36] Isolation and purification of bovine rhodopsin

Abstract

Publisher Summary This chapter describes the isolation and purification of bovine rhodopsin. Rhodopsin is the general name for the visual pigments of both the rod cells in the vertebrate retina and the invertebrate photoreceptor cells. Rhodopsin is an intrinsic membrane glycoprotein, confined to the membranous structures (photoreceptor membranes) of a special part of the rod cell, the rod outer segment (ROS), which is highly specialized for light reception. Rhodopsin is determined by measuring the decrease in absorbance at 500 nm upon illumination, using a molar absorbance of 40,500. Under some conditions, the photoproducts also absorb at 500 nm, but addition of hydroxylamine will convert all free and bound retinaldehyde into retinal oxime, which has virtually no absorption beyond 450 nm. In detergents like the quarternary ammonium derivatives (DTAB, TTAB, CTAB) or the amine oxides (C10DAO, C12DAO), addition of hydroxylamine is not required, because under these conditions retinal is completely released and hardly interferes at 500 nm. Hydroxylamine should be handled with care, because it is highly mutagenic. The assay can be performed on suspensions of photoreceptor membranes, but scattering artifacts may interfere. Most accurate results are obtained after prior solubilization of the membrane in an appropriate detergent, followed by centrifugation or filtration through a membrane filter if some turbidity remains.