Electrophoretic comparison of cereal proteins

Three albumins were isolated from bread wheat seeds by gelfiltration on Sephadex G-100 and differential preparative disc-electrophoresis on polyacrylamide gel. The proteins were obtained in suficient yield and purity to carry out characterisation studies. The electrophoretic mobilities of the three albumins in polyacrylamide discs in glycine- Tris medium, p H 9.5, were 0.28, 0.34 and 0.39 (referred to that of bromophenol blue taken as I ) . The isoelectric points were 6'40, 6.40 and 5.35 and the molecular weights 17,700,18,200 and 18,900, respectively. The amino acid compositions o f the purified albumins were very similar. The correlation of the purified albumins with those isolated from bread wheats by other authors is discussed. Introduction'SOLUBLE' proteins from bread and Triticum durum wheat are usually separated using electrophoretic m e t h o d~. l -~ However, the heterogeneity of protein extracts, even after a preliminary fractionation, seriously limits the evaluation of these results. Therefore, attempts were made to extract and purify species-specific proteins from, for instance, bread wheat in order to prepare immune sera against the proteins. Several varieties of both species could then be tested by immunodiffusion and the specificity of such fractions could be statistically analysed. In this way it was possible to characterise an albumin specific for bread wheat.10-12The albumin fraction from the seeds of Triticum aestivum or Triticum durum, extracted with 0. S M -N~C~ and precipitated with between 0 . 9 3~ and 1.33 M ammonium sulphate, was gelfiltered on Sephadex G-100. Four major fractions of different molecular weights were obtained (A, B, C, D) (Cantagalli, P., unpublished results). In disc-electrophoresis the lowest molecular weight fraction (Db) from bread wheat had a significantly different composition from the same fraction obtained from T. durum wheat (Dd) (Cantagalli, P., unpublished results).Therefore, an attempt was made to isolate and characterise the albumins present in fraction D b . Experimental Extraction and purification of albuminsAlbumins were extracted according to the method of Cantagalli et a1.13 1000 g of bread wheat whole flour (a mixture of varieties Autonomia and Generoso) were suspended in 2000 ml of phosphate buffer, pH 6.6 ( O -O~~M M -N~~H P O~, O .~~I M -K & P O~, 0-48~-NaCi), at 4"c and left overnight. The suspension was then centrifuged at 2700 Y g and the albumins were separated from the supernatant by salting out with between 0 . 9 3~ and 1 ' 33111 ammonium sulphate. The precipitate obtained was collected by centrifuging at 2700 x g and washed three times with 1 *33M ammonium sulphate. The yield of albumins was approximately 1 g. Gel-filtration on SephadexAbout 1 g of albumins was dissolved in 25 ml of buffer solution, pH 7.4 (0.008~-NazHP04, 0.001rvr-KH2P04, 0.137~-NaC1, 0*003~-KC1), applied on a Sephadex G-100 column (90 x 6 cm) and eluted with the same buffer at a rate of 1 .O ml/min, according to the method of Cantagalli et al.13 * Laboratorio Chimico Pro...

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“…of the protein loaded on the column, clearly shows the presence of eight anodic bands at pH 9 . 5 (Fig. 2).…”

Section: Purificationmentioning

confidence: 91%

Purification and properties of three albumins from Triticum aestivum seeds

et al. 1971

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“…It was interesting that the humin, compared with whole wheat, was found to contain less glutamic acid and proline, and more cystine, methionine and basic amino acids. Bran proteins are known to have electrophoretic mobilities similar to those wheat albumins (Elton & Ewart, 1964) and it is known from unpublished work in these laboratories that the amino acid composition of a purified major component of wheat albumin compared with that of whole wheat also shows similar differences. This suggests either that some bran proteins are less accessible or are less easily hydrolysed than endosperm proteins, or that both these differences occur.…”

Section: Hydrolysis Of Whole Seedsmentioning

confidence: 92%

The effect of individual rye chromosomes on the amino acid content of wheat grains

Riley

Ewart²

1970

Genet. Res.

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At the present time attempts are being made to improve the nutritional value of wheat by increasing the content of certain essential amino acids, particularly lysine, in the proteins of the grain. Rye grain has a considerably higher lysine content than that of wheat. Consequently in the present work studies were made of the amino acid contents of the grains of the wheat variety Holdfast, the rye variety King II, the Triticale derived from these parental varieties and the seven lines in which, in turn, each pair of chromosomes of King II are separately added to Holdfast.Rye chromosome I increased the lysine content of wheat by 8-7 % and associated changes in the proportions of other amino acids suggest that this increase is meaningful. Rye chromosome I is in homoeologous group 5 and other reports have indicated a relationship between changed lysine content and another character determined by chromosomes of this group. Consequently there is a suggestion that group 5 chromosomes may be of particular significance in the determination of lysine content in wheat grains. Confirmation of this would lead to a more rational approach to breeding for higher lysine content.

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“…[15][16][17] In this paper some applications of starch-gel electrophoresis (SGEP) to the study of wheat proteins are discussed, particularly as regards the detection of protein-protein interactions.…”

Section: Introductionmentioning

confidence: 99%

The interaction of proteins during gel electrophoresis

Ewart¹

1966

J Sci Food Agric

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Reversible polymerisation and A + B ⇌ C type reactions among proteins during zone electrophoresis may give rise to tailing, extra bands and mobility changes. One or more of these signs are likely to appear with the milder type of interaction, i.e. equilibrium constant in the range 102–108, whether the time to equilibrium is measured in hours or less. Two‐dimensional electrophoresis is more useful for diagnosing interactions. No definite evidence has been obtained for protein‐protein interactions among the slow‐moving gliadins of Bison and Cappelle varieties and the total number of bands probably represents the minimum number of molecular species present. The electrophoretic pattern of a given variety does not appear to alter qualitatively when the year or place of growth is changed. It is estimated that a wheat protein concentration needs to be above ∼0·1 mg/ml to be observable on a starch gel.

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Electrophoretic comparison of cereal proteins

Cited by 26 publications

References 17 publications

Purification and properties of three albumins from Triticum aestivum seeds

Purification and properties of three albumins from Triticum aestivum seeds

The effect of individual rye chromosomes on the amino acid content of wheat grains

The interaction of proteins during gel electrophoresis

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