Bleaching Kinetics of Visual Pigments
Bleaching Kinetics of Visual Pigments
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Date
1977
Authors
Resnik, Judith Arlene
Advisor
Zajac, Felix E. III
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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.