Simplified concanavalin-A sepharose adsorption method for separation of cone visual pigments from rhodopsin.
Batch adsorption of chicken photoreceptor extract using Concanavalin-A Sepharose enables separation of rod and cone pigments and separation of cone pigments of different color sensitivity. In earlier work, column separations using the same adsorption medium, although effective, with high resolution, were slow, demanding and required many differential bleachings of column fractions for analysis. It is shown here that affinity separations can be performed in the batch adsorption mode to purify cattle, frog and chicken rhodopsin. These procedures are more rapid and much more convenient. An extract from the chicken retina can rapidly be separated into four fractions, including three highly enriched (80% or more) visual pigment fractions: (1) extraneous proteins, carotenoids and phospholipids; (2) short wavelength-sensitive pigments; (3) iodopsin; and (4) rhodopsin. While the resolution is not as great as that of the columns, the selectivity is sufficient to produce cone pigments, which are only slightly contaminated with rhodopsin and free of other proteins, either to experiment with directly or to enable heavier loading on high resolution columns. The method is adaptable both to highly labile pigments and to very small quantities, neither of which perform well in column separations.
D000327:Adsorption; D000818:Animals; D002417:Cattle; D002498:Centrifugation; D003208:Concanavalin A; D005260:Female; D008362:Mannosides; D008722:Methods; D010786:Photoreceptor Cells / Q000378:metabolism*; D012168:Retinal Pigments / Q000032:analysis* / Q000302:isolation & purification*; D012243:Rhodopsin / Q000032:analysis*; D012374:Rod Cell Outer Segment / Q000032:analysis; D012685:Sepharose
D008362:Mannosides; D012168:Retinal Pigments; D003208:Concanavalin A; D012243:Rhodopsin; D012685:Sepharose