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Improved Methods for High-Throughput Extraction and Assay of Green Barley Malt Proteinase Activity Facilitating Examination of Proteinase Activity Across Large-Scale Barley Populations

¹ USDA-ARS, Cereal Crops Research Unit, 502 Walnut Street, Madison, WI 53726. Mention of trade names or commercial products in this presentation is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. ² Corresponding author. Phone: 608-262-4480. Fax: 608-890-0302. E-mail address: mark.schmitt@ars.usda.gov

We report efficient sample extraction and assay methods allowing quantitative determinations of proteinase activities from barley malt. The improved methods are used to assay >2,200 developmental lines of malting barley for two subsets of proteinase activity. The distributions of the resulting activities suggest differences in population structures between the two types of proteinases. Comparison of the activities of the green malt proteinases with standard malting quality measurements show highly significant correlations that differ between the proteinase subsets. The pH 4.5 hydrolysis of the artificial substrate Z-Phe-Arg-AMC correlates well with the traditional malting quality measurements, supporting the role of cysteine-class proteinases in mobilization of grain reserves during malting and mashing. Results from assays of gelatin hydrolysis at pH 6.0 suggest that these proteolytic activities may be involved in other aspects of seed C and N dynamics also linked to malting quality measurements. The differences between the pH 4.5 and 6.0 activities assayed here and their association with malting quality measurements suggest different physiological roles for the two proteinase activities in several aspects of seed germination. Either assay could be useful for population surveys, depending on the particular facet of seed metabolism under study.