The process of seeing involves the capture of light photons by specific photopigments (PP) contained within specific photoreceptors (PR) within the retina. Two major classes of PR exist (i.e., rods and cones) with the cones being further divided into three types (i.e., red, green, and blue). These divisions among PR types are based upon their absorption spectra (i.e., the wavelength of light most efficiently absorbed) which is in turn dependent upon the presence of unique PP in each PR. These PP are composed of a glycoprotein (opsin) and an analog of vitamin A (11-cis-retinal) called the chromophore. Since these Pp all contain the same chromophore it is believed that the molecular basis for the varying absorption spectra resides within the opsin molecule. Much is known about the molecular structure of the opsin contained in bovine rods (i.e., rhodopsin). Much less is known about the molecular structure of the opsins associated with the three color-specific PP because it has not been possible to purify them. This application proposes to produce antibodies to the color-specific cones which will then be used to prepare samples of pure color-specific cones through affinity procedures. From these purified color-specific cones the color-specific opsins would then be purified and used as immunogens to stimulate the production of large amounts of anti-color specific opsin antibodies. These latter antibodies will be used, in affinity procedures, to purify large amounts of color-specific opsin from homogenized whole retina which could then be subjected to biochemical analysis. The salient feature of this application is the combination of monoclonal antibody production, immunocytochemistry, and the use of goldfish to permit the production and isolation of antibodies to color-specific cones without their prior purification. Monoclonal antibody production procedures allow for the immunization with impure antigens. However, they do require pure antigen for the clone screening process. This latter requirement for pure antigen will be circumvented through the use of immunocytochemistry on goldfish retina where the three types of color-specific PR are morphologically distinguishable. The overall objective of this procedure is to purify to homogeneity the three color-specific opsins. An understanding of the structural similarities and difference between the color-specific PP opsins would be very helpful in understanding the biochemistry of color vision.
