Purified preparations of cattle rhodopsin have been titrated to various pH, irradiated, and the pH changes followed thereafter until completed. In this way we have obtained the titration curves of rhodopsin, of the immediate product of irradiation, measured within 30 seconds; and of the final product of irradiation (opsin).
The rhodopsin preparations display about 54 titratable groups per mole of pigment: about 34 base-binding and 20 acid-binding groups. In default of an absolute purification, one cannot be sure that all of these go with rhodopsin itself.
Exposure to light induces an immediate rise of pH between pH 2 and 8, maximal at about pH 5. This—followed by its slow partial or complete reversal—is the only change of pH in the physiological range (6–7). It involves the exposure of 1 new acid-binding group per mole of rhodopsin with pK about 6.6, close therefore to that of the imidazole group of histidine.
At acid and alkaline pH this immediate change is followed by slower changes, occupying up to 40 minutes at 20°C. These changes are always in the direction of neutrality. They involve increases of 5 to 6 moles acid bound at acid pH, and 7 moles base bound at alkaline pH. They are associated with the irreversible denaturation of opsin in acid and alkaline solution, as evidenced by loss of its capacity to regenerate rhodopsin. Such frank denaturation procedures as the exposure of rhodopsin to alkali or heat in the dark result in comparable acid-base changes.