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. 2013 Apr 26;288(24):17698–17712. doi: 10.1074/jbc.M112.397257

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© 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 3. — Measurement of the extinction coefficient of the protonated Schiff base. The extinction coefficient of the protonated SB between all-trans-retinal and acid-denatured opsin is higher than that of the protonated SB between 11-cis-retinal and acid-denatured opsin. At room temperature, dark state D190N rhodopsin was acid-denatured with 4 μl of 1 m HCl and measured by UV-visible spectroscopy (black line), giving an extinction coefficient of 32,600 m⁻¹ cm⁻¹ for the 11-cis-retinal SB. A second sample of D190N was light-bleached for 1 min to convert all 11-cis-retinal to all-trans-retinal. The Meta II sample was denatured and measured by UV-visible spectroscopy (green line), giving an extinction coefficient of 41,600 m⁻¹ cm⁻¹ for the all-trans-retinal SB.