Author
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Schmitt, Mark |
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Submitted to: American Society of Brewing Chemists Newsletter
Publication Type: Abstract Only Publication Acceptance Date: 3/8/2004 Publication Date: 7/24/2004 Citation: Schmitt, M. 2004. Improved assay for barley seed and green malt proteases [abstract]. American Society of Brewing Chemists Newsletter. p. 63. Interpretive Summary: Technical Abstract: Protein hydrolysis and remobilization during malting is a critical process in malt production, ultimately contributing to beer quality. However, the process is complex, involving both storage and other grain proteins as substrates and a large number of protease activities representing all four protease classes (cysteine, serine, aspartic, and metalloproteases) in both cellular (aleurone) and acellular (endosperm) tissues. Previous assays have involved following disappearance of seed proteins by HPLC or PAGE as well as by protease activity on artificial substrates. Several years ago, Jones et al (Anal. Biochem.,1998) described an assay system utilizing an artificial substrate (Azogelatin), which provided several improvements over previous assays. However, the Jones Azogelatin protease assay required in-house synthesis of the dye-labeled gelatin substrate, used relatively large volumes (1 mL) of tissue extract, and involved several sample manipulations during the assay (precipitation, centrifugation, and sample transfer) prior to spectrophotometric analysis of azo dye-labeled protein fragments released during protein hydrolysis. Several fluorescently-labeled protease substrates are commercially available which offer significant advantages and can be utilized in an improved protease assay. Upon protease activity and protein hydrolysis, a fluorescent signal generated which can readily be measured and quantified. This assay offers a number of improvements over previous assays in that it can measure protease activity continuously as well as in end-point mode without significant sample handling, it requires microliter rather than milliliter sample volumes, and uses commercially available substrates rather than depending on in-house substrate synthesis. The assay can be routinely run in a microplate-based spectrofluorimeter, reducing reagent volumes and sample requirements while increasing sample throughput capacities. The solution assay is readily quantifiable, shows inhibition by class-specific protease inhibitors, and can be performed over appropriate physiological pH values. It can be adapted for 1- and 2-D PAGE systems. The assay may show utility for in situ analysis of protease localizations in barley and other seeds. Specific assay details and examples will be provided. |
