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Thioredoxin, a ubiquitous 12-kDa regulatory disulfide protein, was found to reduce disulfide bonds of allergens (convert S-S to 2 SH) and thereby mitigate the allergenicity of commercial wheat preparations. Allergenic strength was determined by skin tests with a canine model for food allergy. Statistically significant mitigation was observed with 15 of 16 wheat-sensitive animals. The allergenicity of the protein fractions extracted from wheat flour with the indicated solvent was also assessed: the gliadins (ethanol) were the strongest allergens, followed by glutenins (acetic acid), albumins (water), and globulins (salt water). Of the gliadins, the alpha and beta fractions were most potent, followed by the gamma and omega types. Thioredoxin mitigated the allergenicity associated with the major protein fractions-i.e, the gliadins (including the alpha, beta, and gamma types) and the glutenins-but gave less consistent results with the minor fractions, the albumins and globulins. In all cases, mitigation was specific to thioredoxin that had been reduced either enzymically by NADPH and NADP-thioredoxin reductase or chemically by dithiothreitol; reduced glutathione was without significant effect. As in previous studies, thioredoxin was particularly effective in the reduction of intramolecular (intrachain) disulfide bonds. The present results demonstrate that the reduction of these disulfide bonds is accompanied by a statistically significant decrease in allergenicity of the active proteins. This decrease occurs alongside the changes identified previously-i.e., increased susceptibility to proteolysis and heat, and altered biochemical activity. The findings open the door to the testing of the thioredoxin system in the production of hypoallergenic, more-digestible foods.

Protease inhibitors, including the Kunitz and Bowman-Birk trypsin inhibitorsof soybean, can be reduced by the NADP/thioredoxin system (NADPH, thioredoxin, and NADP-thioredoxin reductase) from either Escherichia coli or wheat germ. In the present study, we report that, when reduced by thioredoxin, the Kunitz and Bowman-Birk soybean trypsin inhibitors lose their ability to inhibit prytsin. Moreover, the reduced form of the inhibitors showed increased susceptibility to heat and proteolysis by either subtilisin or a protease preparation from germinating wheat seeds

Thioredoxin of the h-type — earlier linked to the reduction of wheat (Triticum durum Desf. cv. Monroe) endosperm proteins — was converted from an oxidized to a partially reduced state during germination and seedling development. While the abundance of thioredoxin progressively decreased during this period, the availability of reducing equivalents, defined as the product of the relative abundance of thioredoxin and the percent reduction, increased. The amount of the enzyme catalyzing the reduction of thioredoxin h (NADP-thioredoxin reductase) remained constant. The activities of enzymes generating the NADPH needed for the reduction of thioredoxin (glucose 6-phosphate and 6-phosphogluconate dehydrogenases) increased. The level of thioredoxin h in the endosperm appeared to be controlled by the embryo via hormones. Gibberellic acid enhanced the disappearance of thioredoxin, whereas abscisic acid showed the opposite effect. Moreover, uniconazole, an inhibitor of gibberellic acid synthesis, slowed seedling growth and inhibited the disappearance of thioredoxin in a manner reversible by gibberellic acid. The results are consistent with a role for thioredoxin h in initiating the mobilization of nitrogen and carbon needed for germination and seedling development.

We describe a protease, named "thiocalsin," that is activated by calcium but only after reductive activation by thioredoxin, a small protein with a redox-active disulfide group that functions widely in regulation. Thiocalsin appeared to be a 14-kDa serine protease that functions independently of calmodulin. The enzyme, purified from germinating wheat grain, specifically cleaved the major indigenous storage proteins, gliadins and glutenins, after they too had been reduced, preferentially by thioredoxin. The disulfide groups of the enzyme, as well as its protein substrates, were reduced by thioredoxin via NADPH and the associated enzyme, NADP-thioredoxin reductase. The results broaden the roles of thioredoxin and calcium and suggest a joint function in activating thiocalsin, thereby providing amino acids for germination and seedling development.

Lipoic acid was found to be present at low levels in wheat germ (ca. 0.1 ppm) but was not detected in flour or semolina. The reduced form of lipoic acid provided the reducing equivalents for thioredoxin in the reduction of proteins of wheat endosperm (albumins, globulins, gliadins, and glutenins). The results indicate that, while lipoic acid could function to reduce thioredoxin physiologically, its importance to technologies based on flour or semolina is questionable

Gliadins and glutenins, the major storage proteins of wheat endosperm (Triticum durum, Desf. cv Monroe), were reduced in vitro by the NADP/thioredoxin system (NADPH, NADP-thioredoxin reductase and thioredoxin; in plants, the h type) from either the same source or the bacterium Escherichia coli. A more limited reduction of certain members of these protein groups was achieved with the reduced form of glutathione or glutaredoxin, a protein known to replace thioredoxin in certain bacterial and mammalian enzyme systems but not known to occur in higher plants. Endosperm extracts contained the enzymes necessary to reduce NADP by the oxidative pentose phosphate pathway (hexokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase). The gliadins and glutenins were also reduced in vivo during germination--an event that accompanied their proteolytic breakdown. The results suggest that thioredoxin, reduced by NADPH generated via the oxidative pentose phosphate pathway, functions as a signal in germination to enhance metabolic processes such as the mobilization of storage proteins and, as found earlier, the activation of enzymes.

Thioredoxin, reduced either enzymatically with NADPH and NADP-thioredoxin reductase or chemically with dithiothreitol, reduced alpha-amylase and trypsin inhibitor proteins from several sources. Included were cystine-rich seed representatives from wheat (alpha-amylase inhibitors), soybean (Bowman-Birk trypsin inhibitor), and corn (kernel trypsin inhibitor). This system also reduced other trypsin inhibitors: the soybean Kunitz inhibitor, bovine lung aprotinin, and egg white ovoinhibitor and ovomucoid proteins. The ability of these proteins to undergo reduction by thioredoxin was determined by 1) a coupled enzyme activation assay with chloroplast NADP-malate dehydrogenase or fructose-1,6-bisphosphatase, 2) a dye reduction assay with 5‘,5‘-dithiobis(2-nitrobenzoic acid), and 3) a direct reduction method based on the fluorescent probe, monobromobimane, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Reduction experiments with the seed proteins were carried out with thioredoxin from wheat germ (h-type) or Escherichia coli; the corresponding experiments with the animal trypsin inhibitors were carried out with thioredoxin from calf thymus or E. coli. In all cases, thioredoxin appeared to act catalytically; the reduced form of glutathione was without effect. When considered in conjunction with earlier results on purothionin (confirmed and extended in the current study), the new findings suggest that the NADP/thioredoxin system functions in the reduction of protein inhibitors of seeds and animal tissues. These results also raise the question of the occurrence of glutaredoxin in plants, as E. coli glutaredoxin was found to promote the reduction of some but not all of the proteins tested.

Gas chromatographic and mass spectrometric analyses showed that two low molecular weight proteins, extracted from glutenins of two durum wheat cultivars, contained strongly bound lipids with particular composition in comparison to semolina lipids. The percentages of palmitic acid stearic acid, and oleic acid were high; Various fatty alcohols and hydrocarbons were also present in the purified proteins; the amount of dodecanol, especially in one sample, was very high

Semolina samples were obtained from several durum wheat cultivars. The cooking quality of the spaghetti prepared from the semolina showed both inter and intravarietal differences. Proteins from semolina were extracted sequentially and both the reactive and total SH plus SS content of the protein extracts determined amperometrically. The albumin-globulin fractions contained the highest amounts of both reactive and total SH plus SS groups. The total SH plus SS content was higher in the gliadins than in the glutenins but the latter contained higher amounts of reactive SH plus SS groups. A highly significant correlation was found between the total amount of SH plus SS content of the glutenins and the cooking quality of the samples (r=0.835): This might be a functional relation.

Two low molecular weight durum wheat (Triticum durum Desf) glutenins, DSG-1 and DSG-2 (durum-wheat sulphur-rich glutenin fractions) were isolated from two cultivars, Mondur of good technological quality and Kidur of poor technological quality. The glutenin fraction, composed mostly of DSG protein, was extracted using a low concentration of NA-tetradecanoate and then fractionted by by using molecular sieve chromatography (Bio-Gel P 30). The amino acid compositions and the N-terminal sequences of the pure DSG proteins were determined, and their hydrophobic characteristics, calculated on the basis of these data, showed that DSG-2 is more hydrophobic than DSG-1. The amino acid compositions of DSG-1 and DSG-2 were different. The N-terminal amino acids of DSG-1 and DSG-2 were also different but were identical for each of the two cultivars. In the case of DSG-1, in addition to the main chain a minor chain was found in which the first three amino acids of the main chain were missing. The minor chain represented about 30% int he DSG-1 of mondur and almost 50% in Kidur.