Isolation and identification of the phosphorylated species of rhodopsin

Rhodopsin is phosphorylated in a light-dependent manner by a kinase intrinsic to the rod outer segment. We have used chromatofocusing to separate six phosphorylated species of rhodopsin and have recovered in the pH gradient fractions 60-80% of the initial phosphorylated sample loaded on the column. The isolated species of rhodopsin coincide with the species that are observed in isoelectric focusing gels in the pH range 6.1-4.7. Unphosphorylated rhodopsin focuses at a pI of 6.0. Two species having two phosphates per rhodopsin with isoelectric points of 5.45 and 5.40 have been isolated. The phosphate to rhodopsin ratios for the remaining species are 3.8, 5.0, 6.1, and 8.2 with isoelectric points of 5.16, 4.99, 4.85, and 4.73, respectively. The chromatofocusing profile suggests that there may be multiple forms of rhodopsin with the same number of phosphates among some of the other phosphorylated forms of rhodopsin.