STRUCTURE AND FUNCTION IN RHODOPSIN .11. MEASUREMENT OF THE RATE OF METARHODOPSIN-II DECAY BY FLUORESCENCE SPECTROSCOPY

An increase in fluorescence is observed upon light activation of bovin e rhodopsin. The rate of increase is monoexponential (t(1/2) = 15.5 mi n) at 20 degrees C in 0.1% lauryl maltoside, pH 6.0, and parallels the rate of decay of metarhodopsin II. We show that the increase in fluor escence is due to the release of free retinal, which no longer quenche s the tryptophan fluorescence. An extrinsic fluorescence reporter grou p, pyrene maleimide, attached to bovine rhodopsin also shows an increa se in pyrene fluorescence on illumination. The rate of increase in pyr ene fluorescence matches the rate of increase in tryptophan fluorescen ce. This result has been used to develop a micromethod, requiring on t he order of 1 mu g of rhodopsin, for measurement of metarhodopsin II d ecay. An Arrhenius plot derived from the fluorescence assay shows the energy of activation barrier for retinal release from rhodopsin to be 20.2 kcal/mol in 0.1% dodecyl maltoside at pH 6.0.