The effect of detergent selection on retinal outer segment A280/A500 ratios.

Abstract

A280/A500 purity ratios for 5 different retinal rod outer segment (ROS) preparations were determined without prior centrifugation, in 1-cm light path cuvettes, in 1% concentrations of 5 detergents.Mean ratios were 3.66 + 0.23 for Brij 96, 3.56 ± 0.20 for cetyltrimethylammonium bromide, 3.12 ± 0.14 for Na-cholate, 5.62 ± 0.34 for digitonin, and 1.78 ± 0.07 for Triton X-100.Ratios in Triton were depressed by a large UV absorbance of the detergent, which prevented accurate photodetection of the UV absorbance of the protein.Brij and digitonin also displayed significant UV absorbance.Pu- rity ratios in digitonin were also elevated by UV lightscattering associated with undissolved, sedimentable protein.Differences evidently attributable to interac- tion of a particular detergent with ROS protein or lipoprotein persisted even when these UV effects were minimized by use of 1-mm light path cuvettes and cen- trifugation of nonsolubilized material prior to spectro- photometry.Such differences were minimal, however, among the 3 nonionic detergents octyl glucoside, Emul- phogene and Ammonyx LO.At 11 gM rhodopsin and detergent concentrations, the 3 nonionic detergents displayed negligible A280 and no significant sediment upon centrifugation.In the presence of excess ROS, Emulphogene extracted more protein from the ROS than did Ammonyx LO or octyl glucoside.Despite dif- ferences in color of the unextracted, bleached pellets resulting from extraction by these detergents, Emul- phogene spectra of the pellets revealed qualitatively similar contents.We have selected Emulphogene for A280/A500 ratios because (a) it does not absorb in the 278-nm range; (b) it does not interfere with Lowry protein determinations while all ofthe other detergents except octyl glucoside do; (c) it apparently extracts more ROS protein than do Ammonyx LO or octyl glu- coside when ROS is in excess; and (d) it is much cheaper than octyl glucoside.The purity of rhodopsin and of retinal rod outer segment preparations from which it is derived have long been estimated in detergent solutions by comparing absorbance at the ab- sorption maximum of rhodopsin (about 500 nm, depending on species) with absorbance at some wavelength which indicates the presence of contaminants, typically 280 nm for protein or 400 nm for hemoglobin, cytochromes, or non-rhodopsin pig-