title: Characterization and pharmacological properties of a novel multifunctional Kunitz inhibitor from Erythrina velutina seeds.
abstract: Inhibitors of peptidases isolated from leguminous seeds have been studied for their pharmacological properties. The present study focused on purification, biochemical characterization and anti-inflammatory and anticoagulant evaluation of a novel Kunitz trypsin inhibitor from Erythrina velutina seeds (EvTI). Trypsin inhibitors were purified by ammonium sulfate (30-60%), fractionation followed by Trypsin-Sepharose affinity chromatography and reversed-phase high performance liquid chromatography. The purified inhibitor showed molecular mass of 19,210.48 Da. Furthermore, a second isoform with 19,228.16 Da was also observed. The inhibitor that showed highest trypsin specificity and enhanced recovery yield was named EvTI (P2) and was selected for further analysis. The EvTI peptide fragments, generated by trypsin and pepsin digestion, were further analyzed by MALDI-ToF-ToF mass spectrometry, allowing a partial primary structure elucidation. EvTI exhibited inhibitory activity against trypsin with IC50 of 2.2×10(-8) mol.L(-1) and constant inhibition (Ki) of 1.0×10(-8) mol.L(-1), by a non-competitive mechanism. In addition to inhibit the activity of trypsin, EvTI also inhibited factor Xa and neutrophil elastase, but do not inhibit thrombin, chymotrypsin or peptidase 3. EvTI was investigated for its anti-inflammatory and anti-coagulant properties. Firstly, EvTI showed no cytotoxic effect on human peripheral blood cells. Nevertheless, the inhibitor was able to prolong the clotting time in a dose-dependent manner by using in vitro and in vivo models. Due to anti-inflammatory and anticoagulant EvTI properties, two sepsis models were here challenged. EvTI inhibited leukocyte migration and specifically acted by inhibiting TNF-α release and stimulating IFN-α and IL-12 synthesis. The data presented clearly contribute to a better understanding of the use of Kunitz inhibitors in sepsis as a bioactive agent capable of interfering in blood coagulation and inflammation.
authors.affiliation: Laboratório de Química e Função de Proteínas Bioativas, Departamento de Bioquímica, Centro de Biociências, Universidade Federal do Rio Grande do Norte, Natal, RN, Brasil.
authors.forename: Richele J A
authors.initials: RJ
authors.lastname: Machado
authors.forename: Norberto K V
authors.initials: NK
authors.lastname: Monteiro
authors.forename: Ludovico
authors.initials: L
authors.lastname: Migliolo
authors.forename: Osmar N
authors.initials: ON
authors.lastname: Silva
authors.forename: Michele F S
authors.initials: MF
authors.lastname: Pinto
authors.forename: Adeliana S
authors.initials: AS
authors.lastname: Oliveira
authors.forename: Octávio L
authors.initials: OL
authors.lastname: Franco
authors.forename: Sumika
authors.initials: S
authors.lastname: Kiyota
authors.forename: Marcelo P
authors.initials: MP
authors.lastname: Bemquerer
authors.forename: Adriana F
authors.initials: AF
authors.lastname: Uchoa
authors.forename: Ana H A
authors.initials: AH
authors.lastname: Morais
authors.forename: Elizeu A
authors.initials: EA
authors.lastname: Santos
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000893:Anti-Inflammatory Agents / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D000925:Anticoagulants / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D002465:Cell Movement / Q000187:drug effects; D002846:Chromatography, Affinity; D016207:Cytokines / Q000378:metabolism; D004353:Drug Evaluation, Preclinical; D004895:Erythrina / Q000737:chemistry*; D004926:Escherichia coli / Q000187:drug effects; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007958:Leukocyte Count; D051379:Mice; D008807:Mice, Inbred BALB C; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D010936:Plant Extracts / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D055550:Protein Stability; D012639:Seeds / Q000737:chemistry*; D018805:Sepsis / Q000188:drug therapy / Q000276:immunology; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014357:Trypsin / Q000737:chemistry; D014361:Trypsin Inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*
chemical_list: D000893:Anti-Inflammatory Agents; D000925:Anticoagulants; D016207:Cytokines; C053865:Kunitz-type protease inhibitor, plant; D010455:Peptides; D010936:Plant Extracts; D010940:Plant Proteins; D014361:Trypsin Inhibitors; D014357:Trypsin