Purification and primary structure determination of a Bowman-Birk trypsin inhibitor from Torresea cearensis seeds.
A Bowman-Birk-type trypsin inhibitor (TcTI) was purified from seeds of Torresea cearensis, a Brazilian native tree of the Papilionoideae sub-family of Leguminosae. Three forms of the inhibitor were separated by anion exchange chromatography. The major form with 63 amino acids was entirely sequenced; it shows a high structural similarity to the Bowman-Birk inhibitors from other Leguminosae. The putative reactive sites of the inhibitor are a lysine residue at position 15 and a histidine at position 42 as identified by alignment to related inhibitors, direct chemical modification and specific enzymatic degradation. Immunoprecipitation with antibodies raised in rats is reduced significantly if TcTI is complexed with chymotrypsin and, to a lesser degree, if complexed with trypsin. TcTI forms a ternary complex with trypsin and chymotrypsin. The binary complexes with trypsin or chymotrypsin were isolated by gel filtration. Dissociation constants of the complexes with trypsin, plasmin, chymotrypsin, and factor XIIa are 1, 36, 50, 1450 nM, respectively; human plasma kallikrein, human factor Xa, porcine pancreatic kallikrein and bovine thrombin are not inhibited. TcTI prolongs blood clotting time of the contact phase activation pathway by inhibition of FXIIa.
Tanaka
A S
AS
Departamento de Bioquimica, Universidade Federal de São Paulo, Escola Paulista de Medicina, UNIFESP, Brazil.
Sampaio
M U
MU
Marangoni
S
S
de Oliveira
B
B
Novello
J C
JC
Oliva
M L
ML
Fink
E
E
Sampaio
C A
CA
D000595:Amino Acid Sequence; D000818:Animals; D000925:Anticoagulants / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D001665:Binding Sites; D001777:Blood Coagulation / Q000187:drug effects; D002417:Cattle; D002918:Chymotrypsin / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D007887:Fabaceae / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000302:isolation & purification; D010946:Plants, Medicinal; D051381:Rats / Q000276:immunology; D012639:Seeds / Q000737:chemistry*; D014357:Trypsin / Q000378:metabolism; D014358:Trypsin Inhibitor, Bowman-Birk Soybean / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D014361:Trypsin Inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology
D000925:Anticoagulants; D010455:Peptides; D014358:Trypsin Inhibitor, Bowman-Birk Soybean; D014361:Trypsin Inhibitors; D002918:Chymotrypsin; D014357:Trypsin