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Using rapid mixing techniques and resonance Raman spectroscopy, we have found that the 1H/2H exchange time for the Schiff base proton of bacteriorhodopsin in purple membrane is 4.7 msec, when experiments are carried out at pH 2 or pH 7 at room temperature in the dark. We argue that diffusion of neutral water into the membrane is fast on this time scale. Also, model Schiff bases in solution have a pKa between 6 and 7, and we show that such model Schiff bases have much faster exchange rates. Therefore, we conclude that the Schiff base proton in bacteriorhodopsin is protected from interaction with the medium, probably by interaction with a protein group, and this would account for a pKa considerably higher than 6-7.
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