The effects have been examined of chymotrypsin, pepsin, trypsin, and pancreatic lipase on cattle rhodopsin in digitonin solution. The digestion of rhodopsin by chymotrypsin was measured by the hydrolysis of peptide bonds (formol titration), changes in pH, and bleaching. The digestion proceeds in two stages: an initial rapid hydrolysis which exposes about 30 amino groups per molecule, without bleaching; superimposed on a slower hydrolysis which exposes about 50 additional amino groups, with proportionate bleaching. The chymotryptic action begins at pH about 6.0 and increases logarithmically in rate to pH 9.2. Trypsin and pepsin also bleach rhodopsin in solution. A preparation of pancreatic lipase bleached it slightly, but no more than could be explained by contamination with proteases. In digitonin solution each rhodopsin molecule is associated in a micelle with about 200 molecules of digitonin; yet the latter do not appear to hinder enzyme action. It is suggested that the digitonin sheath is sufficiently fluid to be penetrated on collision with an enzyme molecule; and that once together the enzyme and substrate are held together by intermolecular attractive forces, and by the "cage effect" of bombardment by surrounding solvent molecules. The two stages of chymotryptic digestion of rhodopsin may correspond to an initial rapid fragmentation, such as has been observed with many proteinases and substrates; superimposed upon a slower digestion of the fragments. Since the first phase involves no bleaching, this may mean that rhodopsin can be broken into considerably smaller fragments without loss of optical properties.

The stability of cattle rhodopsin and of its protein moiety opsin toward acids and alkalies and on aging was determined by two criteria: maintenance of absorption spectrum, and capacity to regenerate after exposure to light. On storage at 3°C. at pH near neutrality, the absorption spectrum in the visible region may remain unchanged for as long as 6 months; but the regenerability progressively declines, at very different rates in different preparations. The cause of this decline has not been determined. It may involve denaturation at sites other than the retinene-protein bond, which by the evidence of the absorption spectrum remains intact. Cattle rhodopsin maintains its absorption spectrum at any pH from 3.9–9.6 for at least an hour at 25–27°C. To both sides of this pH range the pigment bleaches, the extinction falling to half in 1 hour at pH 3.3 and 10.5. The exposure of rhodopsin to light greatly increases the vulnerability of the product (opsin) to acids and bases. Opsin rapidly loses its capacity to regenerate rhodopsin to both sides of the range of pH 5.5–7.0. Half the regenerability is lost within 45 seconds at pH 3.4 and 9.1; and within 1 hour at pH 5 and 8.

Purified preparations of cattle rhodopsin have been titrated to various pH, irradiated, and the pH changes followed thereafter until completed. In this way we have obtained the titration curves of rhodopsin, of the immediate product of irradiation, measured within 30 seconds; and of the final product of irradiation (opsin). The rhodopsin preparations display about 54 titratable groups per mole of pigment: about 34 base-binding and 20 acid-binding groups. In default of an absolute purification, one cannot be sure that all of these go with rhodopsin itself. Exposure to light induces an immediate rise of pH between pH 2 and 8, maximal at about pH 5. This—followed by its slow partial or complete reversal—is the only change of pH in the physiological range (6–7). It involves the exposure of 1 new acid-binding group per mole of rhodopsin with pK about 6.6, close therefore to that of the imidazole group of histidine. At acid and alkaline pH this immediate change is followed by slower changes, occupying up to 40 minutes at 20°C. These changes are always in the direction of neutrality. They involve increases of 5 to 6 moles acid bound at acid pH, and 7 moles base bound at alkaline pH. They are associated with the irreversible denaturation of opsin in acid and alkaline solution, as evidenced by loss of its capacity to regenerate rhodopsin. Such frank denaturation procedures as the exposure of rhodopsin to alkali or heat in the dark result in comparable acid-base changes.