Modification of wheat gliadin by γ‐rays

Abstract

Wheat gliadin has been y-irradiated under nitrogen with a dose of 20 Mrad. No significant changes were observed in the amino acid analysis, chymotryptic fingerprints, or elution curves from Sephadex G-200. Discrete bands had been largely replaced by tailing in the starch-gel electrophoretic pattern. It is concluded that random bond scission has occurred, chiefly altering the side chains, and to some degree protein fragments have been grafted on to other sites. There is no definite evidence for the formation of macromolecular structures by inter-protein crosslinks. On average it is likely that of the order of only one or two bonds per molecule were affected. IlltrOdUCtiOnThe production of free radicals is one of the effects of substantial doses of y-radiation on dry proteins. According to Singh & Ormerod the radicals are locaiised on certain preferred sites, probably glycine or cystine.1 The possibility of polymerisation of flour proteins by y-rays was suggested by Maes.2 Lee reported protein and starch breakdown and a deterioration in dough properties when flour was y-irradiated.3-5 The results of Deschreider6 indicated a decrease in the glutenin fraction and an increase in starch solubility on y-irradiation.This paper describes attempts to deduce the nature of changes in gliadin proteins induced by y-rays. Experimental Preparation of gliadinPurified Wichita gliadin, prepared as described earlier,7 was run for 16.5 h in an LKB 3340C column electrophoresis apparatus. The column was packed with Pevikon C-870 powders as a supporting medium. Dilute sodium lactate (pH 3 '3) was the buffer used, and a potential of 300 V was applied between the electrodes. The eluted fractions were dialysed vs. 0.01 N acetic acid and freeze-dried. Fractions showing no sign of glutenin on starch-gel electrophoresis were combined. IrradiationThe glutenin-free gliadin (200 mg), contained in a specimen tube which had been closed with a rubber bung after evacuation and flushing with nitrogen (< 10 ppm OZ), was irradiated to a level of 20 Mrad over 154 h from a SOCo source at Leicester University. Starch-gel electrophoresisThis was carried out as described previo~sly.~ Proteins were reduced as follows: 0.5 ml of 0.067 M phosphate buffer pH 7.4,2.5 % in mercaptoethanol and 6 M in urea was added to > 9 mg of protein in a tube, which was flushed with nitrogen, closed and allowed to stand for 24 h. The pH of the solution was changed by dialysing overnight against dilute sodium lactate buffer9 containing similar levels of urea and mercaptoethanol. To make gels suitable for electrophoresis under reducing conditions, 70 g of starch, suspended in 500 ml of dilute sodium lactate buffer 6 M in urea, were heated with stirring for 4.5 min and a further 50 ml of solution containing 15 ml of mercaptoethanol were added just before the liquid was poured. Electrophoresis was carried out for 20 h at N 7V/cm.

Susceptibility to transglutaminase of gliadin peptides predicted by a mass spectrometry‐based assay

“…For the first time in a wheat flour, this procedure allowed us to identify, as tTG substrates in vitro, a few gliadin peptides having a specific amino acid composition amongst a myriad of other peptides. Contrary to what was expected, only a few gliadin peptides interacted with tTG even if a high content of Q residues was available along the gliadin chains [4,5]. Only one belonged to the Q‐X‐Q consensus sequence (peak 7, Table 1) while Q residues occupying the −2 position with respect to a P residue were preferentially modified, confirming previous data concerning tTG specificity [3].…”

“…In addition, because gliadin proteins are rich in Q and poor in K residues [4,5], in the absence of MDC, tTG may act as an acyl acceptor and form complexes with gliadin peptides. Assays to detect gliadin peptide–tTG complexes were unsuccessful owing to the high heterogeneity of the reaction mixture and the very high molecular weight of the protein–peptide complexes.…”

“…Gluten proteins and derived peptides are also good substrates for tTG‐catalyzed cross‐linking given the high frequency of Q residues, which account for about 40% [4,5]. In addition to forming gliadin–gliadin complexes, tTG can be incorporated into high molecular weight complexes with gliadins [6], giving rise to novel antigenic epitopes [2].…”

Characterization of wheat gliadin proteins by combined two-dimensional gel electrophoresis and tandem mass spectrometry

“…The excised spots were digested with trypsin for protein identification, as described in Section 2, and a few high molecular mass tryptic peptides missing in databases were obtained due to the low level of lysine and arginine residues in parent proteins [22,23]. Chymotrypsin, having a broader specificity than trypsin, provided an efficient gliadin spot digestion with a greater number of medium-sized peptides, better suited for a mass spectrometric identification.…”

Identification of transglutaminase‐mediated deamidation sites in a recombinant α‐gliadin by advanced mass‐spectrometric methodologies

“…According to the amino‐terminal sequence and electrophoretic mobility, the components of gliadin can be divided into α‐, γ‐, and ω‐gliadins, with each subgroup consisting of a mixture of varying proteins slightly differing in their primary structures. Another peculiarity of gliadin is found in its amino acid composition, as it is made up of a high percentage of glutamine (30%–40%) and proline (20%–25%) residues and a very low percentage of basic/acid aminoacid residues (2%–5%; Booth and Ewart 1970; Wieser et al 1987). Hence, because of these structural features, the identification of all potential tTGase‐specific deamidation sites becomes a complex task.…”