ARGININE MODIFICATION IN KUNITZ BOVINE TRYPSIN INHIBITOR THROUGH 1, 2-CYCLOHEXANEDIONE*

Abstract
Arginine residues (5.5 of 6) of the trypsin-kallikrein inhibitor from bovine organs (Kunitz inhibitor) were selectively modified by reaction with 1, 2-cyclohexanedione in sodium borate buffer, pH 9.0. The modified inhibitor is still highly active in inhibiting trypsin and chymotrypsin at 1:1 inhibitor:enzyme molar ratio and full inhibition was acheived at slightly higher molar ratio. The extent of correct refolding, on reoxidation of the reduced, arginine-modified inhibitor is diminished and regeneration of 2 arginines occurred under the reduction conditions. The stability constants and the standard-free energies of binding of the complexes between trypsin, or chymotrypsin, and the native, the arginine-modified and the reduced and reoxidized arginine-modified inhibitor have been determined from inhibitory assays.

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