Hydrogen-tritium exchange of rhodopsin: effect of solvent on the incorporation of slowly exchanging tritium atoms

The hydrogen-tritium exchange technique has been used to demonstrate the presence of conformational changes in proteins. They are visualized as changes in the exchange kinetics of the proteins labile hydrogens. To enable the study of the conformational changes of rhodopsin - the visual pigment of the vertebrate retinal rod outer sigments - upon illumination, it is necessary to ensure that the associated labile hydrogens become tritiated during incubation time. The effect of several incubation media on the rhodopsin exchange-in-kinetics have been studied.. The solubilisation effect by detergent on the exchange-in-kinetics of rhodopsin was also investigated. It is shown that both membrane-bound and detergent-solubilised rhodopsin possess an important number of very slowly exchanging hydrogen atoms. The number of slowly exchanging tritium atoms incorporated in rhodopsin is greatly increased by the presence of phosphate ions in the incubation medium