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Titration of the retinal Schiff base in D85N bacteriorhodopsin. Deprotonation was determined from the amplitudes of the shift of the maximum from near 600 nm to 410 nm. Reconstitution of the hydroxylamine-bleached chromophores was with either authentic (○) or 13-demethyl 12–14 ethano (•) retinal. Titration was in 2 M NaCl, where the apparent pKa of the Schiff base is somewhat lower than at lower NaCl concentrations (4, 33, 37).
