This study investigates the influence of ionic strength on the binding constant (KDOC) between benzo(h)quinoline (BHQ) and LHA by using fluorescence quenching method. Being a basic polycyclic aromatic hydrocarbon, BHQ is the dominated solute as the solution¡¦s pH value is higher than BHQ¡¦s pKb. In contrast, BHQ+ is the major species as the solution¡¦s pH value is lower than BHQ¡¦s pKb. In a salty neutral or basic LHA solution, the cation will bind with the acidic functional groups of LHA, then the conformation of LHA would be coiled up to be small in size. Due to that, leading to the decrease of the corresponding BHQ¡¦s KDOC. Furthermore, the charge density of cation is an important factor in control of the variation of BHQ¡¦s KDOC. The lower charge density of cation is, the less BHQ¡¦s KDOC varied. Besides, SO4 2- may suppress the binding affinity between Na+ and the acidic function groups of LHA, so that lower variation of BHQ¡¦s KDOC was observed than that of Cl- in a Na+ contained LHA solution. In an acidic solution, cation will also bind with the acidic functional groups of LHA, leading to the decrease of the binding sites of BHQ+ on LHA and the corresponding BHQ+¡¦s KDOC. Besides, Mg2+ could provide more binding sites for the acidic functional groups of LHA than Na+, so that the variation of BHQ+¡¦s KDOC with Mg2+ addition is higher than that with Na+ addition.

Polycyclic aromatic hydrocarbons (PAHs) are in general characterized by high toxicity, persistance, and bio-accumulation in environment. It is important to understand the transport and fate of PAHs in environment. In aquatic environment, PAHs are easily bound with dissolved organic matter (DOM), such as humic substance, and KDOC is usually used in denoting the binding constant. Previously, many investigations focused on the binding of a single PAH to DOM. However, few researches considered the competition effect on the binding of multiple PAHs to DOM. This study therefore explored the competition between the binary PAHs, pyrene and phenanthrene, in binding with humic substances. No significant competition effect in KDOC could be observed between these two compounds with LHA (Leonardite Humic Acid) because of the relatively large variation in KDOC determination. Humic substances from different sources might have different properties. Several characteristic indicators of humic substances were used to interprete the variations of KDOC of pyrene and phenanthrene, such as elemental ratio, total acidity, functional group content, etc. Negative correlations of O/C and phenolic group content to the KDOC of pyrene and phenanthrene were observed (p<0.05). In contrast, positive correlation of aromaticity and £`280 of humic substance to the KDOC of pyrene and phenanthrene were observed (p<0.05). Therefore, the contents of phenolic and aromatic groups of humic substance are essential factors in determining their binding constants with PAHs.

Advances in analytical chemistry have resulted in a growing body of literature showing measurable concentrations of pharmaceuticals in both drinking and wastewater. Removal of such chemicals is typically inefficient and often poorly characterized. To characterize one such method of removal (micellar enhanced ultrafiltration, (MEUF)), interactions of a cetyl trimethylammonium bromide (CTABr) surfactant and sulfonamide antibiotics were examined by NMR and semi-equilibrium dialysis (SED). The locus and orientation of binding in a micelle was established for seven sulfonamides by 1H NMR, and it was found that hydrophilic sulfonamides showed weak coordination with the micelle, whereas hydrophobic sulfonamides penetrated into the micellar interior with coordination of the SO2NH group to the charged surface layer. Binding constants were determined by 1H NMR and showed apparent order of magnitude differences between nuclei. Several compounds were unable to be characterized in this manner due to low change in chemical shift with addition of CTABr. SED was performed as an alternative method to determine binding constants. Values determined in this manner were higher than those determined by 1H NMR. Binding constants were converted into changes in Gibbs free energy and used to evaluate and, where necessary, modify the orientation and locus proposed by 1H NMR. Attempts are made to correlate binding constants with octanol-water partition coefficients to determine if a free energy relationship can be derived. Characterization of these systems may allow for a predictive methodology to determine the MEUF removal efficiencies of new sulfonamide and surfactant combinations. It is also hoped that this work may be generalized to predict MEUF efficiency for a wide range of contaminants that might be found in wastewater. / Thesis (Master, Chemistry) -- Queen's University, 2011-01-31 09:46:28.248

Estuary is a complex region due to the mixing of fresh and sea water as well as the mixing of terrestrial and marine dissolved organic matters (DOM), so that the sorption behavior between hydrophobic organic compounds and dissolved organic matters is very complicated to estimate. By applying fluorescence quenching method, we investigated the influence of the mixing of binary dissolved organic matters from different sources on the binding constant (Kdoc) of pyrene and DOM. Results show that the kinetic of mixing of binary DOMs influences the binding constant, that means if there weren¡¦t enough standing time for mixing of binary DOMs, it would cause some bias in estimating the binding constants of pyrene and DOMs mixture. In this study, we also found that there exists a linear relationship on the binding constants between pyrene and DOMs mixture against mixing ratios. In addition, the mixing mechanism of SRNOM and LHA was dominated by diffusion process from the results of initial mixing efficiency. However, the diffusion process might not be the only mechanism of the mixing reaction of SHHA and LHA, other factors should be considered. Further investigations should be done to find out the dominating factors on the mixing of SHHA and LHA system in the future.

Resorcinarenes can be functionalized at their upper and lower rims. In this work, the upper rim of a resorcinarene was functionalized with glutamic acids and the lower rim was functionalized with either methyl or undecyl alkyl groups. The cavitands were characterized by nuclear magnetic resonance (NMR), mass spectrometry (MS), UV-vis spectroscopy, dynamic light scattering (DLS) and electron microscopy. The binding of resorcinarene with amine guests was studied in DMSO by UV-vis titration. The obtained binding constants (K values) were in the range of 12,000-136000 M-1. The resorcinarenes were shown to form aggregates in a variety of solvents. The aggregates were spherical as confirmed by DLS, SEM and TEM experiments. Dynamic light scattering (DLS) experiments revealed the size of the aggregates could be controlled by cavitand concentration, pH, and temperature. The resorcinarene with undecyl alkyl group were adsorbed onto 55% cross-linked styrene-divinylbenzene resin to prepare a new stationary phases for ion chromatography (IC) columns. The new column packing material was applied in determination of uremic toxins and water contaminants. The new IC column afforded separation of the five uremic toxins : guanidinoacetic acid, guanidine, methylguanidine, creatinine, and guanidinobenzoic acid in 30 minutes. Detection and quantification of uremic toxins helps diagnose kidney problems and start patient care. Gradient elutions at ambient temperature with methanesulfonic acid (MSA) as eluent resulted in detection levels in water from 10 to 47 ppb and in synthetic urine from 28 to 180 ppb. Trace levels of creatinine (1 ppt) were detected in the urine of a healthy individual using the columns. The new IC stationary phase separated cationic pharmaceuticals including a group of guanidine compounds in surface water. Detection limits in the range of 5 - 32 µg L-1 were achieved using integrated pulsed amperometric detection (IPAD) for guanidine (G), methylguanidine (MG), 1,1-dimethylbiguanidine (DMG), agmatine (AGM), guanidinobenzoic acid (GBA) and cimetidine (CIM). Suppressed conductivity (CD) and UV-vis detection resulted in limits of detection similar to IPAD, in the range of 1.7 - 66 µg L-1, but were not able to detect all of the analytes. Three water sources, river, lake, and marsh, were analyzed and despite matrix effects, sensitivity for guanidine compounds was in the 100 µg L-1 range and apparent recoveries were 80-96 %. The peak area precision was 0.01 - 2.89% for IPAD, CD and UV-vis detection.

The research presented in this thesis involves the application of chiral cationic and anionic surfactants for simultaneous enantioseparation of structurally similar compounds in capillary electrophoresis (CE) and CE coupled to mass spectrometry (CE-MS). The first chapter briefly introduces the fundamentals of CE and CE-MS, emphasizing the micellar electrokinetic chromatography (MEKC) and MEKC-MS techniques, as well as ionic liquids (ILs) and affinity CE (ACE). In chapter 2, a mixture of five racemic profen (PROF) drugs are simultaneously separated with the combined use of 2,3,6-tri-O-methyl-β-cyclodextrin (TM-β-CD) and IL-type surfactant, N-undecenoxycarbonyl-L-leucinol bromide (L-UCLB). Enantioseparations of these PROFs are optimized using a standard recipe containing 35.00 mM TM-β-CD, 5.00 mM sodium acetate at pH 5.0, and varying the concentration as well as chain length of the IL surfactants. The batch-to-batch reproducibility of L-UCLB is found to be acceptable in terms of enantiomeric resolution, and migration time. A competitive inhibition mechanism is proposed to investigate the ternary interactions among TM-β-CD, ILs, and PROFs. The apparent binding constant of TM-β-CD to L-UCLB is estimated by nonlinear and linear plotting methods. The binding constants of one representative PROF (e.g., fenoprofen) to TM-β-CD and to L-UCLB are estimated by a secondary plotting approach. The R- and S-fenoprofen having different binding constant values, resulting in the enantioseparation due to the synergistic effect of TM-β-CD and L-UCLB. The R- and S-configurations of barbiturates display differences in potency and biological activity. In Chapter 3, a multivariate MEKC-ESI-MS approach for the simultaneous analysis of the racemic mixture of three barbiturates is presented. The chiral selector employed is the polymeric surfactant polysodium N-undecenoxycarbonyl-L-isoleucinate. The central composite design is used to optimize the chiral resolution, decrease the total analysis time, and improve the ESI-MS signal-to-noise ratio for these barbiturates. In preliminary experiments, the ranges of the factors investigated in the multivariate approaches are determined. Then the multivariate optimizations are conducted to determine the best overall chiral resolution with shortest possible run times for barbiturates. The limit of detection of ESI-MS is several folds higher compared to the UV detection. The predicted optimum results are in good agreement with the experimental data.

<p>The goal of this work is to develop novel molecular systems, functionalization techniques, and data collection routines with which to study the binding of immobilized cognate binding partners. Our ultimate goal is the routine evaluation of thermodynamic parameters for immobilized systems through interpretation of the variation of the binary probability of binding as a function of soluble ligand concentration. The development of both data collection routines that minimize non-specific binding and functionalization techniques that produce stable ordered molecular systems on surfaces are of paramount importance towards achievement of this goal. Methodologies developed here will be applied to investigating the thermodynamics of multivalent systems.</p><p>In the first part of this work, the effect of contact force on molecular recognition force microscopy experiments was investigated. Increased contact forces (>250 pN) resulted in increased probabilities of binding and decreased blocking efficiencies for the cognate ligand-receptor pair lactose-G3. Increased contact force applied to two control systems with no known affinity, mannose-G3 and lactose-KDPG aldolase resulted in non-specific ruptures that were indistinguishable from those of specific lactose-G3 interactions. Thus, it is essential to design data collections routines that minimize contact forces to ensure that ruptures originate from specific, blockable interactions.</p><p>In the second part of this work we report the first example of the preparation of stable self assembled monolayers through hydrosilylation of a protected aminoalkene onto hydrogen-terminated silicon nitride AFM probes and subsequent conjugation with biomolecules for force microscopy studies. Our technique can be used as a general attachment technique for other molecular systems.</p><p>In the third part of this work we develop novel molecular systems for tethering oriented vancomycin and its cognate binding partner L-Lys-D-Ala-D-Ala to surfaces and AFM tips. Unbinding experiments demonstrated that traditional methods for forming low surface density amine layers (silanization with APTMS and etherification with ethanolamine) provided molecular constructs which displayed probabilities of binding that were too low and showed overall variability too high to use for probabilistic evaluation of thermodynamics parameters. Instability and heat-induced polymerization of APTMS layers on tips and surfaces also prohibited their utility. Formation of Alkyl SAMs on silicon provides a more reliable, stable molecular system anchored by Si-C bonds that facilitates attachment of vancomycin and is capable of withstanding prolonged exposure to heated organic and aqueous environments. It follows that covalent immobilization of KDADA to silicon nitride AFM tips via Si-C bonds using hydrosilylation chemistry will be similarly advantageous. These methods offer great promise for probabilistic evaluation of thermodynamic parameters characterizing immobilized binding partners and will permit unambiguous determination of the role of multivalency in ligand binding, using an experimental configuration in which intermolecular binding and aggregation are precluded.</p> / Dissertation

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No. of bitstreams: 2 Dissertação- Michelly Christine dos Santos - 2012.pdf: 1834625 bytes, checksum: af88a6d02e2b5362de31315d2c4ff78c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2012-10-11 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Magnetic nanoparticles with two different chemical compositions, magnetite and zinc doped manganese ferrite were synthesized, functionalized with lauric acid bilayers and dispersed into water yielding stable aqueous colloidal suspensions at physiological pH. Bioactive molecules of different sizes, Amphotericin B and curcumin, were associated to the lauric acid bilayers of the suspended nanoparticles. X-ray diffraction analyses reveled a diffraction pattern characteristic of the cubic spinel crystalline phase for both samples. The nanoparticles average size estimated by Debye-Scherrer’s formula was 9 nm for zinc doped manganese ferrite and 8 nm for magnetite. The hysteresis curves showed that the nanoparticles are super paramagnetic at room temperature with saturation magnetization of 44.2 emug-1 for zinc doped manganese ferrite and 51.5 emug-1 for magnetite. The IR analysis showed the presence of lauric acid associated to the nanoparticles, and the TG curves showed a higher amount of lauric acid bonded to zinc doped manganese ferrite. The hydrodynamic diameters of the nanoparticles indicate that they are dispersed into the suspension as aggregates, and that the association of bioactive molecules did not affect the hydrodynamic sizes of the aggregates. The IR spectra confirmed the presence of bioactive molecules in the formulations and the UV-VIS spectra showed that these molecules are solubilized into the lauric acid bilayers in their monomeric forms. The functionalized nanoparticles containing the bioactive molecules present less negative zeta potential than the lauric acid functionalized nanoparticles, indicating that the bioactive molecules interact with the medium of the suspensions. The evaluation of aggregation of the nanoparticles in cell culture medium showed that the nanoparticles aggregation is significantly reduced by the addition of proteins (FBS and BSA) in cell culture medium. The study of interaction of the nanoparticles with bovine serum albumin before and after the association with the bioactive molecules showed that these systems present great affinity for BSA, with binding constants (Kb) in the range of 105 -106M-1. These results suggest that the nanoparticles functionalized with lauric acid bilayers, pure or associated to amphotericin B and curcumin, can be bioconjugated to proteins producing formulations with higher colloidal stability in biological media. / Nanopartículas magnéticas com duas diferentes composições químicas, magnetita e ferrita de manganês dopada com zinco, foram sintetizadas, funcionalizadas com bicamadas de ácido láurico e dispersas em meio aquoso produzindo suspensões coloidais estáveis em pH fisiológico. Posteriormente, foram associadas moléculas bioativas de tamanhos diferentes, Anfotericina B e curcumina, nas bicamadas de ácido láurico funcionalizadas nos dois tipos de nanopartículas em suspensão. A caracterização estrutural e composicional das nanopartículas foi realizada por difração de raios X e análise química dos teores de íons metálicos. Os difratogramas mostraram um padrão de difração referente à formação de material constituído por uma única fase cristalina cúbica do tipo espinélio. Os tamanhos médios estimados pela fórmula de Debye-Scherrer foram 9 e 8 nm para as nanopartículas de ferrita de manganês dopadas com zinco e de magnetita, respectivamente. Os teores dos íons metálicos indicaram um pequeno desvio na estequiometria final dos sólidos em relação à estequiometria inicial. As técnicas utilizadas para caracterizar as nanopartículas funcionalizadas antes e após a associação da Anfotericina B e da curcumina foram IV, UV-VIS, TG, VSM e ainda foram realizadas medidas de potencial zeta e de diâmetro hidrodinâmico. As curvas de histerese indicaram a formação de nanopartículas superparamagnéticas à temperatura ambiente com magnetização de saturação de 44,2 emug-1 para a ferrita de manganês dopada com zinco e 51,6 emug-1 para a magnetita. As análises de IV mostraram que as nanopartículas foram funcionalizadas com ácido láurico e as curvas de TG indicaram que as nanopartículas de ferrita de manganês dopadas com zinco contêm maior quantidade de ácido láurico do que as nanopartículas de magnetita. Os valores de diâmetros hidrodinâmicos das nanopartículas indicaram que as mesmas estão dispersas na suspensão na forma de agregados e que a associação das moléculas bioativas não influenciou nos tamanhos hidrodinâmicos dos agregados. Os espectros de IV comprovaram a presença das moléculas bioativas nas formulações e a análise dos espectros de UV-VIS mostrou que as moléculas bioativas estão solubilizadas nas bicamadas de ácido láurico em suas formas monoméricas. As nanopartículas funcionalizadas associadas às moléculas bioativas apresentaram potencial zeta menos negativo que as nanopartículas funcionalizadas, indicando que as moléculas bioativas interagem com o meio das suspensões. O estudo de agregação de nanopartículas em meio de cultura celular mostrou que a adição de proteínas contribui significativamente para a diminuição da agregação das nanopartículas neste meio. O estudo de interação entre a albumina de soro bovino (BSA) e as nanopartículas funcionalizadas antes e após a associação das moléculas bioativas mostrou que os sistemas têm grande afinidade com a BSA, com constantes de ligação (Kb) da ordem de 105–106 M-1. Esses resultados sugerem que as nanopartículas funcionalizadas com bicamadas de ácido láurico, puras ou associadas à Anfotericina B e à curcumina podem ser bioconjugadas a essa proteína produzindo formulações com maior estabilidade coloidal em meios biológicos.

This thesis presents an investigation of fluorescence resonance energy transfer (FRET) as a reporting signal for protein-protein interactions. Quantitative optical assays to measure protein binding, conjugation and deconjugation are developed and results validated by conventional biochemical techniques. The optical techniques developed provide fast, cheap, quantitative and accurate alternatives to conventional methods. Fluorescent protein fluorophores ECFP and Venus-EYFP were chosen as they are a non-interfering FRET pair and provide an inexpensive and convenient cloning-based labelling method. The small ubiquitin-like modifier SUMO and the SUMOylation pathway leading to its conjugation to target proteins is investigated as a model system. These assays are hence particularly relevant to research on post-translational modification and ubiquitin systems. In protein-protein binding assays we utilise both steady-state and time-resolved FRET detection to measure the equilibrium binding constant of the well-characterised pair SUMO1 and Ubc9. An assay in multi-well plate format is also presented, which uniquely enables repeat measurements under varying conditions and under the addition of further substances. The multi-protein binding interactions of the SUMOylation pathway including RanBP2 are analysed in binding inhibition assays. Our results clarify the role of RanBP2: a covalent SUMO1-Ubc9 link is required for the formation of a trimeric complex, although mutual binding sites are present on all three proteins. Furthermore, the binding of SUMO1 and Ubc9 is disrupted by RanBP2, which may be an essential step in transferring SUMO1 to its target protein. A FRET-based kinetic study of this conjugation process to RanGAP1 is presented. An assay to monitor the deconjugation of SUMO1 by specific proteases is established using a doubly-tagged SUMO construct. This enables a quantitative analysis of protease and substrate specificity based on real-time kinetic data, a characterisation of crude cell extracts and a high-throughput screen for protease inhibitors using FRET. A screen of the National Cancer Institute (NIC) diversity set for SenP1 inhibition reveals nine suitable compounds, which are potential anti-cancer drugs. The results of two further projects, the study of protein-protein binding by measuring small refractive index changes and the autofluorescence of normal and neoplastic cervical tissue models are also presented. In the latter, principal component analysis was used to systematically identify emission regions of significant variation between samples, enabling discrimination between healthy and pre-cancerous tissue models.

Polycyclic aromatic hydrocarbons (PAHs) and nitrogen-containing polycyclic aromatic compound (N-PAC) are widespread toxic pollutants in environments. The fate of PAHs and N-PACs are of great concern because some of these compounds were identified as caricinogenic, mutagenic and teratogenic compounds. As described in literature, dissolved organic matter (DOM) is an important factor in control of their fate; however, the binding behaviors between these compounds and DOM are still not fully understood. The binding constants (KDOC) between humic substances and one selected N-PAC, benzo[h]quinoline, were measured at varying pH levels using fluorescence quenching (FQ) method. As fluorescence characteristics of benzo[h]quinoline change with pH, determination required two optimum sets of excitation and emission wavelength pairs. A simple mixing model was proposed and used to eliminate the inherent fluorescence interference between benzo[h]quinoline (BQ) and its protonated form, benzo[h]quinolinium (BQH+), and to deduce Kmix which represents the overall binding as the sum of that for the individual analogs. The characteristics of humic substances, especially their hydrophobicity and aromaticity, established by principal components analysis of structural and elemental compositions, were the main determinants of their binding affinity with both benzo[h]quinoline and benzo[h]quinolinium (KBQ and KBQH+) across a range of pH values. Hydrophobic interaction is likely to control the binding between humic substance and benzo[h]quinoline and benzo[h]quinolinium, in lower and higher pH ranges (pH<3, pH>6). In contrast, cation exchange seems to control on the binding affinity of benzo[h]quinolinium in the middle range of pH. Determination of PAH concentration is quite essential for investigating the fate of PAHs in environments. Microwave-assisted headspace solid-phase microextraction (MA-HS-SPME) with a polydimethylsiloxane/divinylbenzene (PDMS/DVB) fiber was applied as a single step prior to determination of PAH concentrations in water using GC-MS. To optimize the extraction efficiency of PAHs by MA-HS-SPME, the influence of various parameters, including temperature, duration of thermal desorption, microwave irradiation power and duration, and the temperature of the circulating cooling water system, was studied. The proposed method was demonstrated applicable to environmental water samples. In addition, DOM matrix effect did not influence the determination and extraction efficiency of PAHs. Although the proposed simple mixing model can eliminate the fluorescent interference of hydrophobic organic compounds with acid-base pair forms, it is still limited in using for correcting the KDOC measurement of more than two fluorescent compounds simultaneously. A new alternative protocol, complexation-flocculation combined with MA-HS-SPME/GC-MS method, was proposed to determine the binding constants of seleted PAHs to humic substances. The results obtained are comparable with KDOC data reported in literatures. CF-MA-HS-SPME/GC-MS provides some advantages over other methods, such as applicable not limited to fluorescent compounds, faster in determination and capable in measuring varieties of compounds simultaneously.