Behavior of crayfish rhodopsin and metarhodopsin in digitonin: the 510 and 562 nm "visual pigments" are artifacts.

The visual pigment of the main rhabdom of the crayfish (P533) is unstable in digitonin. While slowly hydrolyzing to N-retinylidene opsin, a portion passes through a long-lived intermediate (P'505) with absorption similar to metarhodopsin but with the retinal still in the cis configuration. Crayfish metarhodopsin (M515) is similarly unstable in digitonin, and a portion converts to M'508 while bleaching slowly in the dark. Both P'505 and M'508 are light sensitive and bleach through an intermediate absorbing at still shorter wavelengths, M'460. The photobleaching of M'508 is likely a two-photon process, possibly involving P'505 as an intermediate. The persistence of these altered forms of the pigment with lambda max near 510 nm has compromised earlier efforts to analyze extracts of crayfish rhodopsin by partial bleaching. First, because of the incomplete decay of M515 (a portion of which liners as M'508), the difference spectrum for a red light exposure followed by dark decay has lambda max at 562 nm, but this difference spectrum does not describe a pigment. Because of the photosensitivity of M'508, a second bleaching exposure reveals the presence of a pigment with lambda max near 510 nm, but it is not a visual pigment and it is not present in the extract initially.