ANTAGONISM OF THE MYOTOXIC EFFECTS OF BOTHROPS-JARARACUSSU VENOM AND BOTHROPSTOXIN BY POLYANIONS

The effects of heparin and other polyanions on the myotoxicity of Bothrops jararacussu venom and purified bothropstoxin (BthTX) were investigated. The release rate of creatine kinase (CK) from isolated extensor digitorum longus muscle and the plasma CK activity of mice were used to quantify the results. The myotoxic effects of B. jararacussu venom or BthTX were inhibited by preincubation of these agents with one of the following: a heterogeneous heparin preparation (designated 'heparin'), low mol. wt heparin (H-4500) or dextran sulfates (DS-8000 and DS-500,000). Non-sulfated dextran (D-40,000) and two chondroitin sulfates were ineffective. The antimyotoxic effects of the polyanions are ascribed to their forming inactive acid-base complexes with the basic myotoxins of Bothrops venoms. Gel-filtration experiments in Sephadex provided direct evidence for complex formation between heparin and BthTX. Intravenous (i.v.) administration of H-4500 or DS-8000 opposed the increase in plasma CK activity induced by a subsequent i.m. injection of venom or BthTX. In contrast, pretreatment with i.v. heparin or DS-500,000 enhanced the venom-induced increase in plasma CK activity. This effect was not observed (1) when the animals were treated with a polyvalent antivenom, which inhibits the coagulation and local stasis induced by Bothrops venoms, and (2) when BthTX, which has no thrombotic or hemorrhagic properties, was the myotoxic agent. The potentiation of the venom-induced increase in plasma CK activity by heparin and DS-500,000 is ascribed to improved washout of the CK released from damaged fibers, because of the anticoagulant properties of the drugs.

Spectrophotometric determination of phosphite in fertilizers in a flow injection system with online sample preparation

A flow injection system with online sample preparation is proposed for the determination of phosphite in liquid fertilizers by spectrophotometry. After loop-based injection, phosphite is oxidized by an acidic permanganate solution (1.0 10(-2) mol L-1 KMnO4 + 1.0 mol L-1 H2SO4) in a heated reactor (50 degreesC). The phosphate generated is then determined by the molybdenum blue method. Influence of flow rates, temperature, and concentration and order of addition of reagents, sample volume, and reactor configuration for the blue complex formation on recorded signals were investigated. The pow system was applied to phosphite determination in commercial samples of liquid fertilizers. The proposed system handles about 80 samples per hour [0.05-0.40% (w/v) H3PO3; R = 0,9998], consuming about 80 muL sample, 1 mg KMnO4, 25 mg (NH)(6)Mo7O24, and Ia mg ascorbic acid per determination. Results are precise [relative standard deviation less than or equal to 3.5% for 0.1% (w/v) H3PO3, n = 12] and in agreement with those obtained by gravimetry at 95% confidence level. (C) 2000 John Wiley & Sons, Inc.

Thermal Characterization and Cytotoxicity of Complexes Formed by Papain and Cyclodextrin

Papain is a proteolytic enzyme with restricted applications due to its limited stability. Cyclodextrins are widely used in pharmaceutical formulations once they are able to form complexes with other molecules, improving their stability and bioavailability. The purpose of the present paper was to analyze complexes formed by papain/hydroxypropyl-beta-cyclodextrin and papain/beta-cyclodextrin by thermal analysis and cytotoxicity tests to verify their possible interactions and toxicological behavior. Complex solutions were prepared at different molar ratios and collected as a function of time to be lyophilized and analyzed. Results showed changes in endothermic events and cytotoxicity profiles. A complex formation for both complexes is observed; nevertheless, beta-cyclodextrin was able to change the enzyme characteristics more efficiently.

Water regulation of actinomycin-D binding to DNA: the interplay among drug affinity, DNA long-range conformation, and hydration

Actiaomycin-D (actD) binds to natural DNA at two different classes of binding sites, weak and strong. The affinity for these sites is highly dependent on DNA se(sequence and solution conditions, and the interaction appears to be purely entropic driven Although the entropic character of this reaction has been attributed to the release of water molecules upon drug to DNA complex formation, the mechanism by which hydration regulates actD binding and discrimination between different classes of binding sites on natural DNA is still unknown. In this work, we investigate the role of hydration on this reaction using the osmotic stress method. We skew that the decrease of solution water activity, due to the addition of sucrose, glycerol ethylene glycol, and betaine, favors drug binding to the strong binding sites on DNA by increasing both the apparent binding affinity Delta G, and the number of DNA base pairs apparently occupied by the bound drug n(bp/actD). These binding parameters vary linearly with the logarithm of the molar fraction of water in solution log(X-w), which indicates the contribution of water binding to the energetic of the reaction. It is demonstrated that the hydration change measured upon binding increases proportionally to the apparent size of the binding site n(bp/uctD). This indicates that n(bp/actD) measured from the Scatchard plod is a measure of the size of the DNA molecule changing conformation due to ligand binding. We also find that the contribution of DNA deformation, gauged by n(bp/act) to the total free energy of binding Delta G, is given by Delta G = Delta G(local) + n(bp/actD) x delta G(DNA), where Delta G(local), = -8020 +/- 51 cal/mol of actD bound and delta G(DNa) = -24.1 +/- 1.7cal/mol of base pair at 25 degrees C. We interpret Delta G(local), as the energetic contribution due to the direct interactions of actD with the actual tetranucleotide binding site, and it n(bp/actB) X delta G(DNA) as that due to change inconformation, induced by binding, of it n(bp/actD) DNA base pairs flanking the local site. This interpretation is supported by the agreement found between the value of delta G(DNA) and the torsional free energy change measured independently. We conclude suggesting an allosteric model for ligand binding to DNA, such that the increase in binding affinity is achieved by increasing the relaxation of the unfavorable free energy of binding storage at the local site through a larger number of DNA base pairs. The new aspect on this model is that the size of the complex is not fixed but determined by solutions conditions, such as water activity, which modulate the energetic barrier to change helix conformation. These results may suggest that long-range allosteric transitions of duplex DNA are involved in the inhibition of RNA synthesis by actD, and more generally, in the regulation of transcription. (C) 2000 John Wiley & Sons, Inc.

Host-guest complexation of a nitroheterocyclic compound with cyclodextrins: a spectrofluorimetric and molecular modeling study

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Spectrophotometric study of binary and ternary systems involving metal ions and benzylidenepyruvates: Equilibria in aqueous solutions

The protonation of 4-dimethylaminobenzylidenepyruvate (DMBP) and 2-chloro-4-dimethylaminobenzylidenepyruvate (2-CI-DMBP) and their complex formation with Mn(II), Co(II), Ni(II), Cu(II), Zn(II), Pb(II), Cd(II) and Al(III) have been studied by potentiometric and spectrophotometric methods at 25 °C and ionic strength 0.500 M, held with sodium perchlorate. The stability order found for 1 :1 complexes of both ligands is Al(III) > Cu(II) > Pb(II) > Ni(II) > Zn(II) > Co(II) > Cd(II) > Mn(II). The stability changes move in the same direction as the pKa of the ligands. The results are compared with literature values reported for metal ion pyruvate systems. Thermodynamic stabilities of ternary complexes formed in Cu(II)-B-L- systems, where B = 2,2′-bipyridyl (bipy), ethylenediamine or glycinate and L = DMBP or 2-CI-DMBP, were also determined. The Cu(bipy)L+ species are more stable than would be expected on purely statistical grounds. The importance of the :t system associated with bipy on the enhanced stability of its mixed ligand complexes is stressed. Analytical applications of the investigated ligands are outlined.

Composition of the first coordination sphere of Ni2+ in concentrated aqueous NiBr2 solutions by x-ray diffraction

Two concentrated solutions of NiBr2 have been examined by x-ray diffraction. The Fourier transformed scattering data indicate inner complex formation between Ni2 + and Br- ions. Average numbers of bonded bromide ions per nickel atom have been determined for each solution and the reliability of the complexation numbers as well as of the other structural parameters has been critically examined. © 1985 American Institute of Physics.

Biophysical Characterization of Interactions Involving Importin-α during Nuclear Import

Proteins containing the classical nuclear localization sequences (NLSs) are imported into the nucleus by the importin-α/β heterodimer. Importin-α contains the NLS binding site, whereas importin-β mediates the translocation through the nuclear pore. We characterized the interactions involving importin-α during nuclear import using a combination of biophysical techniques (biosensor, crystallography, sedimentation equilibrium, electrophoresis, and circular dichroism). Importin-α is shown to exist in a monomeric autoinhibited state (association with NLSs undetectable by biosensor). Association with importin-β (stoichiometry, 1:1; K D = 1.1 × 10 -8 M) increases the affinity for NLSs; the importin-α/β complex binds representative monopartite NLS (simian virus 40 large T-antigen) and bipartite NLS (nucleoplasmin) with affinities (K D = 3.5 × 10 -8 M and 4.8 × 10 -8 M, respectively) comparable with those of a truncated importin-α lacking the autoinhibitory domain (T-antigen NLS, K D = 1.7 × 10 -8 M; nucleoplasmin NLS, K D = 1.4 × 10 -8 M). The autoinhibitory domain (as a separate peptide) binds the truncated importin-α, and the crystal structure of the complex resembles the structure of full-length importin-α. Our results support the model of regulation of nuclear import mediated by the intrasteric autoregulatory sequence of importin-α and provide a quantitative description of the binding and regulatory steps during nuclear import.

fac-[RuCl3(no)(dppb)] (I) and mer-[RuCl3(NO)(diop)] (II) complexes: Syntheses, characterization and x-ray structures

The fac-[RuCl3(NO)(dppb)] complex I has been prepared from solution of the correspondent mer isomer in refluxing methanol (dppb = 1,4-bis(diphenylphosphino)butane). The mer-[RuCl3(NO)(diop)] (II) has been obtained from the mer-[RuCl3(diop)(H2O)] by bubbling NO for 1 h in dichloromethane (diop = 2S,3S-O-isopropylidene-2,3-dihydroxy-1,4-bis(diphenylphosphino)butane). The complexes have been characterized by microanalysis, cyclic voltammetry (CV), IR and 31P{1H} NMR spectroscopies. The crystal and molecular structures of these two compounds have been determined from X-ray studies. The mer-[RuCl3(NO)(dppb)] isomer III was characterized in solution by NMR spectra (31P{1H}, 1H{31P}, 31P-1H HETCORR, COSY 1H-1H, HMQC 1H-13C and HMBC 1H-13C). © 2002 Elsevier Science Ltd. All rights reserved.

Voltammetric determination of dipyrone using a N,N′- ethylenebis(salicylideneaminato)oxovanadium(IV) modified carbon-paste electrode

The preparation and electrochemical characterization of a carbon paste electrode modified with N.N′-ethylenebis(salicylideneiminato) oxovanadium(IV) complex ([VO(Salen)]) as well as its behavior as electrocatalyst toward the oxidation of dipyrone were investigated. The electrochemical behavior of the modified electrode and the electrooxidation of dipyrone were explored using cyclic voltammetry. The voltammetric response of the modified electrode is based on two reactions. One electrochemical related to the oxidation of the metallic center of the [VO(Salen)] and the other involving the chemical redox process involving the oxidized form of the complex and the reduced form of dipyrone. The best voltammetric response was observed for a paste composition of 25% (m/m) [VO(Salen)], KCl solution pH from 5.5 to 8.0 as the electrolyte and potential scan rate of 10 mV s-1 in the presence of dipyrone. A linear voltammetric response for dipyrone was obtained in the concentration range from 9.9 × 106 to 2.8 × 10 -3 mol L-1, with a detection limit of 7.2 × 10 -6 mol L-1. Among of several compounds tested as potential interference, only ascorbic acid presented some interference. The proposed electrode is useful for the quality control and routine analysis of dipyrone in pharmaceutical formulations.

Determination of calcium by atomic absorption spectrophotometry applied to the preparation of alginate:chitosan complexes

Polysaccharicles, as alginate and chitosan, have been used to obtain modified release dosage forms. Alginate, due to its property of building gels during the complex formation with calcium ions, allows the building of capsules containing a core constituted by calcium alginate. This work had for objective to determine the appropriate calcium concentration for the preparation of alginate-chitosan capsules, by means of calcium quantification using atomic absorption spectrophotometry. The methodology of calcium quantification was validated through analysis of the limit of detection, precision, accuracy and recovery of the method. The capsules, containing or not the drug, were prepared by the complex coacervation/ionotropic gelification method. Calcium was quantified after samples mineralization and dilution in lantanium solution. The results showed that the amount of calcium incorporated into the capsules depends on the amount of calcium added to the medium, and this ratio increases until the concentration of 1.5% of initial calcium chloride and above this concentration there is a decrease in the proportion of calcium bonded. It was observed that the proportion of calcium that links to the polymer is inversely proportional to the amount of calcium added. The calcium amount incorporated depends on the concentration of the polymeric dispersions used as well as on the ratio between the two polymers.

Probing the binding of the coumarin drugs using peptide fragments of DNA gyrase B protein

Bacterial DNA gyrase, has been identified as the target of several antibacterial agents, including the coumarin drugs. The coumarins inhibit the gyrase action by competitive binding to the ATP-binding site of DNA gyrase B (GyrB) protein. The high in vitro inhibitory potency of coumarins against DNA gyrase reactions has raised interest in studies on coumarin-gyrase interactions. In this context, a series of low-molecular weight peptides, including the coumarin resistance-determining region of subunit B of Escherichia coli gyrase, has been designed and synthesized. The first peptide model was built using the natural fragment 131-146 of GyrB and was able to bind to novobiocin (K a = 1.8 ± 0.2 × 105/M) and ATP (Ka = 1.9 ± 0.4 × 103/M). To build the other sequences, changes in the Arg136 residue were introduced so that the binding to the drug was progressively reduced with the hydrophobicity of this residue (Ka = 1.3 ± 0.1 × 105/M and 1.0 ± 0.2 × 105/M for Ser and His, respectively). No binding was observed for the change Arg136 to Leu. In contrast, the binding to ATP was not altered, independently of the changes promoted. On the contrary, for peptide-coumarin and peptide-ATP complexes, Mg2+ appears to modulate the binding process. Our results demonstrate the crucial role of Arg 136 residue for the stability of coumarin-gyrase complex as well as suggest a different binding site for ATP and in both cases the interactions are mediated by magnesium ions. Copyright Blackwell Munksgaard, 2005.

Preparação e caracterização inicial de complexo de inclusão entre nitrofurazona e 2-hidroxipropil-β-ciclodextrina

Nitrofurazone (NF), 5-nitro-2-furaldehyde semicarbazone, a broad-spectrum antibiotic, has reported toxic effects and low solubility in water. It would be of great interest to form inclusion complexes between NF and a cyclodextrin, to develop more effective and safer antibiotic formulations. This paper focuses on the preparation of inclusion complexes of NF with 2-hydroxypropyl-β- cyclodextrin (HP-β-CD) and their initial characterization by evaluating rates of complex formation, photostability, solubility isotherms, release rate profiles, stoichiometry of the complexes and their morphology, as revealed by scanning electron microscopy. The kinetic tests of complex formation revealed that 17,3 h is enough for stabilization of the NF-cyclodextrin complex. The solubility isotherm studies showed that the isotherm changes from type A to type B, as a function of temperature. The photostability experiments showed that the insertion of the NF in the HP-β-CD cavity protects the drug from photodecomposition. The release kinetic tests showed that the profile of NF release from the complex is altered by the presence of HP-β-CD in the medium. A Job's plot indicated that the stoichiometry of the complex was 1:1 NF:HP-β-CD. The scanning electron micrographs showed changes in the crystal structure of NF in the complex. This study focused on the physicochemical properties of drug-delivery formulations that could potentially be developed into a novel type of therapy with NF.

Development and application of a highly selective biomimetic sensor for detection of captopril, an important ally in hypertension control

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Exploratory study on sequestration of some essential metals by indigo carmine food dye

Indigo carmine forms a stable complex with different ions, and the stability constant of the complexes were evaluated as log K equal to 5.75; 5.00; 4.89 and 3.89 for complexes with Cu(II), Ni(II), Co(II) and Zn(II) ions, respectively, in 0.1 mol L -1 carbonate buffer solution at pH 10. The interaction between Cu(II) ions and indigo carmine (IC) in alkaline medium resulted in the formation of the Cu 2(IC) complex, measured by the spectrophotometric method, with a stoichiometric ratio between indigo carmine and metal ions of 2:1 (metal-ligand). The reported method has also been successfully tested for determination of copper in pharmaceutical compounds based on copper-gluconate without pre-treatment.