Purification and characterization of biotin-binding protein II from chicken oocytes.
BBP-II, the major biotin-binding protein from chicken oocytes, was purified 12,000-fold with a 22% yield. The purification procedure includes butan-1-ol extraction of yolk lipids, phosphocellulose chromatography of the water-soluble proteins, DEAE-cellulose chromatography at pH 7.4 and hydroxyapatite column chromatography. Final purification was obtained by using a second DEAE-cellulose column chromatography at pH 6.0. BBP-I activity separated from BBP-II activity during elution from the first DEAE-cellulose column. Purified BBP-II was homogeneous on both polyacrylamide-gel electrophoresis and SDS/polyacrylamide-gel electrophoresis under conditions that would detect a 1% impurity. The subunit Mr determined from SDS/polyacrylamide-gel electrophoresis was 18,200 (72,600 for tetramer), which compares favourably with an Mr value of 17,300 (69,100) calculated from the amino acid analysis. A single precipitin line formed when rabbit antiserum to the protein was directed against a crude chicken egg-yolk sample. BBP-II purified by this procedure lacked carbohydrate and phosphate, was stable indefinitely when frozen, and was quite stable at room temperature. The N-terminal amino acid sequence showed polymorphism at three positions in the first 23 residues and was about 45% identical with the N-terminal 22 residues of avidin. Antiserum to BBP-II cross-reacted with BBP-I and similar proteins in the yolk of eggs from various birds and alligator as judged by immunodiffusion and enzyme-linked immunosorbent assays. No cross-reaction was observed with chicken egg-white by either of these methods.
Bush
L
L
Department of Chemistry and Biochemistry, University of Delaware, Newark 19716.
McGahan
T J
TJ
White
H B
HB
D000595:Amino Acid Sequence; D000818:Animals; D001710:Biotin / Q000378:metabolism*; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002645:Chickens; D007700:Kinetics; D008969:Molecular Sequence Data; D009865:Oocytes / Q000032:analysis*
D002352:Carrier Proteins; C017071:biotin-binding proteins; D001710:Biotin