Le présent travail porte sur l’étude de deux protéines de la famille des oxydases à hème-fer par des techniques de spectroscopie et d’électrochimie. Le premier chapitre décrit l’étude du cytochrome bo3 oxydase issue d’E. coli. Nous nous sommes intéressés à l’étude des interactions enzyme-quinone par l’utilisation de quinones avec des longueurs chaines isoprenyl différentes. Notre but est de mieux comprendre le rôle de la longueur de la chaine des quinones sur l’activité catalytique de l’enzyme et sur les propriétés redox des cofacteurs à hème. Dans l’étape suivante, on a étudié les résidus impliqués dans le site de liaison des quinones (haute affinité, QH). Plusieurs mutations de ces résidus sont étudiées pour mieux comprendre l’importance de chacun des résidus dans cette liaison. Dans la dernière partie de ce premier chapitre, la spectroscopie SEIRAS «spectroscopie d’absorption infrarouge exaltée de surface» est introduite comme une technique alternative pour l’étude des protéines membranaires. Dans le second chapitre, la protéine membranaire cNOR issue de P. denitrificans est étudiée. Nous nous sommes focalisés sur l’effet de différents environnements (pH, présence de protéo-liposomes) sur la stabilité de la cNOR. Pour ce faire, trois valeurs de pH (6.5, 7.5 et 8.5) sont choisies et quelques échantillons de cNOR sont reconstitués dans des protéo-liposomes. Enfin, le donneur de proton terminal (au centre binucléaire) dans la protéine cNOR était étudié. De plus, nous avons étudié les ligands des ions Ca2+ puisqu’il est proposé que le donneur de proton est situé proche de cette région. / This thesis is focused on the study of two members of the heme-copper oxidase family by using spectroscopic and electrochemical techniques. In the first chapter cytochrome bo3 oxidase from E. coli was studied. We focused on the quinone-enzyme interactions by using quinones with different isoprenyl chains. Our aim was to better understand the role of isoprenyl chain on the catalytic activity of the enzyme and the redox properties of the heme cofactors. In the next step we studied the residues that are suggested to be in the high-affinity (QH) quinone binding site. Several site-directed mutants of these residues were investigated in order to better understand the position of QH binding site and the importance of each residue. In the last part of this chapter surface-enhanced infrared absorption spectroscopy (SEIRAS) was introduced as an alternative technique to study the membrane proteins. In the second chapter cytochrome c dependent nitric oxide reducates (cNOR) from P. denitrificans was studied. We focused on the effect of different environment (pH, proteoliposomes) on the stability of cNOR. For that purpose three pH values (6.5, 7.5 and 8.5) was selected and some of the cNOR samples were reconstituted in liposomes. Finally, the terminal proton donor (to the binuclear center) in cNOR was investigated. We studied the ligands of the Ca2+ site in cNOR since it was suggested that the proton donor may be close to this area.

This diploma thesis deals with acid-base and electrolytic behavior of hyaluronan solutions at different ionic strength. Acid-base behavior of hyaluronan was investigated by acid-base titrations which were carried out with two different methods, acid and alkaline acid-base titration. Dissociation constants at different ionic strength at zero degree of dissociation and at 50% degree of dissociation were evaluated from the results of acid-base titrations. Dissociation constants obtained from acid acid-base titrations have values between 3,0 and 3,6. Dissociation constants obtained from alkaline acid-base titrations are not very informative because their values are much higher than the expected values. The study of degradation of hyaluronan during acid-base titration was performed to complete study of acid-base behavior. Electrolytic behavior of hyaluronan solution was performed by conductometric titrations in three different environments.

The presented dissertation focuses on non-covalent interactions of hyaluronan of different molecular weights (9–1540 kDa) with basic (oligo)-amino acids (especially arginine) and the antimicrobial peptide cecropin B. High-resolution ultrasonic spectroscopy (HR-US), isothermal titration calorimetry (ITC) and potentiometric titration techniques were chosen to investigate the interactions. The thesis focuses on the characterization of interactions, especially with respect to the used molecular weight of interacting polymers and the ionic strength of the environment. Whether interactions occur or not was determined primarily by the length of the arginine oligomer chain. For monomeric amino acids, the interactions were investigated mainly by potentiometric titrations. Interactions were observable from arginine oligomers with eight monomer units. The molecular weight of hyaluronan mainly affected the intensity of the interactions. The transition between the individual conformations of hyaluronan (rod and random coil) was especially significant. Investigation of interactions was performed in water, in solutions with different concentrations of sodium chloride and in PBS. The sufficiently high ionic strength of the solution was able to suppress the interactions in water between the oligomers of arginine and hyaluronan. The basic antimicrobial peptide cecropin B has been shown to interact with hyaluronan in water but not in PBS. Based on these results, it was possible to conclude that the hyaluronan-cecropin B system would be particularly suitable for topical applications.

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A fundamental analysis performed at LKAB is the determination of the iron (II) content of the company’s iron ore products. Currently, a redox titration method using potassium dichromate as the titrator is used to determine this content. However, LKAB is considering replacing its current method due to the titrator’s harmful effects on the health and the environment. The purpose of this study is to compare different redox titration methodsvalidation parameters, to find the most suitable method for the determination of the iron (II) content. The titrators in focus for this study is potassium dichromate, cerium (IV) sulphate and potassium permanganate. Several experiments were performed to compare the three titration methods. The validation parameters determined for each method were precision, accuracy, linearity, LOD, LOQ and robustness. The titration error was also calculated. The results showed that the redox titration with potassium dichromate as the titrator had the highest accuracy at 98.95 %, while the redox titration with cerium (IV) sulphate as the titrator had the best precision at 0.099 % (for samples with a similar matrix as LKAB’s iron ore products). Based on the acquired results, it was concluded that LKAB’s current method is the most suitable for the determination of the iron (II) content in LKAB’s iron ore products. The redox titration method using cerium (IV) sulphate as a titrator is a good competitor, butdue to the method’s slightly inferior accuracy and vague end point, it fails to outcompeteLKAB’s current method. / En grundläggande analys som utförs på LKAB är fastställandet av järn (II)-halten i företagets järnmalmsprodukter. För närvarande används en redox-titreringsmetod med kaliumdikromat som titrator för bestämmelsen av denna halt. LKAB överväger dock att byta ut sin nuvarande metod på grund av titratorns hälso- och miljöfarliga effekter. Syftet med denna studie är att jämföra olika redox-titreringsmetoders valideringsparametrar, för att hitta den mest lämpligaste metoden för fastställandet av järn (II)-halten. De titratorer som studeras är kaliumdikromat, cerium (IV) sulfat och kaliumpermanganat. Flertalet experiment utfördes, för att kunna jämföra de tre titreringsmetoderna med varandra. De valideringsparametrar som bestämdes för vardera metoden var precision, noggrannhet, linjäritet, LOD, LOQ och robusthet. Titrerfelet beräknades också. Resultaten visade att redox-titrering med kalimdikromat som titrator hade högst noggrannhet på 98,95 %, medan redox-titrering med cerium (IV) sulfat som titrator hade bäst precision på 0,099 % (för prover med liknande matris som LKAB:s järnmalmsprodukter). Utifrån de resultat som erhölls så drogs slutsatsen att LKAB:s nuvarande metod är den mest lämpligaste för fastställandet av järn (II)-halten i företagets järnmalmsprodukter. Redox-titreringsmetoden med cerium (IV) sulfat som titrator är en bra konkurrent, men på grund av metodens något sämre noggrannhet och otydligare slutpunkt så misslyckas den med att konkurrera ut LKAB:s nuvarande metod.

<p>Virtually all biologically relevant functions and processes are mediated by non-covalent, molecular recognition events, demonstrating astonishingly diverse affinities and specificities. Despite extensive research, the origin of affinity and specificity in aqueous solution - specifically the relationship between ligand binding thermodynamics and structure - remains remarkably obscure and is further complicated in the context of multivalent interactions. Multivalency describes the combinatorial interaction of multiple discrete epitopes across multiple binding surfaces where the association is considered as the sum of contributions from each epitope and the consequences of multivalent ligand assembly. Gaining the insight necessary to predictably influence biological processes with novel therapeutics begins with an understanding of the molecular basis of solution-phase interactions, and the thermodynamic parameters that follow from those interactions. Here we continue our efforts to understand the basis of aqueous affinity and the nature of multivalent additivity.</p><p>Multivalent additivity is the foundation of fragment-based drug discovery, where small, low affinity ligands are covalently assembled into a single high affinity inhibitor. Such systems are ideally suited for investigating the thermodynamic consequences of multivalent ligand assembly. In the first part of this work, we report the design and synthesis of a fragment-based ligand series for the Grb2-SH2 protein and thermodynamic evaluation of the low affinity ligand fragments compared to the intact, high affinity inhibitor by single and double displacement isothermal titration calorimetry (ITC). Interestingly, our investigations reveal positively cooperative multivalent additivity - a binding free energy of the full ligand greater than the sum of its constituent fragments - that is largely enthalpic in origin. These results contradict the most common theory of multivalent affinity enhancement arising from a "savings" in translational and rotational entropy. The Grb2-SH2 system reported here is the third distinct molecular system in which we have observed enthalpically driven multivalent enhancement of affinity.</p><p>Previous research by our group into similar multivalent affinity enhancements in protein-carbohydrate systems - the so-called "cluster glycoside effect" - revealed that evaluation of multivalent interactions in the solution-phase is not straightforward due to the accessibility of two disparate binding motifs: intramolecular, chelate-type binding and intermolecular, aggregative binding. Although a number of powerful techniques for evaluation of solution-phase multivalent interactions have been reported, these bulk techniques are often unable to differentiate between binding modes, obscuring thermodynamic interpretation. In the second part of this work, we report a competitive equilibrium approach to Molecular Recognition Force Microscopy (MRFM) for evaluation of ligand binding at the single-molecule level with potential to preclude aggregative associations. We have optimized surface functionalization strategies and MRFM experimental protocols to evaluate the binding constant of surface- and tip-immobilized single stranded DNA epitopes. Surprisingly, the monovalent affinity of an immobilized species is in remarkable agreement with the solution-phase affinity, suggesting the competitive equilibrium MRFM approach presents a unique opportunity to investigate the nature of multivalent additivity at the single molecule level.</p> / Dissertation

Student Number : 0002483R - MSc dissertation - School of Molecular and Cell Biology - Faculty of Science / Isothermal titration calorimetry was used to investigate the forces that drive ligand binding to the active site of rGST M1-1. In an attempt to gain insight into the recognition of non-substrate ligands by GSTs, this study also investigates interactions between rGST M1-1 and ANS, a non-substrate ligand. At 25 °C, complex formation between rGST M1-1 and GSH, GSO3 -, and S-hexylglutathione is characterised by a monophasic binding isotherm with Kd values of 38.5 mM, 2.1 mM and 0.2 mM, respectively. One molecule of each ligand is bound per monomer of rGST M1-1. Binding of these ligands is enthalpically favourable and entropically unfavourable with a resultant favourable Gibbs free energy, overall. The effects of temperature and buffer ionisation on the energetics of binding were studied. The enthalpic and entropic contributions for all three ligands exhibited temperature dependence over the temperature range investigated (5-30 °C). The Gibbs free energy showed negligible changes with increasing temperature due to enthalpy-entropy compensation. The temperature dependence of the binding enthalpy yielded heat capacity changes of – 2.69 kJ/mol/K and –3.68 kJ/mol/K at 25 °C for GSH and S-hexylglutathione binding and –1.86 kJ/mol/K overall for GSO3 -. The linear dependence of DH on temperature for GSO3 - binding to rGST M1-1 suggests the formation of a more constrained complex which limits the fluctuations in conformations of the mu-loop at the active site. The non-linear dependence of DH on temperature for GSH and Shexylglutathione binding to the enzyme suggests the formation of a complex that samples different bound conformations due to the mobility of the mu-loop even after ligand is bound. Calorimetric binding experiments in various buffer systems with different ionisation enthalpies suggest that the binding of GSH to rGST M1-1 is coupled to the deprotonation of the thiol of GSH while GSO3 - binding to rGST M1-1 is independent of the buffer ionisation. At 25 °C, the rGST M1-1#1;ANS association is represented by a monophasic binding isotherm with one molecule of ANS bound per monomer of rGST M1-1. The interaction is both enthalpically and entropically driven with a Kd value of 27.2 mM representing moderate affinity. The effect of temperature on the interaction was investigated over the temperature range of 5-30 °C. The linear dependence of the binding enthalpy on temperature indicates that no significant structural changes occur upon binding of ANS to the enzyme (DCp = -0.34 kJ/mol/K). The change in heat capacity associated with the interaction can be attributed to the burial of the polar sulphonate group of ANS and the exposure of the anilino and naphthyl rings to solvent as well as the possibility of weak electrostatic interactions between ANS and residues at the active site. The effect of ethacrynic acid, GSH, GSO3 - and S-hexylglutathione on the fluorescence of ANS was investigated in order to obtain some idea as to the location of the ANS binding site on rGST M1-1. ANS was displaced by GSO3 -, S-hexylglutathione and ethacrynic acid, while no displacement occurred upon binding of GSH to the active site of rGST M1-1. Displacement studies and molecular docking simulations indicate that ANS binds to the H-site of rGST M1-1 and the possibility of a second binding site for the molecule cannot be ruled out.

O presente trabalho consistiu no desenvolvimento de metodologia simples e de baixo custo para determinação de gás carbônico da atmosféra. O CO2 é absorvido em solução aquosa de NaOH, 10-4M e, a solução resultante é adicionado NaCl para concentração final de 1,0M. A seguir, titula-se com ácido clorídrico 10-3M, potenciometricamente. A fim de se evitar interferência de íons carbonato possivelmente existentes na solução original de NaOH. Uma alíquota desta é titulada préviamente antes da absorção de CO2, constituindo o \"branco\". A solução de hidróxido de sódio e carbonato é titulada a seguir e, considera-se a diferença do volume no ponto final da solução do branco e da amostra; a avaliação é efetuada segundo Gran II, estudado por Pressinotti (9). Foram efetuados estudos experimentais de introdução do gás carbônico em solução de hidróxido de sódio. Estudaram-se os métodos com o uso de: a - com o uso de bomba misturadora; b - com o uso de frascos lavadores; c - com o uso de injeção com seringa em frasco plástico. Esta última mostrou ser simples e rápida para o processo. / This work deals with the development of a simple and low cost methodology the determination of carbonic gas in the atmosphere. The CO2 is absorved in aqueous solution of NaOH 10-4; M and, to t he resulting solution, NaCl is added to a concentratian of 1.0M and the potentiometric titration is done with HCl 10-3; M solution. To avoid the interference of carbonate ions in the original NaOH solution, an aliquot of the NaOH solution is previously titrated just before the absorption af CO2; this previous titration is the \"blank\". The final solulion of NaOH and carbonate ions is titrated and one measures the diference of the volume at the end point of the \"blank\" and the sample itself. The is made by Gran II method according to Pressinotti (9). Studies about introdution of CO2 in the solution of NaOH were made.The following methods were studied: a - by mixer bomb; b - by the cylindrical vessel bubble absorber; c - by the use of syringe injection. This last one showed to be extremaly simple and of low cost.

Made available in DSpace on 2014-06-11T19:22:18Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-12-20Bitstream added on 2014-06-13T20:28:32Z : No. of bitstreams: 1 goncalves_gs_me_arafcf.pdf: 376750 bytes, checksum: d261fb301fa1b4c966236fbf2eb5c9d0 (MD5) / Universidade Estadual Paulista (UNESP) / O presente trabalho consiste no desenvolvimento de um método analítico para determinação de biotina, utilizando-se a potenciometria indireta. O objetivo é apresentar a validação de método analítico como um processo que estime a eficiência do método proposto na rotina do laboratório para garantia da qualidade total. É um método que envolve equipamento simples e pouco dispendioso como o potenciômetro, que possibilita medir com precisão o valor da concentração de biotina. O doseamento quantitativo de biotina baseia-se no estudo das reações oscilantes do analito perante as análises da titulação indireta utilizando potenciômetro automático. A concentração de biotina foi determinada com massa adicionada conhecida numa matriz que simula uma cápsula, contendo aerosil (1%), estearato de magnésio (1%), celulose microcristalina (20%), amido (40%) e lactose q.s.p. Os resultados mostraram um valor médio de biotina (massa adicionada de aproximadamente 25 mg) determinada de 99,4% com desvio-padrão de 0,0345. As condições experimentais como temperatura, vidraria e concentração dos reagentes foram otimizadas. Os parâmetros investigados no processo de validação para demonstrar o desempenho do método foram: especificidade, linearidade, intervalo, precisão tanto repetitividade, quanto intermediária, exatidão e robustez. O tratamento estatístico dos dados da validação do método analítico envolveu a determinação da média, do desvio padrão e do coeficiente de variação. Para obtenção da curva de calibração se fez necessária a determinação da equação da reta, regressão linear e coeficiente de correlação linear. Este método apresenta grande aplicabilidade em soluções turvas, fluorescentes, opacas ou coradas, ou quando não existem, ou não podem aplicar-se indicadores visuais apropriados. Há possibilidade de determinação... / The present work shows the development of an analytical method for the determination of biotin by using indirect potentiometry. The main goal is to present the validation of analytical methods as a process to estimate the efficiency of the proposed methodology in the laboratory routine for the guaranty of total quality. This method involves simple and low cost devices as the potentiometer, which allows the precise measurement of biotin concentration. The quantitative dosing of biotin is based on the study of the oscillating chemical reactions with the analyte by performing the analysis of the indirect titration with an automatic potentiometer. Biotin concentration was determined by adding a known mass to a matrix that simulates a capsule containing aerosol (1%), estearato de magnésio (1%), celulose microcristalina (20%), amido (40%) e lactose q.s.p. The results showed an average value for the determination of biotin (added mass was ca. 25 mg) of 99.4% with a standard deviation of 0.0345. The experimental conditions as temperature, glassware, and concentration of the chemicals were optimized. The investigated parameters of the validation procedure to demonstrate the performance of the method were: specificity, linearity, interval, precision (repeatability and intermediate), exactness and robustness. The statistical treatment of the data for the validation of the analytical method involved the determination of the average value, standard deviation, and the variation coefficient. In order to obtain the calibration curve, the line´s equation, the linear regression and the coefficient of linear correlation were determined. This method shows great applicability for turbid, fluorescent, opaque or color solutions, or if an appropriate visual indicator is not available or cannot be applied. The method also shows the possibility for the determination of a sequence of equivalence... (Complete abstract click electronic access below)

Acute lymphoblastic leukemia, one of the most malignant cancer forms in children is commonly treated with the thiopurine 6-mercaptopurine (6-MP) in combination with a high dose of methotrexate (MTX). 6-Mercaptopurine is in the body metabolized by the enzyme thiopurine S-methyltransferase (TPMT). Polymorphic variants of TPMT express different catalytic activities, and for this reason the dosage of 6-MP needs to be individualized. In order to better optimize the treatment it is important to understand how mutations in TPMT affect its enzymatic activity. In this thesis we have investigated how the wild type and two variants of TPMT interact with different ligands using fluorescence and isothermal titration calorimetry. Experiments with MTX, ANS and furosemide resulted in a similar binding strength for the wild type and the variant TPMT*8, while the other variant TPMT*6 showed a slightly weaker binding. A binding affinity for polyglutamated MTX to TPMTwt was also determined which resulted in an almost twice as strong binding compared to MTX. Today’s methods to determine enzymatic activity are either based on radioactivity, time consuming or expensive. As an alternative the use of a spectrophotometric assay using 5-thio-2-nitrobenzoic acid (TNB) was investigated. The method showed positive results and could hopefully be adapted to plate readers in future experiments. Using 5.5’-dithiobis-(2-nitrobenzoic acid) (DTNB, also known as Ellman’s reagent) the amount of accessible thiol groups on the protein was estimated. This revealed a similar relationship between TPMTwt and TPMT*6, while the result for TPMT*8 was inconclusive.