Temos interesse em processos de inativação de micro-organismos (bactérias, vírus) de importância biotecnológica, objetivando o desenvolvimento de métodos de diagnóstico, vacinas e novas técnicas de esterilização, investigando modificações estruturais e moleculares nesse processo. Utiliza-se principalmente alta pressão hidrostática associada a outras condições físicas e/ou químicas. O efeito desses agentes em proteínas para investigar a termodinâmica da dissociação e desnaturação também é alvo de investigações. Outro foco é o desenvolvimento de modelos de cinética enzimática mais gerais que o modelo de Michaelis Menten. “Spot synthesis” de peptídeos está em fase de implantação objetivando mapeamento de epitopos das preparações de inativação viral no estudo dos produtos vacinais e, em colaboração com outros grupos, aplicações da técnica no estudo de doenças auto-imunes e screening de atividade de proteases, proteínas quinases e proteínas fostatases.
Equipe
Ancelmo Rabelo de Souza (Doutorado)
Ana Luisa (Iniciação Científica)
Juliana Mattoso Gonçalves Geraldi (Técnica Nível Superior)
Atividades Desenvolvidas
Inativação de micro-organismos de interesse biotecnológico com o uso da alta pressão hidrostática.
Termodinâmica de dissociação e desnaturação de proteínas e partículas virais.
Cinética enzimática. Extensão de modelos de Michaelis-Meten.
Mapeamento de epitopos através de “spot synthesis”.
Journal of Food Science (2012), 77, 8, M417 doi: 10.1111/j.1750-3841.2012.02819.x
Effect of High Hydrostatic Pressure on Aeromonas hydrophila AH 191 Growth in Milk
Ricardo Durães-Carvalho, Ancelmo R. Souza, Luciano M. Martins, Adriane C. S. Sprogis, Jose A. C. Bispo, Carlos F. S. Bonafe and Tomomasa Yano
Abstract: Exposure to high pressure is an efficient method of bacterial inactivation that is particularly important for reducing the microbial load present in foods. In this study, we examined the high pressure inactivation of Aeromonas hydrophila AH 191, a virulent strain that produces aerolysin, a cytotoxic, enterotoxic, and hemolytic toxin. High pressure treatment (250 MPa for 30 min at 25 ◦C in 0.1MPBS, pH 7.4) of A. hydrophila grown in milk reduced bacterial viability by at least 9 orders of magnitude. Under these conditions, the enterotoxic, hemolytic, and cytotoxic activities of A. hydrophila culture supernatants were unaltered. These results indicate the need for caution in the use of high pressure for food processing since although truly toxigenic bacteria may be inactivated, their toxins may not be, thus posing a risk to human health. At higher pressure (350 MPa) the inactivation of bacteria was much more effective. Scanning electron microscopy showed a significant decrease in the number of bacteria after higher pressurization (350 MPa for 1 h) and transmission electron microscopy showed irregular shaped bacteria, suggestive of important cell wall and membrane damage, and cytoplasm condensation.
J. Phys. Chem. B (2012), 116, 14817−14828. dx.doi.org/10.1021/jp310219k |
Entropy and Volume Change of Dissociation in Tobacco Mosaic Virus Probed by High Pressure
Jose A. C. Bispo, Carlos F. S. Bonafe, Ines Joekes, Ernesto A. Martinez, Giovani B. M. Carvalho,and Douglas R. Norberto
Abstract: Virus dissociation and inactivation by high pressure have been extensively studied in recent decades. Pressure-induced dissociation of viral particles involves a reduction in the Gibbs free energy of dissociation and a negative change in volume. In this work, we investigated the combined effect of high pressure and temperature on the dissociation of tobacco mosaic virus (TMV). We assumed the presence of two states of TMV with different tendencies to dissociate. Thus one form presents a low tendency (L) and the other a high tendency (H) to dissociate. Based on the model described here, the L−H transition was favored by an increase in pressure and a decrease in temperature. The volume change of dissociation was pressure- and temperature-dependent, with a highly negative value of −80 mL/mol being recorded at 0 °C and atmospheric pressure. The entropy and enthalpy of dissociation were very temperature- and pressure-dependent, with values of entropy of 450 to −1300 kJ/mol and values of enthalpy of 5.5 × 104 to 2.4 × 104 kJ/mol. The dissociation of TMV was enthalpy-driven at all temperatures and pressures investigated. Based on these findings, we conclude that the model presented allows accurate predictions of viral dissociation behavior in different experimental conditions.
Mol Biol (2013) dx.doi.org/10.4172/2168-9547.1000108
Epitope Mapping of Tobacco Mosaic Virus Capsid Protein: Prediction and Experimental Data from Spot Synthesis
Daniel Ferreira de Lima Neto, Clarice Weis Arns, Dagmar Ruth Stach-Machado, Fernando Rosado Spilki, Juliana Mattoso and Carlos Francisco Sampaio Bonafe
Abstract: The immune system is a network of thousands of molecules, cells and regulatory factors that produce many interrelated responses. In this study, we used spot synthesis to map tobacco mosaic virus (TMV) epitopes in a mice animal model (Balb/c) and compared the results with those obtained using immunoinformatic prediction tools. Mice were inoculated with TMV and after immunization the sera were incubated with an array of overlapping pentadecapeptides that corresponded to the full sequence of the TMV capside protein (TMVcp) that had been synthesized on a cellulose membrane for spot synthesis analysis. Six linear epitopes were identified experimentally, as shown by the IgG-elicited immune responses in mice. The data for epitope prediction based on epitope databases agreed with the results obtained by spot synthesis results. Comparison of the findings for spot intensities and those obtained with the prediction software allowed the identification of different responses according to the MHC class I alleles. The results of this work provide a detailed antigenic profile for TMV.
Open Journal of Biophysics (2012) 2, 4-14 doi:10.4236/ojbiphy.2012.21002
Pressure- and Urea-Induced Denaturation of Bovine Serum Albumin: Considerations about Protein Heterogeneity
Douglas Ricardo Norberto, Joelma Mauricio Vieira, Ancelmo Rabelo de Souza, Jose Ailton Conceicao Bispo, Carlos Francisco Sampaio Bonafe
Abstract: Urea denatures proteins at different concentrations, depending on the experimental conditions and the protein. We investigated the pressure-induced denaturation of bovine serum albumin (BSA) in the presence of subdenaturing concentrations of urea based on a two-state equilibrium. Pressure-induced denaturation was enhanced at urea concentrations ([U]) of 3.5 M to 8.0 M, with the free energy of denaturation at atmospheric pressure 
ranging from +5.0 to –2.5 kJ/mol of BSA. The m values appeared to be biphasic, with m1 and m2 of 0.92 and 2.35 kJ mol–1·M–1, respectively. Plots of 
versus ln[U] yielded values of u, the apparent stoichiometric coefficient, of 1.68 and 6.67 mol of urea/mol of BSA for m1 and m2, respectively. These values were compared with the m and u values of other monomeric proteins reported in or calculated from the literature. The very low values of u systematically observed for proteins were suggestive of heterogeneity in the free energy of denaturation. Thus, a u value of 140 mol of urea/mol of BSA may indicate the existence of a heterogeneous molecular population with respect to the free energy of denaturation.
J Math Chem (2013) 51:144–152 doi: 10.1007/s10910-012-0071-1
Substrate and enzyme concentration dependence of the Henri–Michaelis–Menten model probed by numerical simulation
Jose Ailton Conceicao Bispo, Carlos Francisco Sampaio Bonafe, Maria Gabriela Bello Koblitz,
Carlos Geilson Santana Silva, Ancelmo Rabelo de Souza
Abstract: The use of the classic Henry–Michaelis–Menten (HMM) model (or simply, Michaelis–Menten model) to study the substrate and enzyme concentration dependence of enzyme catalysis is a very important step in understanding many biochemical processes, including microbial growth. Although the HMM model has been extensively studied, the conditions in which the substrate concentration is not in excess have still not been adequately defined mathematically. This lack of definition occurs despite at the cellular and molecular levels most systems generally do not operate in a state of substrate excess. In the present work, we describe an approach for studying enzyme reactions in which substrate concentrations are not in excess. Our results show that the use of extent of reactions and numerical simulation of the velocities of reaction provides an important advance in this field and furnishes results not obtained in previous studies involving these aspects. This approach, in association with knowledge of the rate constants, provides a direct and easy means of examining the single substrate–enzyme profile during product formation at any enzyme–substrate ratio. This approach is more direct than previous models that required the use of empirical equations with arbitrary constants.
J Math Chem (2011) 49:1976–1995
DOI 10.1007/s10910-011-9869-5
Extending the kinetic solution of the classic Michaelis–Menten model of enzyme action
Jose Ailton Conceicao Bispo, Carlos Francisco Sampaio Bonafe, Volnei Brito de Souza, João Batista de Almeida e Silva, Giovani Brandao Mafra de Carvalho
Abstract: The principal aim of studies of enzyme-mediated reactions has been to provide comparative and quantitative information on enzyme-catalyzed reactions under distinct conditions. The classic Michaelis–Menten model (Biochem Zeit 49:333, 1913) for enzyme kinetic has been widely used to determine important parameters involved in enzyme catalysis, particularly the Michaelis–Menten constant (KM) and the maximum velocity of reaction (Vmax ). Subsequently, a detailed treatment of the mechanisms of enzyme catalysis was undertaken by Briggs–Haldane (Biochem J 19:338, 1925). These authors proposed the steady-state treatment, since its applicability was constrained to this condition. The present work describes an extending solution of the Michaelis–Menten model without the need for such a steady-state restriction. We provide the first analysis of all of the individual reaction constants calculated analytically. Using this approach, it is possible to accurately predict the results under new experimental conditions and to characterize and optimize industrial processes in the fields of chemical and food engineering, pharmaceuticals and biotechnology.
Drying Technol. in press.
Modeling drying isotherms using a structure transition model
Jose Ailton Conceicao Bispo, Cristina Maria Rodrigues, Carlos Francisco Sampaio Bonafe and Denilson de Jesus Assis
Abstract: Drying introduces structural changes in the target material that modify its interaction with water. In this work, we developed a model based on star fruit drying that considered two forms of interaction with water. This model provided a very good fit with the experimental data and was applicable to drying in other fruit such as apple, barley and coffee. This model yielded better fits for data reported in the literature than did other models. These findings suggest that the model is applicable to a wide range of systems.
Biochem. Bioph. Res. Co. (2000) 275, 955–961
doi:10.1006/bbrc.2000.3402,
Virus Inactivation by Anilinonaphthalene Sulfonate Compounds and Comparison with Other Ligands
Carlos F. S. Bonafe, Michael Glaser, Edward W. Voss, Gregorio Weber, and Jerson L. Silva
Abstract: Bis-(8-anilinonaphthalene-1-sulfonate) (bis-ANS) causes inactivation of vesicular stomatitis virus (VSV) at micromolar concentrations while butyl-ANS and ANS are effective at concentrations one and two orders of magnitude higher, respectively. VSV fully inactivated by the combined effects of 10 mM bis-ANS and 2.5 kbar hydrostatic pressure elicited a high titer of neutralizing antibodies. Incubation of VSV with >2 M urea at atmospheric pressure caused very little virus inactivation, whereas at a pressure of 2.5 kbar, 1 M urea caused inactivation that exceeded by more than two orders of magnitude the sum of the inactivating effects produced by urea and pressure separately. Measurements of bis-ANS fluorescence showed that increasing the urea concentration reduces the pressure required to disrupt the structure. We conclude that anilinonaphthalene sulfonate compounds inactivate VSV by a mechanism similar to that produced by pressure. The most effective antiviral compound was bis-ANS which can be used for the preparation of safe viral vaccines or as an antiviral drug eventually.
Biochemistry (1998) 37, 11097-11105
Tobacco Mosaic Virus Disassembly by High Hydrostatic Pressure in Combination
with Urea and Low Temperature
Carlos F. S. Bonafe, Claudia M. R. Vital, Rosiani C. B. Telles, Maria C. Goncüalves, Maria S. A. Matsuura, Francisco B. T. Pessine, Daniel R. C. Freitas, and Jorge Vega
Abstract: We investigated the effect of low temperature and urea combined with high pressure on tobacco mosaic virus (TMV). The evaluation of its aggregation state and denaturation process was studied using gel filtration, transmission electron microscopy, and spectroscopic methods. The incubation at 2.5 kbar induced 18% dissociation, and decreasing of temperature to -19 °C promoted additional dissociation to 72%, with stabilization of the dissociation products. Under such conditions, extensive denaturation did not occur. The apparent enthalpy and entropy of dissociation (ΔH*dis and TΔS*dis) were -9.04 kcal/mol subunit and -15.1 kcal/mol subunit, respectively, indicating that the TMV association is an entropicly driven process. The apparent free energy of stabilization given by the presence of RNA is at least -1.7 kcal/mol subunit. Urea-induced dissociation of TMV samples and incubation at high-pressure promoted a higher degree of dissociation. The volume change of dissociation decreased in magnitude from -16.3 to -3.1 mL/mol of dissociated subunit, respectively, in the absence and presence of 2.5 M urea, suggesting exposure of the protein-protein interface to the solvent. High-pressure induced remarkable TMV denaturation in the presence of 2.5 M urea, with a volume change of -101 mL/mol of denatured subunit. The apparent enthalpy and entropy of denaturation (ΔH*den and TΔS*den) by 1.75 M urea at 2.5 kbar was -11.1 and -10.2 kcal/mol subunit, respectively, demonstrating that the TMV protein coat presents an apparent free energy of denaturation by urea close to zero. Although the processes could not be assumed to be pure equilibria, these thermodynamic parameters could be derived by assuming a steady-state condition.
J. Biol. Chem. (1999) 274, 1196–1198, 1999
ATP-induced Tetramerization and Cooperativity in Hemoglobin of Lower Vertebrates
Carlos F. S. Bonafe, Adriana Y. Matsukuma, and Maria S. A. Matsuura
Abstract: The importance of intraerythrocytic organic phosphates in the allosteric control of oxygen binding to vertebrate hemoglobin (Hb) is well recognized and is correlated with conformational changes of the tetramer. ATP is a major allosteric effector of snake Hb, since the absence of this nucleotide abolishes the Hb cooperativity. This effect may be related to the molecular weight of about 32,000 for this Hb, which is compatible with the dimeric form. ATP induces a pH-dependent tetramerization of deoxyHb that leads to the recovery of cooperativity. This phenomenon may be partially explained by two amino acid replacements in the β chains (CD2 Glu-43 3 Thr and G3 Glu-101 3 Val), which result in the loss of two negative charges at the β1 β2 interface and favors the dissociation into dimers. The ATP-dependent dimer 7 tetramer may be physiologically important among ancient animal groups that have similar mutations and display variations in blood pH that are governed by these animals* metabolic state. The enormous loss of free energy of association that accompanies Hb oxygenation, and which is also observed at a much lower intensity in higher vertebrate Hbs, must be taken into consideration in allosteric models. We propose that the transition from a myoglobin-like protein to an allosteric one may be of evolutionary significance.