Hydrogen—tritium exchange of rhodopsin: Effect of solvent on the incorporation of slowly exchanging tritium atoms

Abstract

Rhodopsin, the visual pigment of the vertebrate retinal rod outer segments undergoes isomerisation of its chromophoretic group, retinal, on absorption of a photon. It has been widely suggested that after photoisomerisation of the retinal, rhodopsin is responsible for the release of a transmitter into the interdiscal space of the rod outer segment [ 11. However this transmitter has not yet been identified; indeed it is not yet clear to what extent rhodopsin’s polypeptide chain undergoes conformational changes upon photoisomerisation of the retinal. The hydrogen-tritium exchange technique has often been used to demonstrate the presence of conformational changes in proteins [2,3]. They are visualised as changes in the exchange kinetics of the proteins labile hydrogens. However in order that a conformational change can be detected the labile protons of the polypeptide chain, associated with the conformational change considered, must have become tritiated during the exchange-in of incubation time. Parameters such as the pH, temperature, ionic strength, and the composition of the medium will change the freely accessible proton exchange rate [2] and/or the conformation of the protein and hence modify the observed exchange kinetics. Thus in order that the conformational changes of rhodopsin upon illumination can be studied it is necessary to ensure that the associated labile hydrogens have become tritiated during the incubation time. The effect of several incubation