Carlos Francisco Sampaio Bonafe

ATP-induced Tetramerization and Cooperativity in Hemoglobin of Lower Vertebrates

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 → Thr and G3 Glu-101 → Val), which result in the loss of two negative charges at the α1β2 interface and favors the dissociation into dimers. The ATP-dependent dimer ↔ 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.

Entropy and Volume Change of Dissociation in Tobacco Mosaic Virus Probed by High Pressure

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 × 10(4) to 2.4 × 10(4) 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.

Different urea stoichiometries between the dissociation and denaturation of tobacco mosaic virus as probed by hydrostatic pressure

Viruses are very efficient self-assembly structures, but little is understood about the thermodynamics governing their directed assembly. At higher levels of pressure or when pressure is combined with urea, denaturation occurs. For a better understanding of such processes, we investigated the apparent thermodynamic parameters of dissociation and denaturation by assuming a steady-state condition. These processes can be measured considering the decrease of light scattering of a viral solution due to the dissociation process, and the red shift of the fluorescence emission spectra, that occurs with the denaturation process. We determined the apparent urea stoichiometry considering the equilibrium reaction of TMV dissociation and subunit denaturation, which furnished, respectively, 1.53 and 11.1 mol of urea/mol of TMV subunit. The denaturation and dissociation conditions were arrived in a near reversible pathway, allowing the determination of thermodynamic parameters. Gel filtration HPLC, electron microscopy and circular dichroism confirmed the dissociation and denaturation processes. Based on spectroscopic results from earlier papers, the calculation of the apparent urea stoichiometry of dissociation and denaturation of several other viruses resulted in similar values, suggesting a similar virus-urea interaction among these systems.

Effect of High Hydrostatic Pressure on Aeromonas hydrophila AH 191 Growth in Milk

UNLABELLED 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 0.1 M PBS, 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. PRACTICAL APPLICATION High pressure inactivates Aeromonas hydrophila efficiently but is enhanced when combined with moderate temperature (40 °C). The biological activities of toxins from this bacterium are unaltered under these conditions.

Substrate and enzyme concentration dependence of the Henri–Michaelis–Menten model probed by numerical simulation

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.

Pressure- and Urea-Induced Denaturation of Bovine Serum Albumin: Considerations about Protein !! Heterogeneity

Urea denatures proteins at different concentrations, depending on the experimental conditions and the protein. We in-vestigated the pressure-induced denaturation of bovine serum albumin (BSA) in the presence of subdenaturing concen-trations 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 dena-turation.

Modeling Drying Isotherms Using a Structure Transition Model

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 to the experimental data and was applicable to drying of other products such as apple, barley, and coffee. This model yielded better fits for data reported in the literature than other models. These findings suggest that the model is applicable to a wide range of systems.

Synthesis of Peptides from α- and β-Tubulin Containing Glutamic Acid Side-Chain Linked Oligo-Glu with Defined Length

Side-chain oligo- and polyglutamylation represents an important posttranslational modification in tubulin physiology. The particular number of glutamate units is related to specific regulatory functions. In this work, we present a method for the synthesis of building blocks for the Fmoc synthesis of peptides containing main chain glutamic acid residues that carry side-chain branching with oligo-glutamic acid. The two model peptide sequences CYEEVGVDSVEGEG-E(Ex)-EEGEEY and CQDATADEQG-E(Ex)-FEEEEGEDEA from the C-termini of mammalian α1- and β1-tubulin, respectively, containing oligo-glutamic acid side-chain branching with lengths of 1 to 5 amino acids were assembled in good yield and purity. The products may lead to the generation of specific antibodies which should be important tools for a more detailed investigation of polyglutamylation processes.

Epitope Mapping of Tobacco Mosaic Virus Capsid Protein: Prediction and Experimental Data from Spot Synthesis

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.

Applying structural transition theory to describe enzyme kinetics in heterogeneous systems

Enzyme action was investigated by assuming the occurrence of different states of enzyme-substrate affinities. These states were considered to involve enzyme species with distinct abilities to form reaction product. The results obtained showed strong agreement with the experimental data for the action of peroxidase. This approach provides a powerful tool for predicting the kinetic behavior of other enzymatic processes in conditions not described before. An additional feature of this approach is the ability to characterize processes at any enzyme-substrate concentration ratio, including high enzyme-substrate ratios and enzyme inhibition by substrate or product. This proposal can also be used in systems with heterogeneity concerning the investigated enzyme.