Bauhinia bauhinioides plasma kallikrein inhibitor: interaction with synthetic peptides and fluorogenic peptide substrates related to the reactive site sequence.
A serine proteinase inhibitor was purified from Bauhinia bauhinioides seeds after extraction with 0.15M NaCl by ion-exchange column chromatography on DEAE-Sephadex, gel filtration on Superose 12 column, Mono Q chromatography or alternatively by affinity chromatography on trypsin- Sepharose. The inhibitor is a single polypeptide chain with molecular mass 20 kDa by gel filtration on Superose 12, but was resolved into two peaks by ion - exchange chromatography on Mono Q (FPLC system). The main eluted peak inhibits trypsin (Ki = 0.6 nM), plasma kallikrein (Ki = 0.35 nM), plasmin (Ki = 33.1 nM), and weakly chymotrypsin (Ki = 2,700 nM), being the most effective plasma kallikrein inhibitor isolated from Bauhinia seeds. Therefore, it was denominated Bauhinia bauhinioides kallikrein inhibitor (BbKI). Activity is thermolabile and on trypsin inhibition optimum pH is 8.0. BbKI displays high homology to other plant Kunitz inhibitors, except for the absence of disulfide bridges, and the only cysteine residue is at the C-terminal position (residue 154) characterizes a distinct member of the Kunitz family. The affinity of the inhibitor to trypsin was confirmed by adsorption to trypsin-Sepharose resin and by isolation of the trypsin-inhibitor complex by gel filtration. Peptides with variations around the reactive site of BbKI (GLPVRFESPLRINIIKESY) were synthesized containing a quenched fluorogenic group. Trypsin but not plasma kallikrein substrates, these peptides strongly inhibited plasma kallikrein.
Oliva
M L
ML
Departamento de Bioquimica, Universidade Federal de Sao Paulo-Escola Paulista de Medicina, Rua Tres de Maio, São Paulo, Brazil. maysa.bioq@epm.br
Mendes
C R
CR
Santomauro-Vaz
E M
EM
Juliano
M A
MA
Mentele
R
R
Auerswald
E A
EA
Sampaio
M U
MU
Sampaio
C A
CA
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002846:Chromatography, Affinity; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D010940:Plant Proteins / Q000302:isolation & purification / Q000494:pharmacology*; D020842:Plasma Kallikrein / Q000037:antagonists & inhibitors* / Q000378:metabolism; D019685:Rosales / Q000737:chemistry*; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D015842:Serine Proteinase Inhibitors / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology; D014357:Trypsin / Q000737:chemistry; D014361:Trypsin Inhibitors / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology
C434623:BbKI protein, Bauhinia bauhinioides; D005456:Fluorescent Dyes; C053865:Kunitz-type protease inhibitor, plant; D010455:Peptides; D010940:Plant Proteins; D015842:Serine Proteinase Inhibitors; D014361:Trypsin Inhibitors; D020842:Plasma Kallikrein; D014357:Trypsin