Enrichment of rhodopsin in rod outer segment membrane preparations: Biochemical aspects of the visual process—XVIII

Abstract

Substantial, seasonally varying amounts of opsin are present in cattle rhodopsin preparations, which cannot be converted to rhodopsin by prior dark adaptation of the excised eyes. A method is described for the quantitative conversion of the opsin to rhodopsin by treatment of a rod outer segment preparation with 11- cis retinaldehyde, followed by removal of excess retinaldehyde through reduction by endogenous retinol dehydrogenase and added NADPH and elution of the resulting retinol by sucrose gradient centrifugation. The resulting rhodopsin preparation has a high pigment content of 8 nMol/mg dry weight, which is constant within 2.5 per cent over the entire year. The preparation is of high purity, as indicated by theA 278/A 500 ratio of 2.0–2.5 and theA 400/A 500 ratio of 0.20–0.26. The yield is high, representing 50 per cent of the pigment present in the retina. The preparation is free of detergent, has a full regenerating capacity, and can be stored for long periods of time.