Optical Study of the Light‐Induced Protonation Changes Associated with the Metarhodopsin II Intermediate in Rod‐Outer‐Segment Membranes

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Abstract
The kinetics of the protonation changes and of the formation of the metarhodopsin II intermediate were measured in the presence of the pH indicator dye bromocresol purple after 2 consecutive flashes on the same suspension of right-side-out vesicles from cattle rod-outer-segment membranes. A rapid proton binding was followed by partial proton release. The formation of metarhodopsin II was biphasic. The rapid phase of metarhodopsin II formation was slightly faster than the proton binding and the kinetics of the slow phase were similar to proton release. The protein groups which were protonated and deprotonated were situated on the outside of the vesicles. A structural change of the protein involving pK changes apparently occurs without spectral modification before the formation of metarhodopsin III (half-time of the order of seconds).