TY - JOUR
T1 - Structure and function in rhodopsin
T2 - Measurement of the rate of metarhodopsin II decay by fluorescence spectroscopy
AU - Farrens, David L.
AU - Khorana, H. Gobind
PY - 1995/3/10
Y1 - 1995/3/10
N2 - An increase in fluorescence is observed upon light activation of bovine rhodopsin. The rate of increase is monoexponential (t1/2 = 15.5 min) at 20°C in 0.1% lauryl maltoside, pH 6.0, and parallels the rate of decay of metarhodopsin II. We show that the increase in fluorescence is due to the release of free retinal, which no longer quenches the tryptophan fluorescence. An extrinsic fluorescence reporter group, pyrene maleimide, attached to bovine rhodopsin also shows an increase in pyrene fluorescence on illumination. The rate of increase in pyrene fluorescence matches the rate of increase in tryptophan fluorescence. This result has been used to develop a micromethod, requiring on the order of 1 μg of rhodopsin, for measurement of metarhodopsin II decay. 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.
AB - An increase in fluorescence is observed upon light activation of bovine rhodopsin. The rate of increase is monoexponential (t1/2 = 15.5 min) at 20°C in 0.1% lauryl maltoside, pH 6.0, and parallels the rate of decay of metarhodopsin II. We show that the increase in fluorescence is due to the release of free retinal, which no longer quenches the tryptophan fluorescence. An extrinsic fluorescence reporter group, pyrene maleimide, attached to bovine rhodopsin also shows an increase in pyrene fluorescence on illumination. The rate of increase in pyrene fluorescence matches the rate of increase in tryptophan fluorescence. This result has been used to develop a micromethod, requiring on the order of 1 μg of rhodopsin, for measurement of metarhodopsin II decay. 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.
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U2 - 10.1074/jbc.270.10.5073
DO - 10.1074/jbc.270.10.5073
M3 - Article
C2 - 7890614
AN - SCOPUS:0028957661
SN - 0021-9258
VL - 270
SP - 5073
EP - 5076
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 10
ER -