Nature of the thiamin-binding protein from chicken egg yolk.
A simple, rapid and efficient procedure for the purification of thiamin-binding protein from chicken egg yolk was developed. The method involved removal, by exclusion, of lipoproteins from DEAE-cellulose and subsequent elution of water-soluble proteins held on the ion-exchanger with 1 M-NaCl, followed by treatment of the eluted protein fraction with an aqueous suspension of dextran/charcoal to generate apoprotein from the holoprotein. The resultant protein fraction was subjected to bioaffinity chromatography on thiamin pyrophosphate--AE (aminoethyl)-Sepharose. The protein eluted specifically with 10 microM-thiamin at pH 7.0, was homogeneous by the criteria of polyacrylamide-gel disc electrophoresis, had a mol.wt. of 38 000 +/- 2000 and was not a glycoprotein. The purified thiamin-binding protein specifically interacted with riboflavin-binding protein with no detectable deleterious affect on its (14C)thiamin-binding capacity. The protein bound [14C]thiamin with a molar ratio of 1.0, with dissociation constant (Kd) 0.41 microM. This protein-ligand interaction was inhibited by thiamin analogues and antagonists. The absorption spectrum of the protein in the presence of thiamin exhibited significant hypochromism at the 278 nm band, indicating the involvement of aromatic amino acid residues of the protein, during its binding to the ligand. The protein cross-reacted with the monospecific antiserum to egg-white thiamin-binding protein, showing thereby that thiamin-binding proteins present in chicken egg yolk and white are the products of the same structural gene.
D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002645:Chickens; D004527:Egg Proteins / Q000302:isolation & purification* / Q000378:metabolism; D004530:Egg Yolk / Q000032:analysis*; D005260:Female; D026901:Membrane Transport Proteins; D012256:Riboflavin / Q000378:metabolism; D013053:Spectrophotometry; D013831:Thiamine / Q000302:isolation & purification* / Q000378:metabolism
D002352:Carrier Proteins; D004527:Egg Proteins; D026901:Membrane Transport Proteins; C019079:riboflavin-binding protein; C026989:thiamine-binding protein; D012256:Riboflavin; D013831:Thiamine