Effect of photoregeneration on the calculation of the amount of rhodopsin bleached by small flashes.

A sample of rhodopsin that is exposed to a series of small light flashes of equal intensity is expected to bleach in successively smaller decrements in proportion to the remaining unbleached rhodopsin. The exponential depletion law describing this effect has been used as a rapid, convenient, and intuitive method for determining the fraction of rhodopsin bleached per flash. This method is commonly assumed to be free of error provided the amount bleached is small, so that there is no significant photoregeneration. We show here, however, that if there is any photoregeneration, the bleach fraction calculated in this manner can be in error by a factor of two or more, no matter how little rhodopsin is bleached. This flaw occurs insidiously, without perturbing the expected exponentiality of the bleaching decrements, thereby escaping ready notice. The erroneous bleach values readily propagate as underestimates of metarhodopsin and accompanying G-protein equilibrium and kinetic constants. We derive equations for correcting such errors and illustrate how empirical constants can be obtained from experiments that permit the true fraction bleached to be determined.