Purification of Cone Visual Pigments from Chicken Retina

A novel method for purification of chicken cone visual pigments was established by use of a 3-[(3-cholamidopropyl)dimethylammonio]-l-propanesulfonate-phosphatidylcholine (CHAPS–PC) mixture. Outer segment membranes isolated from chicken retinas were extracted with 0.75% CHAPS supplemented with 1.0 mg/mL phosphatidylcholine (CHAPS–PC system). After the extract was diluted to 0.6% CHAPS, it was loaded on a concanavalin AX2013;Sepharose column. Elution from the column with different concentrations of methyl α-mannoside yielded three fractions: the first was composed of chicken violet, blue, and red in roughly equal amounts, the second predominantly contained chicken red, and the third was rhodopsin with a small amount of chicken green, which was separated from rhodopsin by DEAE-Sepharose column chromatography. Since CHAPS has little absorbance at both ultraviolet and visible regions, we could demonstrate the absolute absorption spectra of chicken red (92%) and rhodopsin (›96%) in these regions. The maximum of the difference spectrum between either chicken red or rhodopsin and its photoproduct (all-trans-retinal oxime plus opsin) was determined to be 571 or 503 nm, respectively. Although chicken green was contaminated with a small amount of rhodopsin having a similar spectral shape, the maximum of its difference spectrum was located at 508 nm by taking advantage of the difference in susceptibility against hydroxylamine between these pigments. Although chicken blue and chicken violet were minor pigments present in the first fraction from the concanavalin A column, their maxima in the difference spectra were determined to be at 455 and 425 nm, respectively, by a partial bleaching method. When the difference spectra were plotted on a scale of the fourth root of the wavelength and the maxima were shifted on the scale so as to coincide with each other, the spectra of the pigments except for chicken violet were super-imposable with each other in the longer wavelength region. On the assumption that the difference spectrum of chicken violet was distorted by the absorption of retinal oxime, the difference spectrum was corrected to a maximum of 415 nm. Thus, we could convincingly identify the four kinds of cone pigments in vitro and demonstrate their absorption maxima. These values were in good agreement with those determined microspectrophotometrically rather than those reported in digitonin, suggesting that the conformations of pigments in the CHAPS-PC system may be closer to the native ones than those in the digitonin system.