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, called BN, was isolated from the seeds of Brassica nigra. The purified protein gave a single band on SDS-PAGE, corresponding to a molecular mass of 15 500 +/- 1000 Da. The inhibitor is composed of two disulfide-linked polypeptide chains, consisting of 39 and 90 residues, respectively. The amino acid sequence of the two chains was determined by Edman degradation of peptides, isolated from enzyme hydrolysates with TPCK-trypsin, EndoLysC proteinase and a Glu-specific proteinase of reduced and vinylpyridinated protein samples. A segment of the 'heavy' chain, between residues 65 and 81, showed homology with the reactive site loop region of the 6-kDa trypsin inhibitors from Nicotiana alata. The basic residue in position 39 (N. alata) or 70 (napins) is conserved as arginine or lysine in all inhibitors from N. alata and in all napins hitherto sequenced. Probably, the two families of trypsin inhibitors have structurally similar reactive sites. BN exhibits an extremely high thermostability: 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 absence and presence of neutral or ionic quenchers demonstrated that the intrinsic emission of this protein is dominated by a tryptophyl residue, buried in the interior of the protein matrix. 20% of the light absorbed by Tyr 63 of the 'heavy' chain is transferred to Trp 26 of the 'light' chain.
Genov
N
N
Institute of Organic Chemistry, Bulgarian Academy of Sciences, Sofia.
Goshev
I
I
Nikolova
D
D
Georgieva
D N
DN
Filippi
B
B
Svendsen
I
I
D000595:Amino Acid Sequence; D001937:Brassica / Q000737:chemistry*; D002850:Chromatography, Gel; D002918:Chymotrypsin / Q000037:antagonists & inhibitors / Q000302:isolation & purification; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011489:Protein Denaturation; D012639:Seeds / Q000737:chemistry; D017421:Sequence Analysis; D015842:Serine Proteinase Inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013381:Subtilisins / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D013696:Temperature; D014361:Trypsin Inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology
C108913:BN protein, Brassica nigra; D010940:Plant Proteins; D015842:Serine Proteinase Inhibitors; D014361:Trypsin Inhibitors; D013381:Subtilisins; D002918:Chymotrypsin