Purification and Biochemical Characterization of a Novel Allergenic Protein from Tatary Buckwheat Seeds

Abstract

An allergenic protein from tatary buckwheat seeds, designated TBt, was purified by salting out, anion exchange chromatography and size exclusion chromatography (SEC). Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and SEC showed that the allergenic protein was composed of two subunits linked by disulfide bonds, which we named TBa and TBb. TBa, TBb and TBt have estimated molecular weights of 24, 34 and 56 kDa, and isoelectric points of 6.85, 5.85 and 7.00, respectively. The immunological characteristics of the three proteins as analyzed by enzyme-linked immunosorbent assay revealed that TBa and TBb have comporable IgE binding activity, both higher than that of TBt. Determination of sulfhydryl (-SH) and disulfide (-S-S-) groups in TBt showed that most cysteine residues exist as disulfide linkages rather than free -SH groups. We calculated that TBt has an -S-S- content of 1.978 M/M protein, indicating that TBt contains one disulfide bond and could be the precursor of a 24 kDa allergenic protein from tatary buckwheat seeds.

Fagopyrum tataricum Gaertn. - Polygonaceae - biochemical characterization - immunological activity - allergen

References

• 1 Bonafaccia G, Marocchini M, Kreft I. Composition and technological properties of the flour and bran common and tatary buckwheat.  Food Chem. 2003;  80 9-15
• 2 Bonafaccia G, Gambelli L, Fabjan N, Kreft I. Trace elements in flour and bran from common and tatary buckwheat.  Food Chem. 2003;  83 1-5
• 3 Li S, Zhang Q H. Advances in the development of functional foods from buckwheat.  Food Sci Nutr. 2001;  41 451-64
• 4 Park J W, Kang D B, Kim C W, Ko S H, Yum H Y, Kim K E. et al . Identification and characterization of the major allergens of buckwheat.  Allergy. 2000;  55 1035-41
• 5 Imai T, Iikura Y. The national survey of immediate type of food allergy.  Arerugi. 2003;  52 1006-13
• 6 Urisu A, Kondo Y, Morita Y, Wada E, Tsuruta M, Yasaki T. et al . Identification of a major allergen of buckwheat seeds by immunoblotting methods.  Allergy Clin Immunol. 1994;  6 151-5
• 7 Nair A, Adachi T. Immunodetection and characterization of allergenic proteins in common buckwheat (Fagopyrum esculentum).  Plant Biotechnol. 1999;  16 219-24
• 8 Yoshimasu M, Zhang J, Hayakawa S, Mine Y. Electrophoretic and immunochemical characterization of allergenic proteins in buckwheat.  Int Arch Allergy Immunol. 2000;  123 130-6
• 9 Koyano S, Takagi K, Teshima R, Sawada J. Molecular cloning of cDNA, recombinant protein expression and characterization of a buckwheat 16-kDa major allergen.  Int Arch Allergy Immunol. 2006;  140 73-81
• 10 Matsumoto R, Fujino K, Nagata Y, Hashiguchi S, Ito Y, Aihara Y. et al . Molecular characterization of a 10-kDa buckwheat molecule reactive to allergic patients’ IgE.  Allergy. 2004;  59 533-8
• 11 Wang Z H, Zhang Z, Zhao Z H, Wieslander G, Norback D, Kreft I. Purification and characterization of a 24kDa protein from tatary buckwheat seeds.  Biosci Biotechnol Biochem. 2004;  68 1409-13.
• 12 Fujino K, Funatsuki H, Inada M, Shimono Y, Kikuta Y. Expression, cloning, and immunological analysis of buckwheat (Fagopyrum esculentum Moench) seed storage proteins.  J Agric Food Chem. 2001;  49 1825-9
• 13 Yoshioka H, Ohmoto T, Uris A, Mine Y, Adachi T. Expression and epitope analysis of the major allergenic protein Fag e 1from buckwheat.  J Plant Physiol. 2004;  161 761-7
• 14 Wang Z H, Wang L, Chang W J, Li Y Y, Zhang Z, Wieslander G. et al . Cloning, expression, and identification of immunological activity of an allergenic protein in tatary buckwheat.  Biosci Biotechnol Biochem. 2006;  70 1195-9.
• 15 Laemmli U K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.  Nature. 1970;  227 680-5.
• 16 Beveridge T, Toma S, Nakai S. Determination of SH- and SS-groups in some food proteins using Ellman’s reagent.  J Food Sci. 1974;  39 49-51
• 17 Hjerten S, Elenbring K, Kilar F, Liao L J, Chen A J, Siebert C J. et al . Carrier-free zone electrophoresis, displacement electrophoresis and isoelectric focusing in a high-performance electrophoresis apparatus.  J Chromatogr. 1987;  403 47-61
• 18 Choi S M, Ma C Y. Extraction, purification and characterization of globulin from common buckwheat (Fagopyrum esculentum Moench) seeds.  Food Res Int. 2006;  39 974-81
• 19 Radovic R, Maksimovic V R, Varkonji-Gasic E I. Characterization of buckwheat seed storage proteins.  J Agric Food Chem. 1996;  44 972-4
• 20 Maksimovic V R, Varkonji-Gasic E I, Radovic S R, Savic A P. The biosynthesis of 13S buckwheat seed storage protein.  J Plant Physiol. 1996;  147 759-61