EXPRESSION AND KINETIC CHARACTERIZATION OF BARLEY CHYMOTRYPSIN INHIBITORS 1A AND 1B

The genes for chymotrypsin inhibitors 1a and 1b (CI-1a and CI-1b) from barley have been expressed in E. coli, and the CI-1a and CI-1b protei ns purified. These proteins, although highly homologous, differ in the active site region at P2, P1' and P3' (Schechter and Berger nomenclat ure), and so might be expected to have differing specificities. Despit e this, analysis of the inhibition kinetics showed that each displayed very similar kinetic behaviour when tested against a range of protein ases. The specificity of the CI-1 proteins is different to that of the other main barley inhibitor, CI-2, and K-i values are found to follow the series subtilisin Carlsberg < neutrophil elastase similar to subt ilisin BPN' << chymotrypsin. Only very weak inhibition is found of try psin, and pancreatic elastase is not measurably inhibited. For the pro teinases inhibited most strongly, characteristic slow-binding inhibiti on kinetics were observed, whereas classical inhibition applied to the weaker interactions. The results are consistent with the major determ inant of specificity being the P1 residue of the inhibitor, which is t he same in both CI-1a and CI-1b. Consistent with this, is the similar spectrum of specificity found for the homologous inhibitor eglin c fro m leech, which has the same P1 residue. Both the CI-1 proteins are fou nd to be less stable than CI-2, with CI-1a being significantly less st able than CI-1b as measured by guanidinium hydrochloride unfolding exp eriments. Possible reasons for the reduced stability are discussed in view of the sequence differences between CI-1a, CI-1b and CI-2.