Tryptophan in bovine rhodopsin: its content, spectral properties, and environment

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Abstract
The tryptophan content of purified bovine rhodopsin was obtained by 2 independent methods: direct analysis of hydrolysates prepared by digestion of opsin with methanesulfonic acid containing 0.2% 3-(2-aminoethyl)indole and a computer-assisted analysis of the near-UV spectrum of rhodopsin. Both methods gave a value of 8 tryptophan residues/rhodopsin. The near-UV spectral analysis, the light-induced difference spectrum of rhodopsin and the susceptibility of residues to oxidation by N-bromosuccinimide imply half of the tyrosine and tryptophan residues are shielded to some extent from the aqueous solvent, 2 of the tryptophan residues are in very apolar environments and following light excitation at least 1 of these tryptophan residues and several tyrosines are exposed to an aqueous environment. Analysis of rhodopsin absorption in the far-UV imply that below 240 nm, approximately half of the absorption is due to aromatic residues and the other half is largely due to the peptide bond. The effect of illumination on secondary structure is to induce a loss in helical structure, calculated to involve 35% of the amino acid rsidues in purified rhodopsin. If light-induced changes in secondary structure are specifically excluded, most of these properties extend to bovine rod outer segment membranes.