Journal of Cereal Science 21 (1995) 145-153 Characterization of Germinated Barley EndoproteolyticEnzymesby Two-dimensionalGel Electrophoresls Ningyan Zhangl- and Berne L. Jones*l- *USDA, Agricultural ResearchService, Cereal Crops Research Unit, 501 N. Walnut St, Madison, V~sconsin 53705, U.S.A. and 1Departmentof Agronomy, Universityof Wisconsin, Madison, Wisconsin 53706, U.S.A. ABSTRACT Germinating barley proteolytic enzymes hydrotyze insoluble seed storage proteins into soluble proteins, peptides and amino acids. This process is of vital importance to both seed germination and the commercial malting process. This study reports the development of a two-dimensionalIEF x PAGE separation method utilizing protein substrates incorporated into the PAGE gel to analyze and partially characterize the endoproteinases of germinating barley grain. The method separated 42 different activities, which fell into five groups on the basis of their pI values, PAGE mobilities and biochemical characteristics. Multiple representatives of each of the four proteinase classes were present, but about 64% of the enzymes were cysteine proteinases. The majority of the endoproteinases had pH optima considerably below neutrality, but those of the serine proteinases were generally eight or higher. Most of the endoproteinases hydrolyzed gelatin faster than edestin, but the four aspartic class proteinases and one of an undetermined class only hydrolyzed edestin. These results show that the protein-hydrolyzing system of green malt (4-day-old germinated barley) is very complex. INTRODUCTION The proteinases of germinating barley are re- sponsible for the breakdown of large, generally insoluble, storage proteins into soluble proteins, peptides and amino acids. This hydrolysis is es- sential for mobilizing the storage protein com- ponents for use during germination and, commercially, it is crucial to the malting process. To date, two barley cysteine endoproteinases, with molecular weights of 30 and 37k and maximal activities at acidic pH values, have been purified ~-s. Both increased greatly during seed germination. An aspartic acid endoproteinase has also been purified from resting barley seeds 4. However, there are many additional en- ABBREVIATIONS USED: DIC=3,4-dichloroisocoumarin; E-64 = trans-epoxysuccinyl-L-leucylamido(4- guanidino)butane; [3-ME = ~-mercaptoethanol; PepA = pepstatin A; Phen= 1,10-phenanthroline; PMSF= phenylmethylsulfonyl fluoride. +To whom correspondence should be addressed. doproteinases in germinating barley seeds. At least seven activity bands have been separated on a one- dimensional native electrophoresis gel containing incorporated protein substrate s, and preliminary experiments indicated that there were multiple endoproteinases present in all, or at least most, of those activity bands. These endoproteinase ac- tivities have previously been shown to occur in the barley grain and to not be due to microbial contamination 6. In order to characterize each en- doproteinase of germinated barley without need- ing to purify the individual enzymes, we have developed a two-dimensional (2-D) method for separating the endoproteinases. The first di- mension separation was with isoelectric focusing (IEF), and the second dimension was poly- acrylamide gel electrophoresis (PAGE) with gels containing immobilized protein substrates. This system allowed us to separate many of the pro- teinases into individual activity spots. We then analyzed the separated enzymes, while they were still located in the gels, under various conditions to see how the different treatments affected their 0733-5210/95/020145 + 09 $08.00/0 145 © 1995 Academic Press Limited