Purification and partial characterization of a lutein-binding protein from the midgut of the silkworm Bombyx mori.
A lutein-binding protein was purified from fifth instar larval midgut of Bombyx mori by a combination of ammonium sulfate fractionation and three chromatographic procedures, gel filtration, chromatofocusing, and anion exchange chromatography. The protein has a pI of 5.4 and an apparent molecular mass of 35,000 Da, as determined by a linear gradient SDS-polyacrylamide gel electrophoresis. The lutein-protein complex is water-soluble and more stable than the carotenoid or protein alone. The carotenoid moiety was identified by thin layer chromatography, light absorption spectroscopy, and high performance liquid chromatography as all-trans-lutein. Lutein is specifically and stoichiometrically bound to the protein, with a ratio of 3 mol of lutein per mol of protein. Binding of lutein (absorption maximum in hexane at 454 nm) to the apoprotein results in a marked red spectral shift of about 38 nm, giving rise to absorption maxima at 432, 462, and 492 nm in 20 mM Tris-HCl, pH 7.0. The lutein-protein complex is characterized by fine spectral structure indicating that lutein is in a relatively rigid environment. This protein is distributed in equal amounts throughout the midgut and in all developmental stages of the larval B. mori.
Jouni
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Department of Biochemistry, Biological Sciences West, University of Arizona, Tucson, Arizona 85721-0088, USA.
Wells
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D000645:Ammonium Sulfate; D000818:Animals; D000906:Antibodies; D012831:Bombyx / Q000254:growth & development / Q000378:metabolism*; D002338:Carotenoids / Q000032:analysis; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004064:Digestive System / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D018507:Gene Expression Regulation, Developmental; D007814:Larva; D014975:Lutein / Q000378:metabolism*; D008297:Male; D008970:Molecular Weight; D011817:Rabbits; D013053:Spectrophotometry
D000906:Antibodies; D002352:Carrier Proteins; D002338:Carotenoids; D000645:Ammonium Sulfate; D014975:Lutein