Electrophoretic characterisation of fractions collected from gluten protein extracts subjected to size‐exclusion high‐performance liquid chromatography

Abstract

Electrophoretic characterisation of fractions collected from gluten protein extracts subjected to size-exclusion high-performance liquid chromatographyThe electrophoretic analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; reduced and unreduced) of fractions, collected from a size exclusion-high performance liquid chromatography (SE-HPLC) separation of gluten proteins using a column with pore size of around 400A, showed clear resolution for the seven elution ranges studied in two Australian bread wheat lines. Polymeric proteins -high molecular weight (HMW) glutenin subunits, low molecular weight (LMW) glutenin subunits, HMW albumins and some modified m-gliadins -appeared exclusively in the region within the first peak of the chromatogram (fractions 1 to 5), the limit being a region that resolved as a small peak before the large peak of gliadins and where some o-gliadins eluted. A larger proportion of HMW glutenin subunits and B subunits contributed to polymer formation of higher molecular weight.The polymer size decreased as the proportion of the other protein components increased. IntroductionWheat endosperm proteins have been extensively studied due to their viscoelastic properties that give wheat flours unique baking characteristics. They are usually classified according to their solubility, amino acid composition, size or polymerising behaviour. Wheat proteins may be separated into albumin (soluble in water), globulin (soluble in salt solution), gliadin (70% ethanol soluble) and glutenin (insoluble in ethanol) [l]. From a functional point of view, they are also grouped into polymeric protein (mainly glutenin) held together by interchain disulphide bonds, and monomeric protein (gliadins and albumins/globulins) [2], in which the only disulphide bonds are intrachain. Statistical relationships between these groups (particularly glutenins) and breadmaking quality have been established and, as a general result, close correlations have been found [2,3]. To reach these conclusions, wheat proteins have been characterised in detail using a wide range of methodologies, particularly over the last twenty-five years.Electrophoretic techniques using SDS-PAGE have been essential in determining the importance of glutenin polypeptides in wheat quality [4], but prior to electrophoresis in an SDS-PAGE gel, the glutenin polymer needs to be broken into subunits by the reduction of the disulphide bonds that build the polymeric structure. Four different types of subunits are found: high molecular weight (HMW) glutenin subunits, low molecular weight (LMW) glutenin subunits, HMW albumin subunits (mostly

Developmental and environmental effects on the assembly of glutenin polymers and the impact on grain quality of wheat

“…By analyzing only one variety, it was possible to avoid any effects due to genetic variation. The relationship has been confirmed by collecting fractions of glutenin from SE-HPLC and showing that the HMW-GS/LMW-GS ratio increases, as the MW of glutenin increases (Larroque et al, 1997). Thus, the change in balance of dough properties (higher strength, lower extensibility) which is accentuated as the S deficiency becomes more acute, is caused by the shift in MWD to higher MWs.…”

Study on the interactions between soy and semolina proteins during pasta making

“…1b) show a profile described by Lamacchia et al (2007) and by Lamacchia, Baiano, Lamparelli, La Notte, and Di Luccia (2010), with a polymeric protein peak of glutenin at the extreme left of the profile (Large Polymeric Proteins, LPP > 100,000 Da), followed by a large peak of monomeric gliadin (Large Monomeric Proteins, LMP < 100,000 Da) and finally small peaks of albumins and globulins, known as Small Monomeric Proteins (SMP) (Carceller & Aussenac, 2001;Larroque, Gianibelli, Batey, & MacRitchie, 1996;Larroque, Gianibelli, Batey, & MacRitchie, 1997;Tosi et al, 2005).…”

Protein composition of wheat gluten polymer fractions determined by quantitative two-dimensional gel electrophoresis and tandem mass spectrometry

“…These polymer extracts are suitable for further separation on the basis of size by size-exclusion chromatography (SEC) [9]. Improvements in solvent systems [10] and SEC column efficiency have made SEC a rapid technique for characterizing the intact polymer without disulfide bond reduction [11]. Using 0.5% SDS with and without sonication, Gupta et al [12] separated wheat flour proteins into SDS extractable (EPP) and SDS unextractable (UPP) protein fractions and demonstrated that the amount of polymer in the unextractable fraction liberated by sonication correlated with dough strength.…”