Bleaching Kinetics of Visual Pigments

Abstract

A rapid scanning microspectrophotometer (RMSP) has been developed and utilized to study the photoproducts resulting from the bleaching of rhodopsin in the isolated retina of the frog. The RMSP is capable of measuring absorption spectra at multiple wavelengths within the milliseconds and longer time domain. The unusual characteristic of the instrument is the use of a special cathode ray tube as a measuring light source. Spectral scanning is accomplished electronically, with a sampling interval of 600 microseconds for each waveband. A lock-in amplifier system enables the RMSP to be utilized as either a single or dual beam instrument. The results discussed in this dissertation have shown that hydrogen ion availability is a primary cofactor in determining the relative concentration of the metarhodopsin III photoproduct, with less appearing, in lieu of greater free retinal formation, at low pH levels. Metabolic factors have also been shown to influence the pathways of photoproduct decay. The most significant effect has been observed in nonacidic intracellular environments, with deficiencies in metabolic energy production also favoring the direct formation of free retinal from metarhodopsin II. The half-times of formation and decay of metarhodopsin III have also been observed to vary, depending on the extracellular environment of photoreceptor cells. In general, both halftimes tend to be greater when proportionately more metarhodopsin III 1s formed. The ratio of the two half-times, however, remains relatively constant, except in anoxic conditions, in which the decay half-time is significantly prolonged with respect to the formation half-time. Several problems associated with the control of experimental conditions have been discussed as they relate to photoproduct sequence and kinetics. The elimination of as many metabolic, ionic, and other insufficiently controlled conditions as possible has been pointed out as a necessary requirement for obtaining meaningful quantitative results. In addition, the baseline magnitude of the optical density of the retina, which is, in part, a quantification of light scattering, has been shown to be significantly larger in conditions of low intracellular pH or insufficient substrate supply. The utilization of this parameter as an indirect indicator of the probably sequence of photoproducts has been discussed. In conclusion, this research has provided a greater insight into the mechanisms affecting the later, slow photoproduct processes in isolated retinas. In particular, the interaction of hydrogen ions and metabolic factors influences the pathways of photoproduct decay in isolated retinas, subsequent to metarhodopsin II. The results and methods described here should be useful in establishing a context in which to study the faster mechanisms involved in photochemical and electrical transduction in photoreceptor cells. In addition, these results may become important in understanding the normal and pathological functionings of the eye.