A NOVEL THERMOSTABLE INHIBITOR OF TRYPSIN AND SUBTILISIN FROM THE SEEDS OF BRASSICA-NIGRA - AMINO-ACID-SEQUENCE, INHIBITORY AND SPECTROSCOPIC PROPERTIES AND THERMOSTABILITY

A novel thermostable protein inhibitor of trypsin and subtilisin, call ed BN, was isolated from the seeds of Brassica nigra. The purified pro tein gave a single band on SDS-PAGE, corresponding to a molecular mass of 15 500 +/- 1000 Da. The inhibitor is composed of two disulfide-lin ked polypeptide chains, consisting of 39 and 90 residues, respectively . The amino acid sequence of the two chains was determined by Edman de gradation of peptides, isolated from enzyme hydrolysates with TPCK-try psin, EndoLysC proteinase and a Glu-specific proteinase of reduced and vinylpyridinated protein samples, A segment of the 'heavy' chain, bet ween residues 65 and 81, showed homology with the reactive site loop r egion of the 6-kDa trypsin inhibitors from Nicotiana alata. The basic residue in position 39 (N. alata) or 70 (napins) is conserved as argin ine or lysine in all inhibitors from N. alata and in all napins hither to sequenced, Probably, the two families of trypsin inhibitors have st ructurally similar reactive sites,BN exhibits an extremely high thermo stability: CD measurements showed that during heating to 97 degrees C it preserves a considerable part of the polypeptide backbone folding, Studies on the fluorescence properties of the inhibitor BN in the abse nce and presence of neutral or ionic quenchers demonstrated that the i ntrinsic emission of this protein is dominated by a tryptophyl residue , buried in the interior of the protein matrix, 20% of the light absor bed by Tyr 63 of the 'heavy' chain is transferred to Trp 26 of the 'li ght' chain. (C) 1997 Elsevier Science B.V.