Rhodopsin Extraction and Characterization

A detailed description of the isolated and prepared bovine ROS was reported previously (58 (link)). The washed ROS with isotonic and hypotonic buffers was used, in the dark under a dim red light (>670 nm) for observance, to extract rhodopsin as described previously (59 (link), 60 (link), 61 (link), 62 , 63 (link), 64 (link)). Solubilized rhodopsin at 1.05 mg/ml in 20 mM 1,3-bis(tris(hydroxymethyl)methylamino) propane, pH 6.9, 100 mM NaCl, and 1 mM LMNG buffer was incubated for 5 min at 20 °C with 0.7 μl of 100 mM stock solutions of each of the nine identified hit compounds in DMSO (100 μM final concentration) and centrifuged at 1000g for an additional 5 min. Absorption spectra of rhodopsin (dark, and treated with the nine hit compounds and untreated) were then measured with a Cary 50 UV-visible spectrophotometer (Varian). Next, photobleaching was carried out with a 150-W fiber light delivered through a 480 to 520 nm band pass filter (Chroma Technology) for 10 s; immediately then, absorption spectra were measured again for each sample in triplicate.