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This chapter discusses the preparation of the spin-labeled derivative, I, 4-(2,2,5,5-tetramethyl-1-pyrrolidinyloxy)retinal, and the formation of pigment with this derivative and bleached purple membrane. Preparation of spin-labeled retinal, all-trans-retinal dissolved in carbon tetrachloride is added to a stirred suspension of stoichiometric amounts of recrystallized N-bromosuccinimide. 2,2,5,5-Tetramethyl-1-pyrrolidinyloxy- 3-carboxylic acid carbon tetrachloride is added with stirring under nitrogen for several minutes to initiate bromination and stirred at room temperature. An excess of N,N-dimethylaniline is added and the reaction mixture stirred for 18 hr. After filtering, the solution is washed with cold 5% sulfuric acid, water, 2% sodium bicarbonate, and again with water. After drying, the solvent is evaporated. The spin-labeled retina is isolated in about 25% yield by thin-layer chromatography. For preparation of spin-labeled bacteriorhodopsin, a solution of the SLR in ethanol is added by 2-μl increments to bleached membrane in sodium acetate buffer, shaken, and the absorption spectra recorded using bleached purple membrane as the reference. Spin-labeled retinal is added until the absorption at the hmax shows no further change and free chromophore absorption just begins to appear.

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