Modification of the functional arginine residue in soybean trypsin inhibitor (Kunitz) by immobilized peptidylarginine deiminase.

Abstract

We immobilized peptidylarginine deiminase, a Ca2 +-dependent protein-modifying enzyme which catalyzes the deimination of arginyl residues in protein, on formyl-Sepharose 4B by reductive amination. The immobilized enzyme was quite stable in the presence of reducing reagents and chelators such as DTT and EGTA. The immobilized enzyme was inactivated under the catalytic conditions in the absence of substrate. The inactive enzyme, however, was totally reactivated by replacement of Ca2+ by a mixture of reducing reagent and chelator in the equilibration buffer.Our preceding paper described the specific modification of the functional arginine residue in soybean trypsin inhibitor (Kunitz) (STI) by peptidylarginine deiminase [H. Takahara, H. Okamoto, and K. Sugawara, J. Biol. Chem., 260, 8378 (1985)]. In this work, the rate of the modification of STI by the immobilized enzyme packed in a column was measured by continuous application of STI at constant temperature and flow rate. The extent of the modification of STI...