Molar extinction coefficients and other properties of an improved reaction center preparation from Rhodopseudomonas viridis |
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Authors: | Roderick K Clayton Betty J Clayton |
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Institution: | Division of Biological Sciences, Cornell University, Ithaca, N.Y. 14853 U.S.A. |
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Abstract: | Reaction centers have been purified from chromatophores of Rhodopseudomonas viridis by treatment with lauryl dimethyl amine oxide followed by hydroxyapatite chromatography and precipitation with ammonium sulfate. The absorption spectrum at low temperature shows bands at 531 and 543 nm, assigned to two molecules of bacteriopheophytin b. The 600 nm band of bacteriochlorophyll b is resolved at low temperature into components at 601 and 606.5 nm. At room temperature the light-induced difference spectrum shows a negative band centered at 615 nm, where the absorption spectrum shows only a weak shoulder adjacent to the 600 nm band. The fluorescence spectrum shows a band at 1000 nm and no fluorescence corresponding to the 830 nm absorption band. Two molecules of cytochrome 558 and three of cytochrome 552 accompany each reaction center. The differential extinction coefficient (reduced minus oxidized) of cytochrome 558 at 558 nm was estimated as 20 ± 2 mM?1 · cm?1 through a coupled reaction with equine cytochrome c. The extinction coefficient of reaction centers at 960 nm was determined to be 123 ± 25 mM?1 · cm?1 by measuring the light-induced bleaching of P-960 and the coupled oxidation of cytochrome 558. The corresponding extinction coefficient at 830 nm is 300 ± 65 mM?1 · cm?1. The absorbance ratio in our preparations was 2.1, and there was 190 kg protein per mol of reaction centers. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed three major components of apparent molecular weights 31 000, 37 000 and 41 000. |
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Keywords: | LDAO lauryl dimethyl amine oxide |
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