Abstract
This chapter describes an accurate and user-friendly method for determining amino acid composition of wheat gluten proteins and their gliadin and glutenin fractions. The method consists of hydrolysis of the peptide bonds in 6.0 M hydrochloric acid solution at 110°C for 24 h, followed by evaporation of the acid and separation of the free amino acids by high-performance anion-exchange chromatography with integrated pulsed amperometric detection. In contrast to conventional methods, the analysis requires neither pre- or postcolumn derivatization, nor a time-consuming oxidation or derivatization step prior to hydrolysis. Correction factors account for incomplete release of Val and Ile even after hydrolysis for 24 h, and for losses of Ser during evaporation. Gradient conditions including an extra eluent allow multiple sequential sample analyses without risk of Glu accumulation on the anion-exchange column which otherwise would result from high Gln levels in gluten proteins.
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References
Wieser H (2007) Chemistry of gluten proteins. Food Microbiol 24:115–119
Veraverbeke WS, and Delcour JA (2002) Wheat protein composition and properties of wheat glutenin in relation to breadmaking functionality. Crit Rev Food Sci Nutr 42:179–208
Belitz HD, Grosch W, and Schieberle P (2009) Cereals and cereal products. In: Belitz HD, Grosch W, and Schieberle P (eds) Food Chemistry, 4th edn. Springer-Verlag, Berlin
Rombouts I et al (2010) [beta]-Elimination reactions and formation of covalent cross-links in gliadin during heating at alkaline pH. J Cereal Sci 52:362–367
Ruckman JE, Zscheile FP, and Qualset CO (1973) Protein, Lysine, and Grain Yields of Triticale and Wheat as Influenced by Genotype and Location. J Agric Food Chem 21:697–700
Sikka KC et al (1978) Comparative Nutritive-Value and Amino-Acid Content of Triticale, Wheat, and Rye. J Agric Food Chem 26:788–791
Del Moral LFG et al (2007) Environmentally induced changes in amino acid composition in the grain of durum wheat grown under different water and temperature regimes in a Mediterranean environment. J Agric Food Chem 55:8144–8151
Jiang XL, Hao Z, and Tian JC (2008) Variations in amino acid and protein contents of wheat during milling and northern-style steamed breadmaking. Cereal Chem 85:504–508
Shewry PR et al (2009) Wheat Grain Proteins. In: Khan K and Shewry PR (eds) Wheat: Chemistry and Technology, 4th edn. AACC International, St. Paul, MN, USA
Darragh AJ et al (1996) Correction for amino acid Loss during acid hydrolysis of a purified protein. Anal Biochem 236:199–207
Hanko VP, and Rohrer JS (2004) Determination of amino acids in cell culture and fermentation broth media using anion-exchange chromatography with integrated pulsed amperometric detection. Anal Biochem 324:29–38
Jandik P et al (2001) Simplified in-line sample preparation for amino acid analysis in carbohydrate containing samples. J Chromatogr B 758:189–196
Ding YS, Yu H, Mou SF (2002) Direct determination of free amino acids and sugars in green tea by anion-exchange chromatography with integrated pulsed amperometric detection. J Chromatogr A 982:237–244
Thiele C, Ganzle MG, Vogel RF (2002) Sample preparation for amino acid determination by integrated pulsed amperometric detection in foods. Anal Biochem 310:171–178
Rombouts I et al (2009) Wheat gluten amino acid composition analysis by high-performance anion-exchange chromatography with integrated pulsed amperometric detection. J Chromatogr A 1216:5557–5562
Olcott HS, and Fraenkel-Conrat H (1947) Formation and loss of cysteine during acid hydrolysis of proteins. Role of tryptophan. J Biol Chem 171:583–594
Fountoulakis M, and Lahm HW (1998) Hydrolysis and amino acid composition analysis of proteins. J Chromatogr A 826:109–134
Aitken A, and Learmonth M (2002) Carboxymethylation of Cysteine Using Iodoacetamide/Iodoacetic Acid. In: Walker JM (ed) The Protein Protocols Handbook, 2nd edn. Humana Press, NJ
Barkholt V, and Jensen AL (1989) Amino acid analysis: Determination of cysteine plus half-cystine in proteins after hydrochloric acid hydrolysis with a disulfide compound as additive. Anal Biochem 177:318–322
Koehler P (2003) Amino Acid and Protein Sequence Analysis. In: Shewry PR and Lookhart GL (eds) Wheat Gluten Protein Analysis, AACC Press, Washington
Acknowledgments
This work is a part of the Methusalem programme “Food for the Future” at the K.U. Leuven. B. Lagrain and I. Celus wish to acknowledge the Research Foundation – Flanders (FWO, Brussels, Belgium), and the Agency for Innovation by Science and Technology (IWT-Vlaanderen, Brussels, Belgium), respectively, for functions as postdoctoral researchers. K. Brijs wishes to acknowledge the Industrial Research Fund (K.U. Leuven, Leuven, Belgium) for a position as Industrial Research Fund fellow.
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Rombouts, I., Lagrain, B., Lamberts, L., Celus, I., Brijs, K., Delcour, J.A. (2012). Wheat Gluten Amino Acid Analysis by High-Performance Anion-Exchange Chromatography with Integrated Pulsed Amperometric Detection. In: Alterman, M., Hunziker, P. (eds) Amino Acid Analysis. Methods in Molecular Biology, vol 828. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-445-2_26
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DOI: https://doi.org/10.1007/978-1-61779-445-2_26
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