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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 microM 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.

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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 beta chains (CD2 Glu-43 --> Thr and G3 Glu-101 --> Val), which result in the loss of two negative charges at the alpha1beta2 interface and favors the dissociation into dimers. The ATP-dependent dimer left arrow over right arrow 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.

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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 degreesC 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 (Delta and TDelta) 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 (Delta and TDelta) 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.

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Under physiological conditions, the oxygen-transport protein from the gastropod Megalobulimulus ovatus, an extracellular hemocyanin, is composed of 20 identical subunits organized into a cylindrical structure (M(r) 9 x 10(6); 100 S). It dissociates in the pressure range of 0.4-2.5 kbar, as observed by spectroscopic methods (light scattering and intrinsic fluorescence) and gel filtration. In contrast to what is seen with smaller proteins, especially dimers, the pressure-dissociation curves for hemocyanin show little dependence on concentration, suggesting that native hemocyanin exists as a population of molecules with different free energies of association. The pressure-induced dissociation results from an equilibrium in which each aggregate responds to pressure independently of the others and, at any given pressure, is in one of two states, whole or dissociated, which persists for long times when compared with the duration of the experiment. The subunit-subunit affinity of dissociated hemocyanin is much lower than that of associated subunits, suggesting that a conformational drift of monomers occurs. When hemocyanin undergoes dissociation in the absence of calcium and at high pH (> 7.2), a large fraction of the dissociated products changes to a conformation that generates stable intermediate states of assembly, lacking the ability to fully reassemble into decamers and didecamers. These intermediates consist primarily of dimers (M(r) 900,000), and they bind oxygen reversibly with a higher affinity than the native hemocyanin. The binding of calcium or protons changes the conformation back to the "associable" state, which finally generates the assembled structure. The dissociation process is highly reversible at low pH (6.8-6.0) or in the presence of millimolar concentrations of calcium. At pH 5.7, dissociation is negligible at pressures up to 2.5 kbar. A decrease in pH from 7.6 to 6.6 increases the half-dissociation pressure (p 1/2) by 1.3 kbar, corresponding to a stabilization of 1.35 kcal per mole of subunit. The effects of Ca2+ and H+ may mean that, in vivo, special ionic conditions or other factors are required to be present at the assembly sites of oligomeric proteins such as hemocyanin.

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The effects of cations and glycerol on the dissociation induced by pressure and on the reassembly of Glossoscolex paulistus hemoglobin were examined by light scattering, gel filtration, and electron microscopy. Calcium stabilized the quaternary structure of the hemoglobin against pressure dissociation. In the presence of 50 mM Ca2+, the half-dissociation pressure (p 1/2) increased by 400 bar, which corresponds to an average stabilization of -0.62 kcal/mol of dissociating subunit. Calcium also promoted a large increase in the yield of recovery of fully assembled hemoglobin at the expense of the partially dissociated (one-twelfth subunit) and fully dissociated forms. Glycerol protected the hemoglobin from pressure dissociation, increasing the half-dissociation pressure (p 1/2) and promoted an increase in the yield of recovery of fully assembled hemoglobin by about 40%. Addition of calcium after return to atmospheric pressure increased recovery of the fully associated form only in a long time scale (many days). The existence of time-dependent changes in the conformation of the dissociated subunits is suggested to explain the partial association to one-twelfth subaggregates (drifted forms) that lack the ability to reassemble to native hemoglobin. The promotion of reassembly by nonprotein factors (calcium and glycerol) is suggested to occur by preventing the formation of wrong intermediate forms (drifted one-twelfth subunits).

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The effects of hydrostatic pressure on the extracellular hemoglobin of Glossoscolex paulistus were investigated by studies of light scattering, intrinsic protein fluorescence, filtration chromatography, and oxygen binding. Pressure promoted a large decrease of light scattering consistent with the dissociation of the hemoglobin. Pressures up to 1.7 kbar caused dissociation with reversibility of the light scattering and fluorescence properties after return to atmospheric pressure. Higher pressures provoked additional dissociation with increasing loss of reversibility. After complete dissociation by incubation at 2.5 kbar followed by decompression, the protein continued mostly dissociated. The dissociated forms were distributed in two populations as based on size exclusion chromatography, one corresponding to small dissociated units (average Mr = 33,000) and the other population corresponding to the one-twelfth subunit (260,000 Mr). The pressure dissociation curves showed no significant dependence on protein concentration suggesting that the native hemoglobin population exists in a distribution of free-energies of association. Both the decrease of concentration dependence and the loss of ability to reassemble seem to increase with the complexity and size of the protein aggregate. These findings permit the conclusion that increased heterogeneity of free-energies of association with the size of the aggregate may result in the molecular individuality of large protein complexes such as subcellular particles and viruses.

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A new procedure for the determination of the sedimentation coefficient in the 50-1000 S range was designed and tested. The characteristics of this protocol are: the use of density gradients self-generated by osmosedimentation and the use of a low-speed centrifuge and of visual monitoring of the sedimenting zone. This procedure was used to determine the sedimentation coefficient of erythrocruorin from Glossoscolex paulistus. The value obtained, S20, omega = 58 S, corresponds to a MW of 3.1 x 10(6) Daltons. The minimum MW (heme), determined by the pyridine-hemochromogen method, was 25,250 Da.

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