Purification, inhibitory properties, amino acid sequence and identification of the reactive site of a new serine proteinase inhibitor from oil-rape (itBrassica napus) seed

A new serine proteinase inhibitor, rapeseed trypsin inhibitor (RTI), has been isolated from rapeseed (Brassica napus var. oleifera) seed. The protein inhibits the catalytic activity of bovine β-trypsin and bovine α-chymotrypsin with apparent dissociation constants of 3.0 × 10^-10 M and 4.1 × 10^-7 M, at pH 8.0 and 21°C, respectively. The stoichiometry of both proteinase-inhibitor complexes is 1:1. The amino acid sequence of RTI consists of 60 amino acid residues, corresponding to an M_r, of about 6.7 kDa. The p₁-p_i, reactive site bond has been tentatively identified at position Arg²⁰-Ile²¹. RTI shows no similarity to other serine proteinase inhibitors except the low molecular weight mustard trypsin inhibitor (MTI-2). RTI and MTI-2 could be members of a new class of plant serine proteinase inhibitors.