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Purification and characterization of biotin-binding protein II from chicken oocytes.

ABSTRACT: 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.

SUBMITTER: Bush L

PROVIDER: S-EPMC1135486 | biostudies-other | 1988 Dec

REPOSITORIES: biostudies-other

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Project description:In addition to the previously characterized egg-yolk biotin-binding protein (BBP-I), we have discovered another BBP (BBP-II) in the plasma and yolk from laying hens. BBP-I is stable to 65 degrees C, whereas BBP-II is stable to 45 degrees C. Both proteins are normally saturated with biotin and together they account for most, if not all, of the biotin in hen plasma and yolk, except in hens fed excessive amounts of biotin (greater than 1 mg of biotin/kg of feed). The maximal production of BBP-I is attained at lower levels of dietary biotin (approximately 50 micrograms/kg) than for BBP-II (approximately 250 micrograms/kg); however, the maximal production of BBP-II is severalfold greater than for BBP-I. Consequently, as dietary biotin increases, the ratio of BBP-II to BBP-I increases and becomes constant at dietary intakes of biotin above 250 micrograms/kg. The observation that the amounts of these proteins are limited by biotin in the normal dietary range (less than 250 micrograms/kg) suggests that biotin is required for the synthesis, secretion or stability of these proteins. Although both plasma vitamin-protein complexes are transported to the oocyte and concentrated in the yolk, BBP-II is transferred more efficiently. Thus biotin deposition in the yolk is a function of the amounts and relative concentrations of the two proteins. Dietary biotin above 250 micrograms/kg exceeds the transport capacity of BBP-I and BBP-II in the plasma; however, unbound biotin does not accumulate. Rather it is efficiently scavenged by avidin in the oviduct and transferred to the egg albumen. Only when avidin becomes saturated at high dietary intake does free or weakly bound biotin accumulate in plasma and yolk. The synthesis of avidin is independent of dietary biotin. Small amounts of BBPs with the heat-stability of avidin or BBP-I respectively are present in the plasma of adult males or immature chickens. BBP-II, the major BBP in the plasma and yolk of laying hens, was not detected in the plasma of non-laying chickens.

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Purification and characterization of biotin-binding protein II from chicken oocytes.

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