Properties of Gluten Fractions Prepared by Ion-Exchange Chromatography on Carboxymethyl-Cellulose

Jw, Lee; Tracey, MV; Winzor, DJ; Wrigley, C. W.; Zentner, H.

doi:10.1071/bi9630717

Aust. Jnl. Of Bio. Sci.

1963

DOI: 10.1071/bi9630717

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Properties of Gluten Fractions Prepared by Ion-Exchange Chromatography on Carboxymethyl-Cellulose

Lee Jw¹,

MV Tracey²,

DJ Winzor³

et al.

Abstract: SummarySeven fractions obtained from wheat gluten by chromatography on carboxy~ methyl-cellulose were studied by ultracentrifugation, gel electrophoresis, chemical, and N-terminal amino acid analysis. On rechromatography, five fractions eluted by sodium chloride behaved as distinct entities. illtracentrifuge experiments indicated that four of these were each undergoing rapid, reversible association. Several N-terminal amino acids were found in each of the fractions which, moreover, could be resolved by the gel… Show more

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Cited by 5 publications

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“…. Gel electrophoretic patterns of fractions resulting from the carboxymethyl cellulose (CMC) fractionation of Simmonds and Winzor (1961) have been published by Graham (1963) and by Lee et al (1963). These results showed that each fraction was heterogeneous, with considerable overlapping of electrophoretic bands from neighbouring fractions, and that fractions eluted late in the chromatogram were contaminated because of tailing of earlier fractions.…”

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An Improved Chromatographic Separation of Wheat Gluten Proteins

Wrigley¹

1965

Aust. Jnl. Of Bio. Sci.

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Gel electrophoresis is at present the best procedure for the analytical fractiona-tion of wheat gluten proteins. Procedures more suitable for preparative studies, such as gel ffitration and column chromatography, have provided only partial fractionation on the basis of gel electrophoresis (Graham 1963; Lee et al. 1963; Wright, Brown, and Bell 1964). Gel electrophoretic patterns of fractions resulting from the carboxymethyl cellulose (CMC) fractionation of Simmonds and Winzor (1961) have been published by Graham (1963) and by Lee et al. (1963). These results showed that each fraction was heterogeneous, with considerable overlapping of electrophoretic bands from neighbouring fractions, and that fractions eluted late in the chromatogram were contaminated because of tailing of earlier fractions. The modification of the Simmonds and Winzor procedure described below results in an improved fractionation of gluten proteins as judged by starch-gel electrophoresis.

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“…Gluten proteins were extracted from Gabo flour as described previously (Coates and Simmonds 1961;Lee et al 1963). Columns (1•5 by 25 cm) containing about 3 g CMC (Whatman CM70 powder) were maintained at 40°C and were eluted at 1 ml/min; fractions were collected at lO-min intervals.…”

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An Improved Chromatographic Separation of Wheat Gluten Proteins

Wrigley¹

1965

Aust. Jnl. Of Bio. Sci.

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The reaction of wheat proteins with sulphite. I.—Sulphitolysis of the proteins of flour with cuprammonium sulphite

McDermott

Pace

1965

J Sci Food Agric

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The reaction of wheaten flour and of some flour protein fractions, with cuprammonium sulphite solutions at pH 10.2 has been investigated. Under these conditions, with a reaction time of 2-4 h. a t room temperature, the thiol and disulphide groups of the flour proteins are completely converted to S-sulphocysteine residues and the proteins dissolve, together with some carbohydrate.The effect of the sulphitolysis procedure on the amide groups of the proteins, in flours of widely different protein content, has been examined and there was no evidence that deamidation had occurred. Similarly the amino-acid composition of a flour protein fraction remained unchanged by the sulphitolysis procedure. In these respects the specificity of the reaction confirmed the original observations of Swan with keratin, but evidence obtained by prolonging the reaction time suggests that with flour proteins, some disaggregation or cleavage of their structure, other than that due to the splitting of disulphide bonds, may also occur and this possibility is being investigated in further work. Some general properties of the solubilised material are described.The insoluble residue is a high quality starch. Introduction mixture of cysteine (CySH) and S-sulphocysteine (CySS0,H) :-It was shown by Clarke1 that cystine (CySSCy) reacts with sulphite to give an equilibrium c y s s c y + SO?-+ c y s -+ CySS0,-.-11)Kolthoff & Stricks2 subsequently established that cysteine reacts with cupric ions and sulphite in ammoniacal solution to give S-sulphocysteine :-c y s -+ 2 cu2+ + so,2-+ CySS0,-+ 2 c u +- (2) Thus by the use of cuprammonium sulphite both cystine and cysteine may be quantitatively converted to S-sulphocysteine-the overall reaction for cystine being :-CySSCy + 2 Cu2+ + 2 SO,2+ + zRSS0,-+ z Cu+ . * (3)Swan314 was the first to examine the application of these reactions to the thiol and disulphide groups in proteins, by studying the interaction of cuprammonium sulphite with keratins. He found that the cystine disulphide bonds were broken symmetrically, according to Equation (3), that the reaction went to completion and that with the reaction media at pH values of 9-10'5 a large proportion of keratin became soluble in the form of S-sulpho-keratin.Thus, in a typical instance, 61% of a sample of wool was dissolved in 24 h. at 22'. Swan also provided evidence that amino-acids other than cystine and cysteine were unaffected by the ammoniacal cupric sulphite reagent. Swan's procedure thus provided a method for the disruption of disulphide bonds in proteins, which appeared to be specific and which, under appropriate conditions, could lead to the solubilisation of a protein largely insoluble in its native form.Swan's method, with a number of variations in the detailed procedure, has been applied by other workers to other proteins. Pechere et prepared the S-sulpho derivatives of trypsinogen and cr-chymotrypsinogen. The reaction was carried out (for I h. at room temperature, in solution containing sulphite and urea, brought to, and maintained at, pH 10...

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Isolation and characterisation of wheat flour proteins. I.—separation of salt‐ and acetic acid‐dispersible proteins by gel filtration, polyacrylamide gel electrophoresis, and sucrose gradient ultracentrifugation

Jankiewicz

Pomeranz

1965

J Sci Food Agric

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Proteins extracted from wheat flour with 0.01 M-sodium pyrophosphate buffer, pH 7.0, and with 0.05 M-acetic acid were centrifuged, dialysed against 0.005 hr-sodium acetate buffer pH 4.1, concentrated by ultrafiltration at 4', and lyophylised. The proteins were dispersed in 0.005 M-sodium acetate buffer pH 4.1 and fractionated on Sephadex G-IOO and Sephadex G-zoo columns. Based on separation according to average mol. wt. on Sephadex G-100, the pyrophosphate-dispersible proteins contained eight, and the following acetic acid-dispersible proteins six fractions. Proteins extracted directly with 0.05 M-acetic acid were separated into seven fractions. The distribution of protein moieties of different average molecular-size ranges is detailed and shown to be different in the different extracts. Aggregation and disaggregation phenomena were observed during repeated re-chromatography of high-and intermediate-molecular weight proteins. Ultra-centrifugation in a stepwise sucrose gradient at 45,000 g. at 4' and fractionation by polyacrylamide-gel electrophoresis separated the protein into slow-moving, intermediate-and high-molecular weight moieties. The relative distribution of these moieties was correlated with the mol. wt. of the fractions eluted from Sephadex columns.

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Properties of Gluten Fractions Prepared by Ion-Exchange Chromatography on Carboxymethyl-Cellulose

Cited by 5 publications

References 9 publications

An Improved Chromatographic Separation of Wheat Gluten Proteins

An Improved Chromatographic Separation of Wheat Gluten Proteins

The reaction of wheat proteins with sulphite. I.—Sulphitolysis of the proteins of flour with cuprammonium sulphite

Isolation and characterisation of wheat flour proteins. I.—separation of salt‐ and acetic acid‐dispersible proteins by gel filtration, polyacrylamide gel electrophoresis, and sucrose gradient ultracentrifugation

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