Characterization and pharmacological properties of a novel multifunctional Kunitz inhibitor from Erythrina velutina seeds.

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...

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Autores principales:	Richele J A Machado, Norberto K V Monteiro, Ludovico Migliolo, Osmar N Silva, Michele F S Pinto, Adeliana S Oliveira, Octávio L Franco, Sumika Kiyota, Marcelo P Bemquerer, Adriana F Uchoa, Ana H A Morais, Elizeu A Santos
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2013
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/3d6afa934fdb40c7957a3ab751b00452
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spelling	oai:doaj.org-article:3d6afa934fdb40c7957a3ab751b004522021-11-18T07:43:59ZCharacterization and pharmacological properties of a novel multifunctional Kunitz inhibitor from Erythrina velutina seeds.1932-620310.1371/journal.pone.0063571https://doaj.org/article/3d6afa934fdb40c7957a3ab751b004522013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23737945/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203Inhibitors 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.Richele J A MachadoNorberto K V MonteiroLudovico MiglioloOsmar N SilvaMichele F S PintoAdeliana S OliveiraOctávio L FrancoSumika KiyotaMarcelo P BemquererAdriana F UchoaAna H A MoraisElizeu A SantosPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 8, Iss 5, p e63571 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Richele J A Machado Norberto K V Monteiro Ludovico Migliolo Osmar N Silva Michele F S Pinto Adeliana S Oliveira Octávio L Franco Sumika Kiyota Marcelo P Bemquerer Adriana F Uchoa Ana H A Morais Elizeu A Santos Characterization and pharmacological properties of a novel multifunctional Kunitz inhibitor from Erythrina velutina seeds.
description	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.
format	article
author	Richele J A Machado Norberto K V Monteiro Ludovico Migliolo Osmar N Silva Michele F S Pinto Adeliana S Oliveira Octávio L Franco Sumika Kiyota Marcelo P Bemquerer Adriana F Uchoa Ana H A Morais Elizeu A Santos
author_facet	Richele J A Machado Norberto K V Monteiro Ludovico Migliolo Osmar N Silva Michele F S Pinto Adeliana S Oliveira Octávio L Franco Sumika Kiyota Marcelo P Bemquerer Adriana F Uchoa Ana H A Morais Elizeu A Santos
author_sort	Richele J A Machado
title	Characterization and pharmacological properties of a novel multifunctional Kunitz inhibitor from Erythrina velutina seeds.
title_short	Characterization and pharmacological properties of a novel multifunctional Kunitz inhibitor from Erythrina velutina seeds.
title_full	Characterization and pharmacological properties of a novel multifunctional Kunitz inhibitor from Erythrina velutina seeds.
title_fullStr	Characterization and pharmacological properties of a novel multifunctional Kunitz inhibitor from Erythrina velutina seeds.
title_full_unstemmed	Characterization and pharmacological properties of a novel multifunctional Kunitz inhibitor from Erythrina velutina seeds.
title_sort	characterization and pharmacological properties of a novel multifunctional kunitz inhibitor from erythrina velutina seeds.
publisher	Public Library of Science (PLoS)
publishDate	2013
url	https://doaj.org/article/3d6afa934fdb40c7957a3ab751b00452
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Characterization and pharmacological properties of a novel multifunctional Kunitz inhibitor from Erythrina velutina seeds.

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