?-lactoglobulin (BLG) is a member of lipocalin superfamily of traorters for small hydrophobic molecules such as retinoids, fatty acids, drugs and vitamins. BLG structure has 2 intramolecular disulfide bonds and one free cystein in cys121. BLG is one of the major bovine milk allergen that disulfide bonds are responsible for bovine milk allergy and low digestibility. It seems that the digestibility of BLG will be increased via the reduction of disulfide bonds. The reducing agents such as DTT is able to reduce disulfide bonds and increase the the BLG digestibility. However due to the toxic effect of DTT, the use of this agent is not applicable. Therefore, this can be substituted by natural enzymes. In the present study, we aim to study the effect of plant thioredoxin systems on the digestibility of milk BLG. Thioredoxins (Trxs) are small and abundant disulfide reductase in all organisms. Trxs are characterized with WC (G/P) PC in active site. Trxs are reduced themselves with NADPH via NADPH-dependent thioredoxin reductase (NTR). Previously the genes encoding three rice Trx, OsTrx1, OsTrx20 and OsTrx23 and the gene encoding OsNTR1were cloned and heterologously expressed in E.coli . In this study these proteins were produced and purified in considerable amounts. The activity of Trxs was confirmed by insulin assay. Moreover, the interaction of OsNTR1 with each of OsTrxs was performed using DTNB assay. The result of this assay showed that OsNTR1 was able to reduce OsTrx1 and OsTrx23. However, OsTrx20 was not reduced by OsNTR1. Instead this isoform was reduced by glutathione (GSH). The isoform OsTrx1 can also be reduced by GSH. Therefore, in this study the effect of four systems OsNTR1/OsTrx23, OsNTR1/OsTrx1, GSH/OsTrx20, GSH/OsTrx1 on the digestibility of BLG was studied. To this end BLG was pre-treated by these systems at 4, 25, 37 and 55 °C and then digested with trypsin. The digestability was studied using SDS-PAGE and HPLC. The results revealed that different Trx system significantly affect on the BLG digestability. However, there was no significant difference between the effect of different systems. In addition, the results show that Trx systems work in 4, 25 and 37 °C. Nevertheless, these systems were not affected at 55 C which could be due to the unstability of NTR in this temperature. Key words: NTR/Trx system, glutathione, beta lactoglobulin, digestibility, trypsin, reverse phase -liquid chromatography
