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Characterization of Oat Endoproteinases that Hydrolyze Oat Globulins

¹Department of Food Technology, University of Helsinki, POB 27, 00014 University of Helsinki, Finland. ²Corresponding author. Phone: 358-9-191-58638. Fax: 358-9-191-58463. E-mail: markku.mikola@helsinki.fi ³USDA, ARS, Cereal Crops Research Unit, 501 Walnut St., Madison, WI 53705.

During the germination of oats, the major seed storage proteins (globulins) are hydrolyzed by endoproteinases. We have used two methods to characterize these endoproteinases. A qualitative PAGE method that used oat globulins as gel-incorporated substrates was used to determine which enzymes hydrolyzed the globulins. The proteolytic hydrolysis products were studied by hydrolyzing the globulins in vitro with the endoproteinases and analyzing the products by SDS-PAGE. Class-specific proteinase inhibitors were used to show that the globulin hydrolyzing enzymes were cysteine-class proteinases. The proteinases were active at pH 3.8. Using the gel analysis method, a little activity was present at the beginning of seed germination, but the major activity only appeared on the sixth day of germination. Extracts from four-day germinated oats contained cysteine proteinases that hydrolyzed the globulins in vitro to form a polypeptide of intermediate size (MW ≈34,500). Cysteine proteases from an eight-day germinated sample totally hydrolyzed the globulins in <1 hr. Very little hydrolysis occurred at pH 6.2, the pH of germinated oats endosperm tissue. The fact that hydrolysis occurred quickly at pH 3.8 implies that there is probably pH compartmentalization within the endosperm, with some areas of the seed having a low pH value where the globulins can be degraded.