Orientador: Hélia Harumi Sato / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-27T13:38:26Z (GMT). No. of bitstreams: 1 Castro_RuannJanserSoaresde_D.pdf: 3791426 bytes, checksum: 08b166335935f4a40427f1ddea405399 (MD5) Previous issue date: 2015 / Resumo: O presente trabalho teve como objetivo utilizar a técnica de delineamento experimental de misturas como estratégia para a produção de proteases por Aspergillus niger LBA02 em fermentação semissólida utilizando formulações contendo diferentes resíduos agroindustriais e produção de hidrolisados proteicos com atividades biológicas e funcionais utilizando a hidrólise enzimática simultânea de proteínas de diferentes fontes. Efeitos sinérgicos e significativos entre as misturas quaternárias de farelo de trigo, farelo de soja, farelo de algodão e casca de laranja foram observados durante a fermentação de A. niger LBA02, atingindo aumentos de 33,7, 7,6, 30,8 e 581,7%, respectivamente, na produção de proteases em comparação com os substratos utilizados de forma isolada. O estudo das características bioquímicas das preparações enzimáticas mostrou que a linhagem de A. niger LBA02 foi capaz de secretar diferentes tipos de proteases em resposta a cada substrato. De um modo geral, as proteases apresentaram atividade ótima a 50 °C e na faixa de pH de 3 a 4. As maiores diferenças entre as preparações de proteases foram observadas para os parâmetros cinéticos e termodinâmicos de ativação e inativação térmica. Na hidrólise enzimática de misturas contendo proteína isolada de soja, proteínas do soro de leite e da clara de ovo utilizando a preparação comercial Flavourzyme® 500L foram observados efeitos sinérgicos entre as formulações contendo misturas binárias ou ternárias, para vários parâmetros. Para atividade antioxidante determinada pelo método DPPH, a mistura contendo proteínas do soro de leite e proteínas da clara de ovo apresentaram aumentos de 45,1 e 37,3% na atividade, quando comparada aos hidrolisados obtidos com as duas fontes de forma isolada, respectivamente. Entre as propriedades funcionais, a capacidade emulsificante foi a que apresentou maior efeito sinérgico, onde os hidrolisados contendo a mistura ternária de proteína isolada de soja, proteínas do soro de leite e da clara de ovo, alcançaram valores 2 a 12 vezes superiores, em relação aos hidrolisados obtidos de forma isolada. A determinação da atividade anti-adipogênica dos hidrolisados revelou que o tratamento de células pré-adipócitas 3T3-L1 com a mistura binária de proteínas do soro de leite e da clara de ovo na concentração de 1.200 ppm reduziu o acúmulo relativo de lipídeos nas células em até 47,9%. Em relação à atividade antimicrobiana, a linhagem de Staphylococcus aureus ATCC 6538 foi a única que apresentou inibição do crescimento quando cultivada em meio suplementado com uma mistura binária de proteína isolada de soja e proteínas da clara de ovo não hidrolisadas, resultando em inibição de 16,82%. Os hidrolisados obtidos com misturas binárias de proteínas do soro de leite e da clara de ovo ou proteína isolada de soja e soro de leite estimularam o crescimento de bactérias lácticas e probióticas, resultando em aumentos de 29,4 a 100% comparados aos meios não suplementados. A utilização de composições contendo diferentes preparações comerciais de proteases para hidrólise de proteína isolada de soja e estudo da atividade antioxidante mostrou diferentes resultados para cada método utilizado. Para inibição dos radicais DPPH, os hidrolisados obtidos com Flavourzyme® 500L combinada com Alcalase® 2.4L mostraram o maior efeito sinérgico, com aumentos de 10,9 e 13,2% da atividade antioxidante, em comparação aos hidrolisados produzidos com as enzimas isoladas. Os hidrolisados obtidos utilizando a mistura ternária de Flavourzyme® 500L, Alcalase® 2.4L e YeastMax® A apresentaram o maior poder de inibição da auto-oxidação do ácido linoleico / Abstract: This study aimed to use the mixture experimental design technique as a strategy to produce proteases by Aspergillus niger LBA02 under solid state fermentation, using formulations containing different agroindustrial wastes. It also aimed to produce protein hydrolysates with biological and functional activities using the simultaneous enzymatic hydrolysis of proteins from the different sources. Synergistic and significant effects between the quaternary mixtures of wheat bran, soybean meal, cottonseed meal and orange peel were observed during fermentation by A. niger LBA02, reaching increases of 33.7, 7.6, 30.8 and 581.7%, respectively, for the production of proteases as compared to the isolated substrates. The study of the biochemical properties of the enzyme preparations showed that the strain of A. niger LBA02 was able to secrete different types of proteases in response to each substrate. In general, the proteases showed optimal activity at 50 °C in the pH range from 3 to 4. The major differences between the protease preparations were observed for the kinetic and thermodynamic parameters of thermal activation and inactivation. In the enzymatic hydrolysis of mixtures containing soy protein isolate, bovine whey protein and egg white protein using the commercial preparation FlavourzymeTM 500L, synergistic effects were observed for various parameters between formulations containing binary or ternary mixtures,. For antioxidant activity as determined by the DPPH assay, the mixture containing bovine whey protein and egg white protein showed increases of 45.1 and 37.3% in their activities as compared to hydrolysates obtained with the isolated proteins, respectively. Of the functional properties, the emulsifying capacity showed the greatest synergistic effect, the hydrolysates containing the ternary mixture of soy protein isolate, bovine whey protein and egg white protein, showing increases ranging from 2 to 12-fold as compared to the hydrolysates obtained using isolated substrates. The determination of the anti-adipogenic activity of the hydrolysates indicated that the treatment of 3T3-L1 preadipocyte cells with 1200 ppm of the mixture containing bovine whey protein and egg white protein reduced the relative lipid accumulation to 47.9%. With respect to antimicrobial activity, the strain of Staphylococcus aureus ATCC 6538 was the only one which showed growth inhibition when cultivated in a medium supplemented with a non-hydrolyzed binary mixture of soy protein isolate and egg white protein, resulting in inhibition of 16.82%. The hydrolysates obtained with binary mixtures of bovine whey protein and egg white protein or soy protein isolate and bovine whey protein stimulated the growth of probiotic and lactic acid bacteria, reaching increases from 29.4 to 100% when compared to non-supplemented media. The use of formulations containing various commercial preparations of proteases for the hydrolysis of soy protein isolate and the study of antioxidant activities showed different results for each method. For DPPH radical scavenging, the hydrolysates obtained with FlavourzymeTM 500L combined with AlcalaseTM 2.4L showed greater synergistic effects, with increases of 10.9 and 13.2% in antioxidant activity as compared to the hydrolysates produced with individual enzymes. The hydrolysates obtained from ternary mixtures of FlavourzymeTM 500L, AlcalaseTM 2.4L and YeastMaxTM A showed the greatest power of inhibition of linoleic acid autoxidation / Doutorado / Ciência de Alimentos / Doutor em Ciência de Alimentos

O envenenamento ofídico é considerado uma condição tropical negligenciada pela OMS, e no Brasil, o gênero Bothrops está envolvido na maioria dos casos. Primeiramente, estudamos a atividade hidrolítica do veneno de B. jararaca sobre peptídeos biologicamente ativos que podem estar relacionadas com o envenenamento. A hidrólise dos peptídeos que foram substratos para as serinopeptidases não foi eficientemente bloqueada pelo soro antibotrópico produzido pelo Instituto Butantan e, portanto, as causas dessas falhas foram investigadas. Para isso, purificamos quatro serinopeptidases não bloqueadas pelo soro e, por estudos imunoquímicos, observamos que apesar deste não bloquear as atividades destas enzimas, o mesmo é capaz de reconhecê-las. Portanto, decidimos obter soros experimentais contra estas moléculas utilizando camundongos, a fim de compará-los com o soro comercial. Os soros experimentais contra as serinopeptidases mostraram capacidade de reconhecimento e alta afinidade contra elas, e mais importante, capacidade de neutralizar suas atividades in vitro. / Snakebite is considered a neglected tropical condition by WHO, and in Brazil, the Bothrops genus is involved in most of the cases. Initially, we have studied the B. jararaca venom activity over bioactive peptides which could be related with the envenomation. The hydrolysis of the peptides substrate for serinepeptidases were not efficiently blocked by the Butantan Institute bothropic antivenom, therefore, the causes of this flaw were investigated. Thereafter, we purified four serinepeptidases not blocked by the antivenom and, by immunochemistry analysis, we observed that although it could not neutralize the activity, it could well recognize these proteins. Thus, we decided to obtain experimental sera against these serinepeptidases in mice, in order to compare it with the commercial antivenom. The experimental sera against these enzymes demonstrated recognition capability and high affinity, and most important, the ability to neutralize their activity in vitro.

La mosca mediterránea de la fruta, Ceratitis capitata, es la principal plaga de la fruticultura en el mundo. El desarrollo de métodos ambientalmente seguros de control de plagas, como Bacillus thuringiensis (Bt), adquiere cada vez mayor importancia. Esta tesis doctoral evalúa la validez de Bt y sus delta-endotoxinas como agentes de control de C. capitata. El análisis de la biodiversidad de Bt ha reflejado la gran riqueza presente en el agroecosistema de los cítricos. Sin embargo, ninguna de las cepas ensayadas (905) muestra alta toxicidad sobre C. capitata cuando esporas/cristales o sobrenadantes de su cultivo son ensayados. En cambio, la solubilización de los cristales de una selección de 42 cepas de Bacillus sp. ha demostrado que, para las cepas de la subespecie israelensis (Bti), este tratamiento produce una ganancia de función biológica. Adicionalmente, la predigestión de dichas protoxinas con proteasas de otro díptero, Culex pipiens, aumenta su actividad larvicida (CL50 31.26 µg/cm2). Por otro lado, se ha demostrado que, entre las delta-endotoxinas producidas por Bti, la protoxina Cyt1Aa es el factor determinante, con una CL50 sobre larvas de 4.93 µg/cm2. Sobre adultos, Cyt1Aa produce efectos subletales. Complementariamente, esta tesis propone un nuevo método de control basado en el desarrollo de toxinas recombinantes de fusión Cry-anticuerpo. Se ha puesto en práctica un sistema modelo para evaluar esta estrategia: se han desarrollado 4 variantes proteicas por la fusión entre partes de la protoxina Cry1Ab y un anticuerpo específico contra GFP (VHH anti-GFP) y éstas se han ensayado sobre larvas transgénicas de Drosophila melanogaster que expresan GFP en su intestino. Deficiencias en la unión de las toxinas de fusión a GFP, han impedido demostrar, por el momento, la estrategia propuesta. Por último, se han detectado al menos 160 proteínas distintas en las membranas intestinales de C. capitata y se han identificado las siguientes: V-ATPasa subunidades A y B, y alfa-tubulina. / Vidal Quist, JC. (2010). Estrategias para la utilización de la bacteria entomopatógena Bacillus thuringiensis (Berliner) en el control de Ceratitis capitata (Wiedemann) [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/8336 / Palancia

Visceral adipose tissue derived serine protease inhibitor (vaspin) or SERPINA12 according to the serpin nomenclature was identified together with other genes and gene products that  were specifically expressed or overexpressed in the intra abdominal or visceral adipose tissue  (AT) of the Otsuka Long-Evans Tokushima fatty rat. These rats spontaneously develop visceral  obesity, insulin resistance, hyperinsulinemia and ‐glycemia, as well as hypertension and thus represent a well suited animal model of obesity and related metabolic disorders such as type  2 diabetes.  The follow-up study reporting the cloning, expression and functional characterization of  vaspin suggested the great and promising potential of this molecule to counteract obesity induced insulin resistance and inflammation and has since initiated over 300 publications, clinical and experimental, that have contributed to uncover the multifaceted functions and molecular mechanisms of vaspin action not only in the adipose, but in many different cells, tissues and organs. This review will give an update on mechanistic and structural aspects of vaspin with a focus on its serpin function, the physiology and regulation of vaspin expression, and will summarize the latest on vaspin function in various tissues such as the different adipose tissue depots as well as the vasculature, skin, bone and the brain.

and keywords Abstract and keywords The ubiquitin-proteasome system (UPS) is a tightly and specifically regulated system of protein degradation in eukaryotic cells. Inhibition of an UPS component might represent a strategy to control human diseases, including cancer. Modulation of the UPS can also be employed in basic research strategies. This thesis deals with two independent yet methodologically connected research aims - first, to search for the target of the newly identified UPS inhibitor CBU79, and second, to develop a fluorescent cell-based reporter exploiting proteasomal degradation. In the first part of my work, previous findings regarding the molecular mechanisms of CBU79 inhibiton on the UPS were confirmed. In the next step, I characterized how the UPS inhibitor CBU79 affects protein synthesis using the metabolic labelling of proteins based on click chemistry. I also examined the cytotoxic effect of CBU79 treatment on different cell lines. Finally, I performed a CRISPR/Cas9 whole-genome enrichment screen with the aim to find a potential target of the inhibitor. I found out that CBU79 probably decreases levels of protein synthesis by triggering cellular signalling via the unfolded protein response (UPR). Using the screen, I found 22 potential targets of the CBU79 inhibitor that will be...

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<p>Molecular interactions lie at the heart of myriad biological processes. Knowledge of molecular recognition processes and the ability to model and predict interactions of any biological molecule to any chemical compound are the key for better understanding of cell functions and discovery of more efficacious medicines.</p><p>This thesis presents contributions to the development of a novel chemo-bioinformatics approach called proteochemometrics; a general method for interaction space analysis of biological macromolecules and their ligands. In this work we explore proteochemometrics-based interaction models over broad groups of protein families, evaluate their validity and scope, and compare proteochemometrics to traditional modeling approaches.</p><p>Through the proteochemometric analysis of large interaction data sets of multiple retroviral proteases from various viral species we investigate complex mechanisms of drug resistance in HIV-1 and discover general physicochemical determinants of substrate cleavage efficiency and binding in retroviral proteases. We further demonstrate how global proteochemometric models can be used for design of protease inhibitors with broad activity on drug-resistant viral mutants, for monitoring drug resistance mechanisms in the physicochemical sense and prediction of potential HIV-1 evolution trajectories. We provide novel insights into the complexity of HIV-1 protease specificity by constructing a generalized IF-THEN rule model based on bioinformatics analysis of the largest set of HIV-1 protease substrates and non-substrates.</p><p>We discuss how proteochemometrics can be used to map recognition sites of entire protein families in great detail and demonstrate how it can incorporate target variability into drug discovery process. Finally, we assess the utility of the proteochemometric approach in evaluation of ADMET properties of drug candidates with a special focus on inhibition of cytochrome P450 enzymes and investigate application of the approach in the pharmacogenomics field.</p>

L’identification de marqueurs pronostiques et prédictifs du cancer du sein est un facteur important pour une meilleure compréhension du processus évolutif et le développement de thérapies ciblées. Les récepteurs des oestrogènes (RE) représentent ainsi à la fois un marqueur pronostique mais aussi prédictif du traitement par le tamoxifène ou les anti-aromatases. Cependant, un certain nombre de patientes vont évoluer en dépit de traitements anti-hormonaux adaptés. L’objectif de notre travail, a été d’évaluer la méthode de mesure des RE, l’apport des protéases dans la distinction de profils tumoraux pronostiques et prédictifs. Nous avons démontré l’influence du mode de mesure des RE et en particulier de l’expression quantitative sur l’interprétation pronostique et sur une meilleure détermination du bénéfice du traitement en fonction du niveau d’expression des RE. Nous avons montré l’intérêt de l’évaluation des protéases tissulaires uPA, PAI-I et cathépsine-D, pour caractériser l’hétérogénéité des tumeurs en complément des RE. Particulièrement, chez les patientes RE+, des taux élevés de cathépsine-D et de PAI-1 étaient un indicateur de mauvais pronostic. Nous avons développé un nomogramme combinant RE et le statut ganglionnaire à 3 types de protéases : PAI-1, cathépsine-D et la thymidine kinase, pour déterminer la probabilité de survie à 2 et 5 ans. De plus, ces protéases évaluées dans les tumeurs infectées par l’Epstein-Barr virus (EBV), témoignaient de tumeurs biologiquement agressives avec des taux plus élevés de thymidine kinase. Notre travail a contribué à améliorer l’identification de profils des tumeurs en fonction des RE et des protéases et de caractériser les tumeurs viro-induites. / The identification of prognostic and predictive markers is important for a better understanding of the evolutionary process and the development of targeted therapies. Thus estrogen receptors (ER) represent both an important prognostic marker but also predictive of therapies using tamoxifen or aromatase inhibitors. However, a number of patients will evolve despite hormonotherapy. The objective of our work was to evaluate the method for measuring ER, the contribution of proteases in the distinction of prognostic and predictive tumor profiles. In our work, we demonstrated the influence of the mode of measure of ER and in particular its quantitative expression on the prognostic interpretation and a better determination of benefit of treatment depending on the level of expression of ER. We show the interest of the evaluation of tissue proteases uPA, PAI-I and cathepsin-D, to characterize the heterogeneity of tumors in addition to ER. Specifically, in ER + patients, high levels of cathepsin-D and PAI-1 are an indicator of poor prognosis. We developed a nomogram combining ER and nodal status, to 3 types of proteases: PAI-1, cathepsin-D and thymidine kinase, to determine the probability of survival at 2 and 5 years. In addition, these proteases are evaluated in tumors infected with the Epstein-Barr virus (EBV) and shows high rates of thymidine kinase in EBV + BC, reflecting biologically aggressive tumors. Our work has helped to improve the identification of profiles of tumors according to ER and proteases and characterize virus-associated tumors.

Résumé: Chaque année, les épidémies saisonnières d’influenza causent de 3 à 5 millions de cas sévères de maladie, entraînant entre 250 000 et 500 000 décès mondialement. Seulement deux classes d’antiviraux sont actuellement commercialisées pour traiter cette infection respiratoire : les inhibiteurs de la neuraminidase, tels que l’oseltamivir (Tamiflu) et les inhibiteurs du canal ionique M2 (adamantanes). Toutefois, leur utilisation est limitée par l’apparition rapide de résistance virale. Il est donc d’un grand intérêt de développer de nouvelles stratégies thérapeutiques pour le traitement de l’influenza. Le virus influenza dépend de l’activation de sa protéine de surface hémagglutinine (HA) pour être infectieux. L’activation a lieu par clivage protéolytique au sein d’une séquence d’acides aminés conservée. Ce clivage doit être effectué par une enzyme de l’hôte, étant donné que le génome du virus ne code pour aucune protéase. Pour les virus infectant l’humain, plusieurs études ont montré le potentiel de protéases à sérine transmembranaires de type II (TTSP) à promouvoir la réplication virale : TMPRSS2, TMPRSS4, HAT, MSPL, Desc1 et matriptase, identifiée récemment par notre équipe (Beaulieu, Gravel et al., 2013), activent l’HA des virus influenza A (principalement H1N1 et H3N2). Toutefois, il existe peu d’information sur le clivage de l’HA des virus influenza B, et seulement TMPRSS2 et HAT ont été identifiées comme étant capables d’activer ce type de virus. Les travaux de ce projet de maîtrise visaient à identifier d’autres TTSP pouvant activer l’HA de l’influenza B. L’efficacité de clivage par la matriptase, hepsine, HAT et Desc1 a été étudiée et comparée entre ces TTSP. Ces quatre protéases s’avèrent capables de cliver l’HA de l’influenza B in vitro. Cependant, seul le clivage par matriptase, hepsine et HAT promeut la réplication virale. De plus, ces TTSP peuvent aussi supporter la réplication de virus influenza A. Ainsi, l’utilisation d’un inhibiteur de TTSP, développé en collaboration avec notre laboratoire, permet de bloquer significativement la réplication virale dans les cellules épithéliales bronchiques humaines Calu-3. Cet inhibiteur se lie de façon covalente et lentement réversible au site actif de la TTSP par un mécanisme slow tight-binding. Puisque cet inhibiteur cible une composante de la cellule hôte, et non une protéine virale, il n’entraîne pas le développement de résistance après 15 passages des virus en présence de l’inhibiteur dans les cellules Calu-3. L’inhibition des TTSP activatrices d’HA dans le système respiratoire humain représente donc une nouvelle stratégie thérapeutique pouvant mener au développement d’antiviraux efficaces contre l’influenza. / Abstract: Seasonal influenza epidemics cause between 3 and 5 millions severe cases of disease, leading to 250 000 to 500 000 deaths worldwide. Only two classes of drugs are currently available to treat influenza infections: neuraminidase inhibitors, such as oseltamivir (Tamiflu) and M2 channel inhibitors (adamantanes). However, the use of these antivirals is restricted by rapid emergence of viral resistance. It is therefore of great interest to develop new therapeutic strategies for the treatment of influenza disease. The influenza virus requires activation of its surface protein hemagglutinin (HA) to become infectious. This activation is achieved by proteolytic cleavage in a highly conserved amino acid sequence of the protein. Host cell proteases are responsible for this cleavage since the viral genome doesn’t encode any protease. For viruses that infect humans, many studies have shown the potential of type II transmembrane serine proteases (TTSP) to promote viral replication: TMPRSS2, TMPRSS4, HAT, MSPL, Desc1 and matriptase, recently identified by our team (Beaulieu, Gravel et al., 2013), activate HA of influenza A viruses (mainly H1N1 and H3N2). However, little is known about cleavage of influenza B virus HA, and only TMPRSS2 and HAT have been identified as being capable of activating this type of virus. This project aimed to identify other TTSPs able to activate influenza B HA. Cleavage efficacies of matriptase, hepsin, HAT and Desc1 were studied and compared. These four proteases were shown to be able to cleave influenza B HA using in vitro assays. However, only cleavage by matriptase, hepsin and HAT promoted viral replication. Moreover, these TTSPs also supported the replication of influenza A viruses. Thus, the use of a slow, tight-binding inhibitor (developed in collaboration with our laboratory) that binds to the TTSP active site, forming a covalent and reversible bond, significantly blocked viral replication in human bronchial epithelial Calu-3 cells. Since this inhibitor targets a host cell component, instead of a viral protein, viruses did not develop resistance after 15 passages in presence of the inhibitor in Calu-3 cells. Thus, inhibition of HA-activating TTSPs in the human respiratory tract represents a novel therapeutic strategy against influenza.

A presença de inibidores no plasma e soro de animais resistentes ao veneno de serpentes, assim como no plasma ou soro de serpentes imunes a seu próprio veneno, é amplamente conhecida. Um grupo destes inibidores são os inibidores de metaloproteases do veneno de serpente (SVMPs), eles têm sido caracterizados do ponto de vista físico químico, porém a falta de informações estruturais e termodinâmicas acerca da sua inibição sobre as SVMPs, tem permanecido como um grande obstáculo para sua aplicação terapêutica ou desenvolvimento como ferramentas moleculares. Por tanto, isolamos um novo inibidor de metaloproteases a partir do soro de C. d. terrificus, por nós denominado crotaini, e realizamos a caracterização cinética de sua interação com a bothropasina, uma das principais metaloproteases do veneno de B. jararaca, e da mesma forma, caracterizamos cineticamente a interação desta metaloprotease com dos inibidores já conhecidos, DM40 e DM43, isolados do soro de D. marsupialis. Determinando as constantes cinéticas mediante cinética enzimática e ressonância plasmônica de superfície (SPR) no Biacore T200. Os valores obtidos permitiram estabelecer que a interação entre o crotaini e a bothropasina, é de alta afinidade, além de evidenciar a sua ligação de forma equimolar e estável. Assim mesmo, o crotaini apresentou uma constante de inibição menor às constantes determinadas para o DM40 e DM43. Adicionalmente, estudos de interação após tratamento quelante da enzima permitem prever que existem diferenças respeito aos domínios estruturais envolvidos na interação do crotaini com a bothropasina, em comparação aos inibidores DM40 e DM43. / The presence of inhibitors in plasma or serum from snake venom - resistant animals is well known. These inhibitors are also present in plasma and serum of snakes that are immune to their own venom. A group of these molecules are snake venom metalloprotease inhibitors (SVMPIs). Although the physicochemical properties of these inhibitors are well established, the lack of structural and thermodynamic information has been a bottleneck for their therapeutic use and development as molecular tools. In this work a new metalloprotease inhibitor was isolated from C. d. terrificus, and named crotaini. The kinetic interaction of crotaini with bothropasin, one of the main metalloproteases from the B. jararaca venom, was investigated. Similarly, the kinetic interactions of crotaini with the inhibitors DM40 and DM43, isolated from D. marsupialis serum, were also studied. The kinetic constants were determined by enzymatic kinetics and surface plasmonic resonance (SPR) in a Biacore T200. The obtained values enabled us to demonstrate the high affinity of the inhibitors toward the metalloprotease, achieved through an equimolar and stable complex formation. In addition, studies of interactions of the metaloprotease bothropasin with its inhibitors in the presence of chelant agents revealed differences between that established with crotaini as compared with those made with DM40 and DM43.