Abstract
Purpose :
Obtaining clean rhodopsin spectra with UV/Vis spectrophotometry often requires extensive purification of pigment from ROS membranes, including the use of detergents for efficient solubilization. Using an integrating sphere accessory, rhodopsin spectra can be obtained from a crude, turbid retina prep that are comparable to high purity, detergent-solubilized rhodopsin samples. This opens the possibility of measuring rhodopsin kinetics from crude human donor tissue preps, thus providing insight into rhodopsin function in disease-affected retinas.
Methods :
Crude ROS preps were obtained from whole bovine, pig, and human retinas. These solutions were loaded into a conventional, fully transparent cuvette without the use of detergent solubilization. The cuvette was placed in a spring-loaded mount accessory that places the sample in the center of a 150mm integrating sphere in a Perkin Elmer Lambda 1050 spectrophotometer.
Results :
UV/Vis absorbance spectra were collected and demonstrated the characteristic 500nm absorption peak for rhodopsin, without the loss of resolution normally seen from light-scattering solutions of turbid retinal preps.
Conclusions :
The ability to accurately measure rhodopsin bleaching and regeneration in its native membrane-bound state opens the possibility of studying pigment function in diseased tissue of individual human donors. This could be tremendously insightful for studies of possible changes in rhodopsin function diseases
This is a 2020 ARVO Annual Meeting abstract.