In situ microspectrophotometric studies on the pigments of single retinal rods.

Three spectral entities have been observed in single intact frog rod outer segments at 506 mmu, 480 mmu and 380 mmu. It is likely that the peak of 506 mmu was somewhat altered by bleaching reactions and originated at about 510 mmu. This is identified with the 502 mmu frog rhodopsin of digitonin extracts. Spectra in polarized light have the same maximum, identifying the dichroism of rods with rhodopsin. The dichroic ratio is around 6, giving the outer segment an axial density of 0.09/5mu or 0.9 OD total, with a pigment concentration of 2 to 3 mM. The dichroism data are used to compute the angle separating the rhodopsin molecular absorption vectors in rods from perfect restriction to a plane. This angle is 16 degrees or 23 degrees depending on which of two assumptions one chooses for the type of molecular ordering. The spectral peaks at 480 mmu and 380 mmu are thought to correspond respectively to metarhodopsin and retinene. Disappearance of the former is accompanied by accumulation of the latter. This reaction seems to occur more slowly in the intact outer segment than the corresponding reaction in solution. Spread of bleaching spectra from illuminated to dark areas of the same rod did not occur over distances of 2 mu or greater. Spectra were similar from rod to rod and from point to point on the same rod showing that frog rods are spectrally homogeneous both individually and collectively.