Effects of two protease inhibitors from Bauhinia bauhinoides with different specificity towards gut enzymes of Nasutitermes corniger and its survival.

Two recombinant protease inhibitors from Bauhinia bauhinioides, rBbKI (kallikrein inhibitor) and rBbCI (cruzipain inhibitor) were evaluated for insecticidal activity against workers and soldiers of Nasutitermes corniger (order: Isoptera; family: Termitidae) through the inhibitors' effect on the insect's gut enzymes. The inhibitor rBbKI was more effective than rBbCI in inhibiting the termite's gut enzymes. The kallikrein inhibitor showed termiticidal activity in workers with an LC50 of 0.9 mg mL-1 after 4 days. Conversely, rBbKI did not affect the survival of soldiers and rBbCI did not show termiticidal activity against N. corniger. The two inhibitors showed different specificity towards the termite's gut enzymes, representing interesting tools to characterize N. corniger enzymes. The different effects of rBbKI and rBbCI on the termite's enzymes and survival may be linked to slight structural differences between these inhibitors.

Compulsión glucídica en mujeres adultas y su relación con el consumo de psicofármacos / Carbohydrate compulsion in adult women and its relationship with psychotropic drug

En la mujer adulta declina la función ovárica y la producción de estrógenos, con sintomatología física y psíquica, alteración en la regulación del apetito y tendencia a la ingesta de hidratos de carbono. Los psicofármacos empleados para el manejo del estado anímico podrían provocar efectos secundarios, como modificación de la ingesta y peso corporal y efectos neuroendocrinos como regulación de la serotonina, entre otros. Objetivos: Identificar la asociación entre el consumo de psicofármacos y la compulsión glucídica en mujeres adultas. Materiales y Método: Se realizó un estudio analítico de corte transversal sobre 114 mujeres mayores de 40 años que asistieron al consultorio de climaterio del Hospital General de Agudos “Bernandino Rivadavia”. Se analizaron variables biológicas, antropométricas, conducta alimentaria y consumo de psicofármacos, recabadas mediante encuestas estructuradas, voluntarias y anónimas, y mediciones antropométricas. Para el análisis estadístico se utilizó SPSS 15.0, calculando medidas de tendencia central,comparación de medias y Odds Ratio (OR) con intervalo de confianza (IC) del 95%, test chi cuadrado (x2) y correlación de Pearson con nivel de significación p< 0,05. Resultados: El 67,5% de la muestra presentó sobrepeso u obesidad y el 48,2% circunferencia de cintura (CC) aumentada. El 69,3% refirió compulsión glucídica, siendo los panificados y el chocolate los alimentos de mayor elección en estos episodios. No se observó significación estadística entre las mujeres que consumían psicofármacos para mejorar la calidad de vida, y la presencia de compulsión o estado nutricional alterado. Conclusión: Las mujeres que consumieron psicofármacos no presentaron diferencias en la prevalencia de compulsión glucídica o el estado nutricional alterado...

Effect of bauhinia bauhinioides kallikrein inhibitor on endothelial proliferation and intracellular calcium concentration.

OBJECTIVES: Proteinase inhibitors act as a defensive system against predators e.g. insects, in plants. Bauhinia bauhinioides kallikrein inhibitor (BbKI) is a serine proteinase inhibitor, isolated from seeds of Bauhinia bauhinioides and is structurally similar to plant Kunitz-type inhibitors but lacks disulfide bridges. In this study we evaluated the antiproliferative effect of BbKI on endothelial cells and its impact on changes in membrane potential and intracellular calcium. MATERIALS AND METHODS: HUVEC proliferation was significantly reduced by incubation with BbKI 50 and 100 µM 12% and 13%. Furthermore, BbKI (100 µM) exposure caused a significant increase in intracellular Ca2+ concentration by 35% as compared to untreated control. RESULTS: The intracellular rise in calcium was not affected by the absence of extracellular calcium. BBKI also caused a significant change in the cell membrane potential but the antiproliferative effect was independent of changes in membrane potential. CONCLUSIONS: BBKI has an antiproliferative effect on HUVEC, which is independent of the changes in membrane potential, and it causes an increase in intracellular Ca2+.

Influyen los valores de tirotrofina sobre el descenso de peso corporal en mujeres adultas con sobrepeso y obesidad? / Do thyrotropin values influence on the decrease of body weight in adult women with overweight and obesity

Introducción: . Objetivos: Estudiar si los valores de TSH influyen sobre el descenso del peso corporal en mujeres adultas con sobrepeso u obesidad. Metodología: Diseño de intervención sobre muestra no probabilística de mujeres >30 años y con IMC ≥25 Kg/m2, que asistieron a un Centro Endocrinológico de la Ciudad Autónoma de Buenos Aires. Resultados: Se estudiaron 105 mujeres (50.8±12.2 años) con un IMC medio de 30.0±4.9 Kg/m2. El 57.1% eran hipotiroideas bajo tratamiento con LT4. Según valor de TSH: Grupo A (27.6%) ≥2.5 mUI/L y Grupo B (72.4%) <2.5 mUI/L. A partir del programa de intervención, la mayoría descendió menos del 1% semanal con respecto a su peso inicial (51.7% grupo A y 56.6% Grupo B), con un grado de adherencia al tratamiento entre el 60 y 70% (51.7% grupo A y 50.1% Grupo B) sin observarse diferencias significativas entre los grupos. No se encontró asociación entre la pérdida de peso semanal con la función tiroidea (OR:0.64; IC:0.29-1.39; p:0.32) ni con valor de TSH (OR:1.21; IC:0.51-2.86; p:0.66). Se encontró correlación directa entre la pérdida de peso semanal y el grado de adherencia al tratamiento (r:0.464; p: 0.000) e inversa con el tiempo transcurrido hasta el primer control (r:-0.30; p: 0.002) y rangos del IMC (r: -0.282; p: 0.004), independientemente de la edad, la función tiroidea o el valor de TSH que presentaban las pacientes. Conclusiones: Los valores de TSH y la función tiroidea no influyeron sobre la pérdida de peso semanal. El descenso de peso se relacionó con la adherencia al tratamiento en forma directa independientemente de la edad, la función tiroidea o el valor de TSH.

Low-dose gamma irradiation of food protein increases its allergenicity in a chronic oral challenge.

Few chronic food protein models have described the relationship between allergenicity and the molecular structure of food protein after physical processing. The effect of γ-radiation on the structure of food protein was measured by fluorescence, circular dichroism and microcalorimetry. BALB/c mice were intraperitoneally sensitized and then given non-irradiated and irradiated Con-A by daily gavage for 28days. The tendency to form insoluble amorphous aggregates and partially unfolded species was observed after irradiation. The administration of non-irradiated and irradiated samples at low-dose significantly increased weight loss as well as plasma levels of eotaxin in animals repeatedly exposed to Con-A. Significant lymphocytic infiltrate filling completely the stroma of microvilli and tubular glands was observed in the small intestinal of the group given Con-A irradiated at a low dose. This phenotype was not observed in animals treated with Con-A irradiated at a high dose.

Coagulant and antibacterial activities of the water-soluble seed lectin from Moringa oleifera.

AIMS: The aim of this work was to analyse the coagulant and antibacterial activities of lectin isolated from Moringa oleifera seeds that are used for water treatment. METHODS AND RESULTS: The water-soluble M. oleifera lectin (WSMoL) was separated from nonhemagglutinating components (NHC) by chitin chromatography. WSMoL fluorescence spectrum was not altered in the presence of ions that are often present in high concentrations in polluted waters. Seed extract, NHC and WSMoL showed coagulant activity on a turbid water model. Both NHC and WSMoL reduced the growth of Staphylococcus aureus, but only WSMoL caused a reduction in Escherichia coli. WSMoL was also more effective in reducing the growth of ambient lake water bacteria. CONCLUSIONS: Data obtained from this study indicate that WSMoL is a potential natural biocoagulant for water, reducing turbidity, suspended solids and bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Moringa oleifera seeds are a material effective in the treatment of water.

Effect of plant neutrophil elastase inhibitor on leucocyte migration, adhesion and cytokine release in inflammatory conditions.

BACKGROUND AND PURPOSE: The serine and cysteine peptidase inhibitor, BbCI, isolated from Bauhinia bauhinioides seeds, is similar to the classical plant Kunitz inhibitor, STI, but lacks disulphide bridges and methionine residues. BbCI blocks activity of the serine peptidases, elastase (K(iapp) 5.3 nM) and cathepsin G (K(iapp) 160.0 nM), and the cysteine peptidase cathepsin L (K(iapp) 0.2 nM). These three peptidases play important roles in the inflammatory process. EXPERIMENTAL APPROACH: We measured the effects of BbCI on paw oedema and on leucocyte accumulation in pleurisy, both induced by carrageenan. Leucocyte-endothelial cell interactions in scrotal microvasculature in Wistar rats were investigated using intravital microscopy. Cytokine levels in pleural exudate and serum were measured by elisa. KEY RESULTS: Pretreatment of the animals with BbCI (2.5 mg·kg(-1)), 30 min before carrageenan-induced inflammation, effectively reduced paw oedema and bradykinin release, neutrophil migration into the pleural cavity. The number of rolling, adhered and migrated leucocytes at the spermatic fascia microcirculation following carrageenan injection into the scrotum were reduced by BbCI pretreatment. Furthermore, levels of the rat chemokine cytokine-induced neutrophil chemo-attractant-1 were significantly reduced in both pleural exudates and serum from animals pretreated with BbCI. Levels of interleukin-1ß or tumour necrosis factor-α, however, did not change. CONCLUSIONS AND IMPLICATIONS: Taken together, our data suggest that the anti-inflammatory properties of BbCI may be useful in investigations of other pathological processes in which human neutrophil elastase, cathepsin G and cathepsin L play important roles.

Effect of plant neutrophil elastase inhibitor on leucocyte migration, adhesion and cytokine release in inflammatory conditions

The serine and cysteine peptidase inhibitor, BbCI, isolated from Bauhinia bauhinioides seeds, is similar to the classical plant Kunitz inhibitor, STI, but lacks disulphide bridges and methionine residues. BbCI blocks activity of the serine peptidases, elastase (Kiapp 5.3 nM) and cathepsin G (Kiapp 160.0 nM), and the cysteine peptidase cathepsin L (Kiapp 0.2 nM). These three peptidases play important roles in the inflammatory process. We measured the effects of BbCI on paw oedema and on leucocyte accumulation in pleurisy, both induced by carrageenan. Leucocyte–endothelial cell interactions in scrotal microvasculature in Wistar rats were investigated using intravital microscopy. Cytokine levels in pleural exudate and serum were measured by elisa.Pretreatment of the animals with BbCI (2.5 mg·kg−1), 30 min before carrageenan-induced inflammation, effectively reduced paw oedema and bradykinin release, neutrophil migration into the pleural cavity. The number of rolling, adhered and migrated leucocytes at the spermatic fascia microcirculation following carrageenan injection into the scrotum were reduced by BbCI pretreatment. Furthermore, levels of the rat chemokine cytokine-induced neutrophil chemo-attractant-1 were significantly reduced in both pleural exudates and serum from animals pretreated with BbCI. Levels of interleukin-1â or tumour necrosis factor-á, however, did not change.Taken together, our data suggest that the anti-inflammatory properties of BbCI may be useful in investigations of other pathological processes in which human neutrophil elastase, cathepsin G and cathepsin L play important roles.

Perfil lipídico y riesgo cardiovascular en mujeres pre y postmenopáusicas / Lipid profile and cardiovascular risk in pre and postmenopausal women

Objetivo: Describir el perfil lipídico de las mujeres pre y postmenopáusicas y estimar el riesgo cardiovascular (RCV) por el Score de Framingham (SF) y circunferencia de la cintura (CC) en ambas etapas biológicas. (pre y postmenopausicas). Metodología: Muestra por conveniencia de mujeres de 40 a 65 años que concurrieron por primera vez a la consulta nutricional, de enero de 2005 a junio de 2006. Variables: Perfil lipídico (COLT>200, HDL<50, LDL>100 y TG>150), Índice aterogénico (IA)>4,5, Categorías de RCV según Score de Framingham (I, II y III). Se realizó medición directa de Peso, Talla y CC (>80 cm y >88 cm), según técnicas estandarizadas. El análisis estadístico se realizó con el programa Epi 6.04d, con intervalo de confianza (IC) del 95%. Se calculó la significación estadística por Chi2 para las variables correspondientes con valor p<0,05. Resultados: Se incluyeron 216 mujeres, 34,7% premenopáusicas (PM) con edad promedio de 47 ± 4,6 años y 65,3 % postmenopáusicas (PoM) con edad promedio de 57 ± 4,7 años. La edad promedio de menopausia fue de 48 ± 5 años. El 34,6% de PM (IC: 24,5-45,9) presentó IMC = 27,3 y el 70,6% (IC: 59,6-80,1) una CC >80 cm. El 56% de PoM (IC: 47,7-64,0) presentó IMC = 27,3 y 80,8 % (IC: 73,7- 86,7) CC > 80cm. El 100% de la muestra se encontró en Categoría III de RCV según SF. La mayor prevalencia de parámetros lipídicos alterados se observó en el Colesterol total y LDL-C, sin diferencias significativas al comparar ambas etapas biológicas. Conclusiones: Mientras por medición de CC la mayoría de la muestra presenta riesgo aumentado, por determinación del SF, la totalidad presentó bajo riesgo cardiovascular proyectado a 10 años.

Perfil lipídico y riesgo cardiovascular en mujeres pre y postmenopáusicas / Lipid profile and cardiovascular risk in pre and postmenopausal women

Objetivo: Describir el perfil lipídico de las mujeres pre y postmenopáusicas y estimar el riesgo cardiovascular (RCV) por el Score de Framingham (SF) y circunferencia de la cintura (CC) en ambas etapas biológicas. (pre y postmenopausicas). Metodología: Muestra por conveniencia de mujeres de 40 a 65 años que concurrieron por primera vez a la consulta nutricional, de enero de 2005 a junio de 2006. Variables: Perfil lipídico (COLT>200, HDL<50, LDL>100 y TG>150), Indice aterogénico (IA)>4,5, Categorías de RCV según Score de Framingham (I, II y III). Se realizó medición directa de Peso, Talla y CC (>80 cm y >88 cm), según técnicas estandarizadas. El análisis estadístico se realizó con el programa Epi 6.04d, con intervalo de confianza (IC) del 95%. Se calculó la significación estadística por Chi2 para las variables correspondientes con valor p<0,05. Resultados: Se incluyeron 216 mujeres, 34,7% premenopáusicas (PM) con edad promedio de 47 ± 4,6 años y 65,3 % postmenopáusicas (PoM) con edad promedio de 57 ± 4,7 años. La edad promedio de menopausia fue de 48 ± 5 años. El 34,6% de PM (IC: 24,5-45,9) presentó IMC = 27,3 y el 70,6% (IC: 59,6-80,1) una CC >80 cm. El 56% de PoM (IC: 47,7-64,0) presentó IMC = 27,3 y 80,8 % (IC: 73,7- 86,7) CC > 80cm. El 100% de la muestra se encontró en Categoría III de RCV según SF. La mayor prevalencia de parámetros lipídicos alterados se observó en el Colesterol total y LDL-C, sin diferencias significativas al comparar ambas etapas biológicas. Conclusiones: Mientras por medición de CC la mayoría de la muestra presenta riesgo aumentado, por determinación del SF, la totalidad presentó bajo riesgo cardiovascular proyectado a 10 años.(AU)

Purification and primary structure determination of two Bowman-Birk type trypsin isoinhibitors from Cratylia mollis seeds.

Two Bowman-Birk type trypsin inhibitors (CmTI(1) and CmTI(2)) were purified from Cratylia mollis seeds by acetone precipitation, ion exchange, gel filtration and reverse-phase chromatography. CmTI(1) and CmTI(2), with 77 and 78 amino acid residues, respectively, were sequenced in their entirety and show a high structural similarity to Bowman-Birk inhibitors from other Leguminosae. The putative reactive sites of CmTI(1) are a lysine residue at position 22 and a tyrosine residue at position 49. Different reactive sites, as identified by their alignment with related inhibitors, were found for CmTI(2): lysine at position 22 and leucine at position 49. The dissociation constant K(i) of the complex with trypsin is 1.4 nM. The apparent molecular mass is 17 kDa without DDT and 11 kDa with reducing agent and heating.

Insularinase A, a prothrombin activator from Bothrops insularis venom, is a metalloprotease derived from a gene encoding protease and disintegrin domains

The first low-molecular-mass metalloprotease presenting prothrombin activating activity was purified from Bothrops insularis venom and named insularinase A. It is a single-chain protease with a molecular mass of 22 639 Da. cDNA sequence analysis revealed that the disintegrin domain of the precursor protein is post-translationally processed, producing the mature insularinase A. Analysis of its deduced amino acid sequence showed a high similarity with several fibrin(ogen)olytic metalloproteases and only a moderate similarity with prothrombin activators. However, SDS-PAGE of prothrombin after activation by insularinase A showed fragment patterns similar to those generated by group A prothrombin activators, which convert prothrombin into meizothrombin independently of the prothrombinase complex. In addition, insularinase A activates factor X and hydrolyses fibrinogen and fibrin. Chelating agents fully inhibit all insularinase A activities. Insularinase A induced neither detachment nor apoptosis of human endothelial cells and was also not able to trigger an endothelial proinflammatory cell response. Nitric oxide and prostacyclin levels released by endothelial cells were significantly increased after treatment with insularinase A. Our results show that, although its primary structure is related to class P-I fibrin(ogen)olytic metalloproteases, insularinase A is functionally similar to group A prothrombin activators.

Purification and preliminary characterization of a plasma kallikrein inhibitor isolated from sea hares Aplysia dactylomela Rang, 1828.

An inhibitor active against pancreatic trypsin was found in the crude extract from the sea hares Aplysia dactylomelaRang, 1828. A stronger inhibitory activity against human plasma kallikrein was detectable after treating this extract at 60 degrees C, for 30 min. The plasma kallikrein inhibitor (AdKI) purification was achieved by acetone fractionation (80%) v/v, ion-exchange chromatography on Mono Q column and gel filtration chromatography on Superdex 75 column (FPLC system). By the latter a molecular mass of 2900 Da was estimated. The purified inhibitor strongly inhibits human plasma kallikrein with a K(i) value of 2.2 x 10(-10)M, while human plasmin and pancreatic trypsin were inhibited with K(i) values of 1.8 x 10(-9) and 4.7 x 10(-9)M, respectively. Chymotrypsin, pancreatic elastase, pancreatic kallikrein and thrombin are not inhibited. The effect of AdKI on plasma kallikrein was confirmed by the prolongation of activated partial thromboplastin time, using a clotting time assay. The inhibitor did not affect prothrombin time or thrombin time. AdKi is a more specific inhibitor than other serine proteinase inhibitors from marine invertebrates.

Kinetic characterization of factor Xa binding using a quenched fluorescent substrate based on the reactive site of factor Xa inhibitor from Bauhinia ungulata seeds.

The specific Kunitz Bauhinia ungulata factor Xa inhibitor (BuXI) and the Bauhinia variegata trypsin inhibitor (BvTI) blocked the activity of trypsin, chymotrypsin, plasmin, plasma kallikrein and factor XIIa, and factor Xa inhibition was achieved only by BuXI (K(i) 14 nM). BuXI and BvTI are highly homologous (70%). The major differences are the methionine residues at BuXI reactive site, which are involved in the inhibition, since the oxidized protein no longer inhibits factor Xa but maintains the trypsin inhibition. Quenched fluorescent substrates based on the reactive site sequence of the inhibitors were synthesized and the kinetic parameters of the hydrolysis were determined using factor Xa and trypsin. The catalytic efficiency k(cat)/K(m) 4.3 x 10(7) M(-1)sec(>-1) for Abz-VMIAALPRTMFIQ-EDDnp (lead peptide) hydrolysis by factor Xa was 10(4)-fold higher than that of Boc-Ile-Glu-Gly-Arg-AMC, widely used as factor Xa substrate. Lengthening of the substrate changed its susceptibility to factor Xa hydrolysis. Both methionine residues in the substrate influence the binding to factor Xa. Serine replacement of threonine (P(1)') decreases the catalytic efficiency by four orders of magnitude. Factor Xa did not hydrolyze the substrate containing the reactive site sequence of BvTI, that inhibits trypsin inhibitor but not factor Xa. Abz-VMIAALPRTMFIQ-EDDnp prolonged both the prothrombin time and the activated partial thromboplastin time, and the other modified substrates used in this experiment altered blood-clotting assays.

Isolation of a trypsin inhibitor from Echinodorus paniculatus seeds by affinity chromatography on immobilized Cratylia mollis isolectins.

A highly purified trypsin inhibitor was obtained from Echinodorus paniculatus when an extract prepared from E. paniculatus seed flour (25 gl(-1), with 0.1 M ammonium acetate buffer, pH 8.3, under agitation for 6 min at 28 degrees C) was chromatographed on Sephadex G-25 (12 mlh(-1)), followed by affinity chromatography on immobilized Cratylia mollis isolectins (Cra Iso 1,2,3-Sepharose). The column chromatography was performed at 24 degrees C; the matrix was washed (30 mlh(-1)) with 0.1 M sodium phosphate buffer, pH 7.4 or with the same buffer containing 0.2 M glucose, followed by application of inhibitor sample and elution with 0.015 M sodium borate buffer, pH 7.4, or 1.0 M NaCl. A purified fraction of inhibitor was obtained by gel filtration chromatography (GF-450/HPLC column). Trypsin inhibitory activity was eliminated when the inhibitor was treated with metaperiodate showing that the carbohydrate moiety was important for trypsin inhibition. Binding of inhibitor was also evaluated on immobilized concanavalin A (Con A-Sepharose) using previously described chromatographic conditions with results similar to Cra Iso 1,2,3-Sepharose chromatography.

Structure of cruzipain/cruzain inhibitors isolated from Bauhinia bauhinioides seeds.

The saline extract of Bauhinia bauhinioides dry seeds was shown to inhibit cruzipain, a cysteine proteinase from Trypanosoma cruzi. The inhibitory activity was assigned to a protein with 164 amino acid residues and molecular mass of 18 034 Da that was purified by chromatography on DEAE-Sephadex, trypsin-Sepharose (removal of trypsin inhibitors), Mono Q and a reversed-phase C4 column. The primary structure is homologous to other plant Kunitz-type inhibitors, but it lacks cysteine residues and therefore the disulfide bridges. No methionine residue was identified by amino acid sequencing. The inhibition of cruzipain fits into a slow-tight binding mechanism with a low dissociation constant (Ki 1.2 nM). The studied Bauhinia protein also inhibits cruzain (Ki 0.3 nM), a C-terminally truncated recombinant species of cruzipain. Cathepsin L, a cysteine proteinase with high homology to cruzipain, is also inhibited (Ki 0.22 nM), but not cathepsin B, papain, bromelain or ficin.

In vivo characterization of Lopap, a prothrombin activator serine protease from the Lonomia obliqua caterpillar venom.

Increasing occurrence of hemorrhagic syndrome in man, caused by contact with Lonomia obliqua caterpillars, has been reported in Southern Brazil in the past 10 years. The L. obliqua venom causes a severe consumptive coagulopathy, which can lead to hemorrhagic syndrome. L. obliqua prothrombin activator protease (Lopap) is a 69-kDa prothrombin activator serine protease isolated from L. obliqua caterpillar bristle extract, which is able to evoke thrombus formation, unclottable blood, and fibrinogen depletion when injected into the blood stream of rats. The purified protein generated thrombin from prothrombin, able to clot purified human fibrinogen and plasma. A decrease in platelet count and inhibition of collagen-induced platelet aggregation were observed, as well as leukocyte infiltration in the lungs. In addition, we observed congestion and hemorrhage in renal glomeruli and necrosis in renal distal tubules. These data support the hypothesis that Lopap contributes to the clinical syndrome caused by human contact with L. obliqua, most probably through prothrombin activation, resulting in a consumption coagulopathy.

Primary sequence determination of a Kunitz inhibitor isolated from Delonix regia seeds.

A serine proteinase inhibitor was purified from Delonix regia seeds a Leguminosae tree of the Caesalpinioideae subfamily. The inhibitor named DrTI, inactivated trypsin and human plasma kallikrein with K(i )values 2.19x10(-8) M and 5.25 nM, respectively. Its analysis by SDS-PAGE 10-20% showed that the inhibitor is a protein with a single polypeptide chain of M(r) 22 h Da. The primary sequence of the inhibitor was determined by Edman degradation, thus indicating that it contained 185 amino acids and showed that it belongs to the Kunitz type family; however, its reactive site did not contain Arg or Lys at the putative reactive site (position 63, SbTI numbering) or it was displaced when compared to other Kunitz-type inhibitors.

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.

Synthetic peptides and fluorogenic substrates related to the reactive site sequence of Kunitz-type inhibitors isolated from Bauhinia: interaction with human plasma kallikrein.

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.