Studies on trypsin inhibitor in barley. I. Purification and some properties

To clarify the properties and functions of a trypsin inhibitor from Japanese barley in comparison with the inhibitor from Pirkka barley, an inhibitor was isolated from the barley Hordeum distichum L var. emend Lamark by extraction with 1% NaCl, ammonium sulfate fractionation and repeated chromatography on DEAE-cellulose and CM-cellulose. The final purified preparation of the inhibitor was found to be homogeneous by both chromatographic and electrophoretic analysis. The inhibitor was thermostable and was stable over the broad pH range from 2 to 11. No inhibition was observed by heavy metal ions and many reagents at 10(-2) M, except that p-chloromercuribenzoate caused a 69% loss of activity. The inhibitor was subjected to isoelectric focusing at pH 7.51 and its molecular weight was calculated to be 14,200+/-900 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The apparent dissociation constant for the complex between the inhibitor and trypsin[EC 3.4.21.4] was 1.64 X 10(-7)M with casein as a substrate. One microgram of purified inhibitor inhibited 1.5 mug of pure trypsin in the hydrolysis of alpha-N-benzoyl-DL-arginine-p-nitroanilide. By chemical modification of arginyl residues in the inhibitor with 1,2-cyclohexanedione, the inhibitor was shown to be an arginine inhibitor. The inhibitor contained relatively many basic amino acids and few half cystines as compared with Pirkka barley trypsin inhibitor.