The conformation of wheat gluten proteins. The secondary structures and thermal stabilities of α-, β-, γ- and ω-Gliadins (1)
Wheat gluten is the visco-elastic mass that remains when dough is washed to remove starch and components soluble in water and dilute solutions of salt. It is composed mainly of proteins (typically about 70%), which are usually classified into two groups. Gliadins are readily soluble in aqueous alcohols, and are monomers associated by hydrogen bonding and hydrophobic interactions. Although they are classically separated into four groups (a-, b, g- and w-gliadins). Glutenin aggregates may not be soluble in aqueous alcohols, and the individual subunits, after reduction, only soluble at low pHS. Two major groups of subunits are present: the low molecular weight and high molecular weight subunits.
Purification of gliadins
Whole untreated grains of wheat (cv. Maris Butler) were milled to pass a 0·7 mm sieve. Milled grain (200 g) was stirred for 1 h with 2000 ml of 70% (vIv) aqueous ethanol at 20 0c. The supernatant, after centrifugation, was mixed with 2 vols of 1’5 MNaCI, allowed to stand for 18 h at 4 °C and the precipitated gliadin removed by centrifugation. The precipitated gliadin (1 g) was dissolved in 6 M urea, 0·01 M acetic acid, and separated into aggregates and monomers by gel filtration in the same solvent on a 90 x 4·4 em column ofSephacryl S-300 . Fractions containing monomers were bulked, dialysed against water and freeze-dried. The monomer fraction (1 g) was dissolved in 0·1 M acetic acid and separated by gel filtration in the same solvent on a 90 x 4·4 ern column of Biogel P100. Fractions containing a-, b- and g-gliadins were bulked, freeze-dried and separated by ion exchange chromatography on CM cellulose in 10mM glycine/acetate buffer, pH 4’6, containing 3 M urea, and eluted with a linear gradient of 0 – 0.25 M NaCl. To prepare ro-gliadins the crude gliadin preparation was separated by ion-exchange chromatography on CM cellulose, at pH 3·6. slight contamination of the b-gliadins with g-gliadins, but little or no contamination of the a- and g-gliadins. The SDS-PAGE showed that most of the o-gliadins had lower relative molecular masses (MrS) and the g-gliadins higher, although the MrS of the three groups overlapped. Two w-gliadin fractions were prepared by ion-exchange chromatography. The first gave a major slow doublet on lactate-PAGE and a single major band on SDS-PAGE. The second gave two major faster bands on lactate-PAGE and several lower Mr bands on SDS-PAGE.
The far-UV cd spectra of the a-, b- and g-gliadins in 70% (v/v) aqueous ethanol show typical a-helix-rich conformations, with minima around 208 and 222 nm, and maxima in the range190-200 nm. The two to-gliadin fractions had spectra that were almost identical to each other, but distinctly different from those of the a-, b- and g-gliadins, with none of the characteristics associated with the a-helical or b-sheet conformation. The spectra of the gliadins show characteristic phenyl-alanine band structure, with two vibronic components centred at 262 and 268 nm. The band at 275-276 nm arises probably from the tyrosyl side chain. The w-gliadin fractions contained no cysteine residues, whereas the a-, b- and g-gliadins contain about 2 mol % cysteine. The broad absorbance between 260 and 320 nm is probably associated with the rigid cysteine disulphide chrornophore. The a-, b- and g-gliadins were found to have similar a-helical contents of 33-34% , 34-35 % and 30-31%, respectively. Analysis of the a-gliadin spectrum estimated 36-37% a-helix, 11-12% b-sheet and 52-53 % aperiodic structure which would include the random coil and the b-turn conformation.
With native and modified g-gliadin, increases in temperature resulted in monotonic decreases in regular structure content. The effects of temperature were found to be reversible for both native and modified g-gliadin. The near-UV spectra of the native and modified y-gliadin show marked differences, the broad absorbance associated with the disulphide chromophore being absent from the spectrum of the modified gliadin.
Increasing temperature resulted in an increase in absorption of the 204 nm minimum, with a simultaneous decrease in the intensity of a shoulder centred at 228-230 nm. the conformational change that was occurring was reaching completion near 60C.
The spectrum corresponds to a class B, b-turn spectrum, with minima in the region 220-230 nm and maxima in the region 200-210 nm. This class of p-turn spectrum is the most common associated with any b-turn conformation.
The w-gliadins are rich in b-turns, with no detectable a-helix or b-sheet; these b-turns are probably interspersed with random coil. In contrast the a-, b- and g-gliadins contain 30-35% a-helix and 10-20% b-sheet.