Electrophoretic and 'in solution' analyses of endoproteinases extracted from germinated oats

During the malting of oats, the insoluble seed storage proteins are hydroly sed by endoproteinases. These germinated oat seed endoproteinases are to be characterised. A qualitative 2-D (IEF x PAGE) method using the gel-incorpo rated substrate gelatin was used to study the heterogeneity of the enzymes and a quantitative 'in solution' assay employing azogelatin was utilised to measure the different proteinase classes present. Proteinase development d uring malting was monitored at pH 3.8, 6.2 (the pH of the germinated oat en dosperm) and 8.0. The numbers and intensities of the proteolylic activities increased during the first three days of germination, but not after that. Electrophoretic studies using class specific endoproteinase inhibitors indi cated that the predominant enzymes were serine and metalloproteinases. No a spartic or cysteine proteinases were detected. The 'in solution' analyses g ave somewhat different results from those obtained with the 2-D method. The concomitant addition of 10 mM calcium and 8 mM cysteine raised the measure d activity by 2.5 fold and, in the presence of these activators, a third of the 'in solution' activity was due to cysteine proteinases, the serine and metalloproteinases each contributed 15% of the activity and the other thir d of the activity was impervious to all of the inhibitors. These different 2-D and 'in solution' results may simply reflect the fact that the enzymes hydrolyse the substrate differently, depending on whether it is dissolved ( 'in solution') or in a bound (2-D analysis) form. (C) 2000 Academic Press.