Preparative Fractionation of Gliadins by Electrophoresis at pH 3.1 (A-PAGE)

Abstract

Purification of gliadin subclasses has been difficult since they share many biochemical and physicochemical properties. In this report, the optimization of a preparative electrophoretic method to fractionate gliadins is described. Separation was performed in preparative 7% polyacrylamide gels at pH 3.1. The separation performance was tested using analytical electrophoresis at pH 3.1 and capillary electrophoresis. Preparative gels of different lengths were employed. Using 5-cm preparative gels, several fractions of alpha-, beta-, and gamma-gliadins and fast-mobility and slow-mobility omega-gliadins were collected in 40 h of separation. Resolution was maintained at a protein load of up to 30 mg in each run. The highest efficiency of recovery was achieved using aluminum lactate as the collecting buffer. Fractionation with 10 cm in length gels improved resolution but increased operation times. Gels of 2 cm in length did not separate alpha/beta- and gamma-gliadins efficiently but were useful to separate the two main fractions of omega-gliadins in shorter times. In conclusion, preparative electrophoresis at low pH allowed the separation of alpha-, beta-, gamma-, and omega-gliadin fractions from crude material under nondenaturing conditions.

Recent developments in high-performance capillary electrophoresis of cereal proteins

“…5). The migration order of the gliadin subclasses was verified to be the same for both FZCE and acidic slab-gel electrophoresis [21], confirming earlier reports [13]. These studies demonstrate the high resolution of FZCE and its usefulness in purity screening preparative fractions.…”

“…The use of ACN as a buffer additive provided a buffer with good UV transmission properties, low viscosity, and no potential for crystallization as often found when using high levels of urea as a buffer modifier. Rumbo and Giorgieri [21] investigated the effects of varying the separation voltage and HPMC level in acidic sodium phosphate buffers. Reducing the level of HPMC to 0.3% was found to lead to faster separations without significantly compromising separation resolution (Fig.…”

“…HPCE has the advantage of analyzing samples unattended and produces digital output which can be easily stored, utilized, and quantified. Recent papers have reported on the ability to differentiate maize genotypes [15][16][17], sorghum genotypes [17], rice cultivars [18,32), wheat cultivars [15,18,21,[33][34][35], spelt wheat lines [22], oats cultivars [18], and barley cultivars [18,20]. An example of the ability of FZCE to differentiate U.S. hard red winter wheats is shown in Fig.…”

“…HPCE was utilized to monitor the purity of subclasses prepared by preparative gel electrophoresis [36]. Gliadin subclasses were collected from preparative slab-gel separations at acidic pH and analyzed by FZCE in an acidic sodium phosphate buffer to monitor the purity of the preparative fractions.…”

Expression and Development of Wheat Proteins during Maturation of Wheat Kernel and the Rheological Properties of Dough Prepared from the Flour of Mature and Immature Wheat

“…The gliadins are further divided into 4 subgroups, namely α-, β-, γ-, and ω-gliadins depending on their mobility during electrophoresis at low pH (Bushuk and Zillman, 1978;Rumbo et al, 1999). Later studies on amino acid sequences, however, have shown that electrophoretic mobility does not always reflect protein relationships and that α-and β-gliadins fall into one group (α-gliadin or α/β-gliadin) (Wieser, 2007).…”