20 Spin Free
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.
More Details 20 FREE SPIN
Get 500 SPIN Clik Here
Done
ReplyDelete