RESUMO
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.
Assuntos
Proteínas de Plantas/farmacologia , Calicreína Plasmática/antagonistas & inibidores , Rosales/química , Inibidores de Serina Proteinase/isolamento & purificação , Inibidores da Tripsina/isolamento & purificação , Sequência de Aminoácidos , Animais , Sítios de Ligação , Bovinos , Cromatografia de Afinidade , Corantes Fluorescentes/metabolismo , Humanos , Hidrólise , Cinética , Dados de Sequência Molecular , Peso Molecular , Peptídeos/síntese química , Peptídeos/química , Peptídeos/farmacologia , Proteínas de Plantas/isolamento & purificação , Calicreína Plasmática/metabolismo , Análise de Sequência de Proteína , Homologia de Sequência de Aminoácidos , Inibidores de Serina Proteinase/genética , Inibidores de Serina Proteinase/farmacologia , Tripsina/química , Inibidores da Tripsina/genética , Inibidores da Tripsina/farmacologiaRESUMO
We have previously described Kunitz-type serine proteinase inhibitors purified from Bauhinia seeds. Human plasma kallikrein shows different susceptibility to those inhibitors. In this communication, we describe the interaction of human plasma kallikrein with fluorogenic and non-fluorogenic peptides based on the Bauhinia inhibitors' reactive site. The hydrolysis of the substrate based on the B. variegata inhibitor reactive site sequence, Abz-VVISALPRSVFIQ-EDDnp (Km 1.42 microM, kcat 0.06 s(-1), and kcat/Km 4.23 x 10(4) M(-1) s(-1)), is more favorable than that of Abz-VMIAALPRTMFIQ-EDDnp, related to the B. ungulata sequence (Km 0.43 microM, kcat 0.00017 s(-1), and kcat/Km 3.9 x 10(2) M(-1) s(-1)). Human plasma kallikrein does not hydrolyze the substrates Abz-RPGLPVRFESPL-EDDnp and Abz-FESPLRINIIKE-EDDnp based on the B. bauhinioides inhibitor reactive site sequence, the most effective inhibitor of the enzyme. These peptides are competitive inhibitors with Ki values in the nM range. The synthetic peptide containing 19 amino acids based on the B. bauhinioides inhibitor reactive site (RPGLPVRFESPL) is poorly cleaved by kallikrein. The given substrates are highly specific for trypsin and chymotrypsin hydrolysis. Other serine proteinases such as factor Xa, factor XII, thrombin and plasmin do not hydrolyze B. bauhinioides inhibitor related substrates.
