Competitive binding assays for riboflavin and riboflavin-binding protein

Lotter, Sharon E.; Miller, Mark S.; Bruch, Richard C.; White, Harold B.

doi:10.1016/0003-2697(82)90390-6

Cited by 26 publications

(10 citation statements)

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“…Data shown are representative of three experiments. Free thiol content (᭹) was estimated by the method of Ellman [20] and riboflavin binding activity (᭺) was measured by fluorescence quenching [29]. centrifuged again at 13000 g to sediment the inclusion bodies.…”

Section: Methodsmentioning

confidence: 99%

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Refolding of native and recombinant chicken riboflavin carrier (or binding) protein : evidence for the formation of non‐native intermediates during the generation of active protein

Pattanaik

Adiga

Visweswariah

1998

European Journal of Biochemistry

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Riboflavin carrier (or binding) protein (RCP) is a phosphoglycoprotein originally purified from the egg white, yolk and serum of laying hens. The 18 cysteine residues present in RCP form nine disulfide bridges, allowing the protein to form a compact structure to generate a hydrophobic pocket in which riboflavin sits. We studied the refolding of totally reduced and denatured egg white RCP and found that the protein initially folded to generate a molecule that did not possess riboflavin-binding activity, despite near-complete oxidation of the cysteine residues. Riboflavin-binding activity was then slowly regained, but the final refolded form of the protein was less compact in structure than the native molecule, due to incomplete oxidation of all the cysteine residues. Denatured and reduced dephosphorylated RCP refolded as efficiently as the native protein, with similar rates of disulfide-bond oxidation and generation of riboflavin binding, showing that the phosphoserine stretch of RCP has little role to play during refolding. In order to study the role of glycosylation in the refolding process, the cDNA for full-length RCP was expressed in Escherichia coli and purified. Recombinant RCP refolded only in the presence of redox buffers, demonstrating that glycosylation of RCP could allow the formation of high yields of productive intermediates in the folding pathway. Using a panel of conformation-specific monoclonal antibodies to RCP, it appeared that the folding intermediates of RCP possessed a structure distinctly different to the native protein, indicating that the correct folding pathway of RCP passed through conformation(s) generated by non-native disulfide bridges.Keywords : riboflavin carrier protein; refolding; monoclonal antibodies.Riboflavin carrier protein (RCP) is a phosphoglycoprotein found in the egg white, yolk and serum of laying hens and other birds [1,2]. The egg-white protein is synthesized in the oviduct of the laying hen [3]. The liver, under the influence of estrogen, synthesizes serum RCP [3,4] which is then deposited through receptor-mediated uptake into the yolk of the developing egg [4]. RCP delivers riboflavin to the developing embryo in the egg, and birds that harbor a mutant RCP gene fail to lay eggs that hatch, as a result of insufficient deposition of the vitamin in the egg [5,6]. Egg-white RCP differs from the yolk and serum RCP only in the pattern of glycosylation [7] and removal of the C-terminal 11Ϫ13 amino acids in yolk RCP, presumably as a result of proteolytic cleavage during oocyte uptake [8]. A stretch of phosphoserine residues is present towards the C-terminal end of RCP [9]. We and others have shown the critical importance of these residues in mediating oocyte-receptor-binding and uptake of RCP during egg development [10,11].RCP is a complex protein with 18 cysteine residues all involved in disulfide-bridge formation [12]. Early studies had indicated that a single disulfide bridge appears to be critical for riboflavin binding [13], but the specific disulfide bridge involved ha...

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Section: Methodsmentioning

confidence: 99%

“…This was used to estimate the fraction of protein in the refolding mixture capable of binding riboflavin. The dissociation constant (K d ) was calculated from the titration curves as described by Hu et al [29].…”

Section: Methodsmentioning

confidence: 99%

Refolding of native and recombinant chicken riboflavin carrier (or binding) protein : evidence for the formation of non‐native intermediates during the generation of active protein

Pattanaik

Adiga

Visweswariah

1998

European Journal of Biochemistry

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“…Samples for biotin analysis were not diluted before freezing. Assays for BBP-I BBP-I was assayed by a radioligand-exchange procedure analogous to that described for assaying riboflavin-binding protein (Lotter et al, 1982). A series of tubes containing 0.1 1sCi of D-[8,9-3H(n)]biotin (lot 2169-146, 35.0 Ci/mmol; New England Nuclear Corp., Boston, MA, U.S.A.) and 20-240 pl of diluted plasma or yolk in the above sodium acetate buffer (total volume 1.0 ml) was incubated at 65°C for 40 min to equilibrate free and BBP-I-bound biotin.…”

Section: Sample Preparationmentioning

confidence: 99%

“…White, T. McGahan & M. A. Letavic, unpublished work). The graphical analysis of this assay yields the amount of endogenous ligand (Lotter et al, 1982). Although there are still some technical problems with this assay that cause underestimates of the amount of binding proteins, it is now possible to analyse BBP in samples not accessible with the previous assay.…”

Section: Introductionmentioning

confidence: 99%

Role of avidin and other biotin-binding proteins in the deposition and distribution of biotin in chicken eggs. Discovery of a new biotin-binding protein

White¹,

Whitehead²

1987

Biochemical Journal

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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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“…A general principle of competitive protein-binding assay has been frequently exploited in analytical biochemistry of vitamins: biotin (Hood 1979), riboflavin (Lotter et al 1982), vitamin BI2 (Lee & Griffiths 1985) and folates (Herbert & Colman 1985). Thiamine radioassay has not yet been suggested, probably because specific soluble thiamine-binding proteins are rare and poorly characterized.…”

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confidence: 99%

Competitive Protein-binding Radioassay of Thiamine in Simple Solutions and in Multivitamin Pharmaceuticals

Kozik

Wrobel

Turyna

1991

Journal of Pharmacy and Pharmacology

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A competitive inhibition radioassay of thiamine is described using a gel obtained by coupling a buckwheat-seed thiamine-binding protein to CNBr-activated Sepharose. The sample to be analysed is incubated with gel suspension and [14C]thiamine and after centrifugation the radioactivity of the supernatant is measured. The method is simple and specific, and applicable over a thiamine concentration range 0.5-5 microM with a coefficient of variation typically below 5%. The gel is reusable and stable for several months. Applicability of the method for direct determination of thiamine in multivitamin pharmaceuticals is demonstrated.

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Competitive binding assays for riboflavin and riboflavin-binding protein

Cited by 26 publications

References 16 publications

Refolding of native and recombinant chicken riboflavin carrier (or binding) protein : evidence for the formation of non‐native intermediates during the generation of active protein

Refolding of native and recombinant chicken riboflavin carrier (or binding) protein : evidence for the formation of non‐native intermediates during the generation of active protein

Role of avidin and other biotin-binding proteins in the deposition and distribution of biotin in chicken eggs. Discovery of a new biotin-binding protein

Competitive Protein-binding Radioassay of Thiamine in Simple Solutions and in Multivitamin Pharmaceuticals

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