Study of the isoelectric precipitation of porcine insulin in aqueous solutions with carbon dioxide

Orientadores: Everson Alves Miranda, Pedro de Alcantara Pessoa FilhoTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia QuimicaResumo: A técnica de precipitação é freqüentemente utilizada na recuperação de proteínas de soluções aquosas. Uma das formas de se realiza-la é através do processo de precipitação isoelétrica, que consiste no ajuste do pH do meio ao valor em que a proteína tem carga global nula, ou seja, no ponto isoelétrico, pI. Ácidos e bases minerais são normalmente utilizados para ajustar o pH do meio ao pI e provocar a precipitação de proteínas. Contudo, para se obter proteínas de alta pureza e evitar a poluição do meio com resíduos indesejáveis, há a necessidade de um processo adicional para a neutralização da solução e remoção dos sais gerados. Além disso, concentrações locais elevadas dos ácidos podem causar a desnaturação da proteína de interesse. Estudos recentes vêm apontando a utilização de eletrólitos voláteis como uma alternativa promissora aos agentes de precipitação convencionalmente empregados na recuperação de proteínas. Eletrólitos voláteis são obtidos pela dissolução de gases como o dióxido de carbono em solução aquosa; nesta dissolução formam-se íons, cujas concentrações dependem da temperatura e pressão do sistema. Biomoléculas de interesse farmacêutico, como a insulina, por exemplo, podem ser potencialmente recuperadas por processos de precipitação isoelétrica com o CO2. Assim, neste projeto realizou-se um estudo experimental e teórico da precipitação isoelétrica da insulina suína com dióxido de carbono, avaliando-se a influência dos parâmetros de processo como a temperatura, pressão e concentrações de eletrólitos e de proteínas sobre a precipitação. A cinética de variação de pH devida à acidificação das soluções contendo a insulina foi determinada, assim como a cinética de precipitação da proteína. A utilização do bicarbonato de sódio como agente tamponante, em conjunto com o ácido carbônico formado em solução, permitiu que os estudos de equilíbrio fossem realizados sem a necessidade de nenhum componente adicional para o controle do pH do sistema. Dados de solubilidade da insulina suína foram obtidos entre as temperaturas de 5 e 25oC, até a pressão de 16 bar de CO2. Estes dados foram correlacionados por um modelo termodinâmico em que a proteína em solução foi considerada como um eletrólito, o que permitiu uma análise do efeito de forças eletrostáticas sobre a solubilidade da proteínaAbstract: Precipitation is a technique frequently employed in downstream process for the recovery of proteins from aqueous solutions. One example of such technique is the isoelectric precipitation, which consists of the adjustment of solution pH to the value where the protein has zero net charge, the isoelectric point, pI. Commonly, mineral acids and bases are used for pH adjustment and to induce protein precipitation. However, in order to obtain high purity proteins and avoid environmental pollution, an additional process is needed to neutralize and remove residual salts. It is also necessary to take into account the fact that local pH extremes can cause denaturation of the targeted protein. Recent works have pointed the use of volatile electrolytes as promising alternatives to the precipitating agents conventionally utilized in protein recovery. Volatile electrolytes are obtained by dissolution of gases as carbon dioxide in aqueous solutions. In this process, the volatile electrolyte dissociates into ions, whose concentrations depend on system pressure and temperature. Biomolecules of pharmaceutical interest as insulin, for instance, may be potentially recovered by isoelectric precipitation process with carbon dioxide. Thus, in this project an experimental and theoretical study of the isoelectric precipitation of porcine insulin with carbon dioxide was conducted, in order to evaluate the influence of parameters such as temperature, pressure and concentrations of electrolytes and protein over the precipitation process. Kinetics of pH variation due to acidification of the insulin solutions was determined, as well as protein precipitation kinetics. The use of sodium bicarbonate as buffer agent with carbonic acid produced in solution allowed the equilibrium studies be conducted without the need of any additional component to control system pH. Solubility data of porcine insulin were obtained in the temperature range of 5 to 25oC and up to CO2 pressures of 16 bar. These data were correlated by a thermodynamic model in which the protein in solution was considered as an electrolyte, which allowed an analysis of electrostatic force effects over protein solubilityDoutoradoEngenharia de ProcessosDoutor em Engenharia Químic

Cotiarinase is a novel prothrombin activator from the venom of Bothrops cotiara

Snake venom serine proteinases (SVSPs) may affect hemostatic pathways by specifically activating components involved in coagulation, fibrinolysis and platelet aggregation or by unspecific proteolytic degradation. in this study, we purified and characterized an SVSP from Bothrops cotiara venom, named cotiarinase, which generated thrombin upon incubation with prothrombin. Cotiarinase was isolated by a two-step procedure including gel-filtration and cation-exchange chromatographies and showed a single protein band with a molecular mass of 29 kDa by SDS-polyacrylamide gel electrophoresis under reducing conditions. Identification of cotiarinase by mass spectrometric analysis revealed peptides that matched sequences of viperid SVSPs. Cotiarinase did not show fibrinogen-clotting, platelet-aggregating, fibrino-genolytic and factor X activating activities. Upon incubation with prothrombin the generation of thrombin was detected using the peptide substrate D-Phe-Pip-Arg-pNA. Moreover, mass spectrometric identification of prothrombin fragments generated by cotiarinase in the absence of co-factors (phospholipids, factor Va, factor Xa and Ca2+ ions), indicated the limited proteolysis of this protein to release prothrombin 1, fragment 1 and thrombin. Cotiarinase is a novel SVSP that acts on prothrombin to release active thrombin that does not match any group of the current classification of snake venom prothrombin activators. (C) 2013 Elsevier Masson SAS. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Inst Butantan, CAT Cepid, Lab Especial Toxinol Aplicada, BR-05503000 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Ciencias Exatas & Terra, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Ciencias Exatas & Terra, São Paulo, BrazilFAPESP: 98/14307-9FAPESP: 11/08514-8FAPESP: 2011/11308-0Web of Scienc

Supercritical CO2 recovery of caffeine from green coffee oil: new experimental solubility data and modeling

The caffeine solubility in supercritical CO2 was studied by assessing the effects of pressure and temperature on the extraction of green coffee oil (GCO). The Peng-Robinson¹ equation of state was used to correlate the solubility of caffeine with a thermodynamic model and two mixing rules were evaluated: the classical mixing rule of van der Waals with two adjustable parameters (PR-VDW) and a density dependent one, proposed by Mohamed and Holder² with two (PR-MH, two parameters adjusted to the attractive term) and three (PR-MH3 two parameters adjusted to the attractive and one to the repulsive term) adjustable parameters. The best results were obtained with the mixing rule of Mohamed and Holder² with three parameters.13191323Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Proteomic study revealed cellular assembly and lipid metabolism dysregulation in sepsis secondary to community-acquired pneumonia

Sepsis is a life-threatening disorder characterized by organ dysfunction and a major cause of mortality worldwide. The major challenge in studying sepsis is its diversity in such factors as age, source of infection and etiology. Recently, genomic and proteomic approaches have improved our understanding of its complex pathogenesis. In the present study, we use quantitative proteomics to evaluate the host proteome response in septic patients secondary to community-acquired pneumonia (CAP). Samples obtained at admission and after 7 days of follow-up were analyzed according to the outcomes of septic patients. The patients' proteome profiles were compared with age-and gender-matched healthy volunteers. Bioinformatic analyses of differentially expressed proteins showed alteration in the cytoskeleton, cellular assembly, movement, lipid metabolism and immune responses in septic patients. Actin and gelsolin changes were assessed in mononuclear cells using immunofluorescence, and a higher expression of gelsolin and depletion of actin were observed in survivor patients. Regarding lipid metabolism, changes in cholesterol, HDL and apolipoproteins were confirmed using enzymatic colorimetric methods in plasma. Transcriptomic studies revealed a massive change in gene expression in sepsis. Our proteomic results stressed important changes in cellular structure and metabolism, which are possible targets for future interventions of sepsis.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico, CNPqFAPESPUniv Fed Sao Paulo, Hosp Sao Paulo, Div Infect Dis, Escola Paulista Med, BR-04039032 Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Biochem, Escola Paulista Med, BR-04023900 Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Dept Microbiol Immunol & Parasitol, Escola Paulista Med, BR-04023062 Sao Paulo, BrazilUniv Fed Sao Paulo, Intens Care Unit, Hosp Sao Paulo, Escola Paulista Med, BR-04024002 Sao Paulo, BrazilHosp Israelita Albert Einstein, Intens Care Unit, BR-05652900 Sao Paulo, BrazilHosp Sirio Libanes, Intens Care Unit, BR-01409001 Sao Paulo, BrazilUniv Fed Sao Paulo, Hosp Sao Paulo, Div Infect Dis, Escola Paulista Med, BR-04039032 Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Biochem, Escola Paulista Med, BR-04023900 Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Dept Microbiol Immunol & Parasitol, Escola Paulista Med, BR-04023062 Sao Paulo, BrazilUniv Fed Sao Paulo, Intens Care Unit, Hosp Sao Paulo, Escola Paulista Med, BR-04024002 Sao Paulo, BrazilFAPESP: 2011/20401-4FAPESP: 2013/15636-8CNPq: 305685/2011-2Web of Scienc

Unraveling snake venom complexity with 'omics' approaches: Challenges and perspectives

The study of snake venom proteomes (venomics) has been experiencing a burst of reports, however the comprehensive knowledge of the dynamic range of proteins present within a single venom, the set of post-translational modifications (PTMs) as well as the lack of a comprehensive database related to venom proteins are among the main challenges in venomics research. the phenotypic plasticity in snake venom proteomes together with their inherent toxin proteoform diversity, points out to the use of integrative analysis in order to better understand their actual complexity. in this regard, such a systems venomics task should encompass the integration of data from transcriptomic and proteomic studies (specially the venom gland proteome), the identification of biological PTMs, and the estimation of artifactual proteomes and peptidomes generated by sample handling procedures. (C) 2014 Elsevier B.V. All rights reserved.Universidade Federal de São Paulo, Dept Ciencia & Tecnol, ICT UNIFESP, BR-12231280 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Bioquim, BR-12231280 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Ciencia & Tecnol, ICT UNIFESP, BR-12231280 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Bioquim, BR-12231280 São Paulo, BrazilWeb of Scienc

Journal of Supercritical Fluids

Acesso restrito: Texto completo.p. 186-192.Supercritical CO2 extraction was used to refine coffee oil obtained by mechanical pressing. Extractions were carried out using 50–70 ◦C and pressures ranging from 15.2 to 35.2MPa, with a CO2 flow rate of 1 standard L/min using a semi-continuous high-pressure extraction apparatus. Green coffee oil fractions were collected at fixed time intervals and the composition of each fraction was determined by HPLC analyses. Caffeine and traces of chlorogenic acid were detected in the first fractions while waxes remained in the extraction vessel. Compared with the original oil the triglyceride composition remained almost unchanged in the fractions. The results also indicate an increase of triglyceride and caffeine extraction with pressure. An increase in extraction temperature results in a retrograde behavior over the pressure range of 15.2–28.3MPa. At pressures higher than 30MPa the solubility behavior of coffee oil was apparently independent of the temperature. A good correlation of the solubility data of green coffee oil was obtained using the Chrastil equation

Química Nova

p. 1319-1323.The caffeine solubility in supercritical CO2 was studied by assessing the effects of pressure and temperature on the extraction of green coffee oil (GCO). The Peng-Robinson1 equation of state was used to correlate the solubility of caffeine with a thermodynamic model and two mixing rules were evaluated: the classical mixing rule of van der Waals with two adjustable parameters (PR-VDW) and a density dependent one, proposed by Mohamed and Holder2 with two (PR-MH, two parameters adjusted to the attractive term) and three (PR-MH3 two parameters adjusted to the attractive and one to the repulsive term) adjustable parameters. The best results were obtained with the mixing rule of Mohamed and Holder2 with three parameters.São Paul

Data in support of quantitative proteomics to identify potential virulence regulators in Paracoccidioides brasiliensis isolates

Paracoccidioides genus are the etiologic agents of paracoccidioidomycosis (PCM), a systemic mycosis endemic in Latin America. Few virulence factors have been identified in these fungi. This paper describes support data from the quantitative proteomics of Paracoccidioides brasiliensis attenuated and virulent isolates [1]. The protein compositions of two isolates of the Pb18 strain showing distinct infection profiles were quantitatively assessed by stable isotopic dimethyl labeling and proteomic analysis. The mass spectrometry and the analysis dataset have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with identifier PXD000804