Peptide Hydrolase C in Germinating Barley

The presence of peptide hydrolases in cereal grains has been known for many years (5) but the detailed properties of only a few highly purified enzymes of this type from cereals are known (1, 6, 8). We have reported recently the presence in germinated barley (2) and in wheat embryo (7) of at least four esterases which hydrolyze a-naphthyl acetate. One of these (ANA'-ase IV) from germinated barley, and present also in wheat embryo, has been purified extensively, examined for peptide hydrolase activity, and found to possess broad dipeptidase activity. Methods for the assay of esterase activity using ANA as substrate, and the chromatography of barley proteins on carboxymethyl cellulose are described (2) as are the procedures for gel filtration through Sephadex G-100, protein measurement (1), and the assay of endopeptidase activity with hemoglobin substrate (3). A unit ofANA-ase is defined as that amount of enzyme which hydrolyzes 1 mumole ofANA per minute under the assay conditions. Barley (Hordeum vulgare L., var. Trophy) was germinated in the dark and 150 g of the lyophilized whole seedling were extracted as described previously (1). The dialyzed extract was chromatographed on a 2.5X 40-cm column of CMC by means of a sodium acetate buffer gradient (0.0050.5 M) at pH 5.5 and filtered once through a 2.5X 40-cm column of Sephadex G-100. The eluate was concentrated by ultrafiltration through No. 8 Visking dialysis tubing to 49 ml containing 36,800 units of ANA-ase and 235 mg of protein. The enzyme was purified further by electrofocusing on the LKB 8100 column (LKB Instruments, Inc.) described by Haglund (4) with a 1% ampholyte solution of isoelectric range pH 4 to 6 and the 49 ml of sample. A potential of 500 v was applied to the column for 48 hr with the anode at the bottom of the column. Fractions of approximately 1 ml were collected from the developed column by displacement with water at a pumping rate of 0.6 ml/min. The effluent was not monitored for protein at 280 nm with a flow cell because this decreased the resolution of the isoelectric protein bands. The peak for the ANA-ase activity (Fig. 1) corresponded to an isoelectric point of 5.90, measured at 4 C. Fractions 71 to 74 inclusive (Fig. 1) were pooled. This solution, 4.6 ml containing 11,600 units of ANA-ase IV, was dialyzed for 20 hr on a revolving wheel (1 rpm) at 4 C against two 175-ml portions of 0.05 M phosphate buffer (pH 7.0), containing 0.1 M