by green, blue-dashed, and red lines, respectively. (d,e) G protein activation rate in the complete dark by bRh-n (green closed square), bRh-7mr (green open square, n = 5), cG-n (red closed square), cG-7mr (red open square) and buffer (black open circle). The western blotting data were cropped and shown in the inset (left lane: bRh-n or cG-n, right lane: bRh-7mr or cG-7mr). (f) G protein activation rate in the complete dark with 10 mM NH₂OH by bRh-n (green closed square), bRh-7mr (green open square), bRh-opsin (red closed square), and buffer (black open circle). The initial rate of G protein activation by bRh-opsin was estimated from the linear fitting (red line) to be 126-fold higher than v_dark of bRh-n in (d). (g) Measurements of v_light and k_d of bRh-n by monitoring the change of intrinsic tryptophan fluorescence after flash light irradiation with (blue line) or without (black line) Gt. Intensities were normalized to the fluorescence increase of Gt in the presence of aluminium fluoride (h) Measurement of k_d of bRh-n by change of intrinsic tryptophan fluorescence after light irradiation. (i) Comparison of the v_dark, v_light, and k_d of visual pigments measured by the same methods as shown in (d, g, h). (j) Comparison of the k_th of visual pigments estimated from data in (i). * indicates a significant difference of relative rate constants between visual pigments connected with a line (p < 0.05; Student’s t- test, two-tailed). All error bars in Fig. 1 represent the S.E.M. of more than three independent measurements.