Effect of pH on the formation and decay of the metarhodopsins of the frog.

1. Suspensions of membrane vesicles were prepared by sonication of dark-adapted frog rod outer segments. The pH was adjusted to one of twelve values between 5.0 and 9.8; the temperature was 15 degrees C. Each preparation was exposed to a single yellow flash of 120 microseconds duration, and rapid slow changes of absorbance were measured at 475 nm wavelength. 2. Rapid changes consist of a transient rise followed by a diphasic decay to a new level of absorbance which is lower than that before the light exposure. The level of absorbance reached at the end of the rapid changes is lower at lower pH. 3. Kinetic analysis reveals that three reactants take part in the rapid reactions, viz. metarhodopsin I and two isochromic forms of metarhodopsin II named metarhodopsin II' and metarhodopsin II". The kinetics of the reversible transition from metarhodopsin I to metarhodopsin II' are not measurably influenced by the pH of the medium. However, the reversible reaction of metarhodopsin II' with metarhodopsin II" is dependent on pH because metarhodopsin II" is encountered either as a protonated or as a deprotonated compound. 4. Slow reactions are due to metarhodopsin I and to metarhodopsin III. A combined quantitative analysis of both rapid and slow reactions involves a reversible reaction between metarhodopsin II' and metarhodopsin III and a hydrolysis of metarhodopsin II' to retinal and opsin. The scheme employed accounts for the pH dependence of all equilibria although only one of the metarhodopsins reacts directly with protons.