Nesta tese são apresentados os resultados de síntese e caracterização dos nanocompósitos de poli(anilina) (PANI) e de poli(benzidina)(PBZ) com argilas. Através do emprego de diferentes técnicas espectroscópicas e de microscopia eletrônica foi possível confirmar a intercalação dos monômeros e sua polimerização. Quando a polimerização foi feita a partir do monômero intercalado na argila (polimerização in situ) são obtidos nanocompósitos no qual a maior parte das cadeias poliméricas está intercalada. Entretanto, quando a polimerização é feita sem a prévia intercalação do monômero (polimerização ex situ), ocorre a formação dos polímeros na superfície externa da argila. Usando a espectroscopia Raman ressonante e de Absorção de raios-X na borda K do nitrogênio (N K XANES) foi possível elucidar a estrutura dos segmentos cromofóricos da PANI e da PBZ intercaladas, e pela primeira vez foi mostrado a presença de um segmento cromofórico na PANI intercalada, não presente na PANI livre, que possui em sua estrutura ligações azo e anéis cíclicos do tipo fenazina. A síntese da PANI intercalada em diferentes argilas e em diferentes condições de síntese permitiu verificar a influência destes fatores na formação desses novos segmentos. / The present work shows the synthesis and characterization of the nanocomposites of poly(aniline) (PANI) and poly(benzidine) (PBZ) formed inside the montmorillonite (MMT)clay galleries. Through the use of a large number of spectroscopic and microscopic techniques was possible to confirm the monomer intercalation and polymerization between the clay layers. When the synthesis is carried out after previous monomer intercalation (in situ route) the formation of polymer occurs mainly between the clay layers. On the other hand, the formation of polymer in external clay surface (ex situ route) occurs when the polymerization is carried out without previous monomer intercalation. Using resonance Raman spectroscopy and X-ray near nitrogen K-edge absorption (N K XANES) was possible to elucidate the structure of chromophoric segments in intercalated PANI and PBZ. For the first time, we show the presence of a new chromophoric segment, having phenazinic and azo groups, in the PANI intercalated by in situ route, indicating that the confinement of monomer is one of the factors leading to the formation of these new segments. The synthesis of intercalated PANI in different clays and experimental reaction conditions show that these factors, not only the confinement, have played an important role in formation of the new chromophoric segments in the structure of intercalated PANI.

Neste trabalho foi realizado o estudo espectroscópico (UV-VIS-NIR, FTIR, Raman e RMN) dos produtos de polimerização da anilina com persulfato de amônio em condições de síntese diferentes da utilizada na síntese padrão da polianilina (pH inicial = 1). A reação foi realizada em meio de HCl (pH inicial = 3) e em meio tamponado (pH = 3). No primeiro caso foi observada a formação majoritária de oligômeros de anilina tipo cabeça-cauda, enquanto que em meio tamponado são formadas macromoléculas com segmentos do tipo 1,4 de Michael de anilina e benzoquinona monoimina com diferentes graus de hidrólise/oxidação, além de unidades tipo fenazina. Esta caracterização está de acordo com as propostas distintas de dois grupos (Surwade et al. e Stejskal et al.), que a princípio pareciam contraditórias. Ainda com relação aos produtos obtidos em meio tamponado (pH = 3), é observada a formação de microesferas com diâmetro entre 3-7 µm. A microscopia Raman juntamente com a espectroscopia Raman ressonante revelou uma correlação entre estrutura tipo fenazina e a formação das microesferas. / In this work it was performed the spectroscopic study (UV-VIS-NIR, FTIR, Raman e NMR) of the polymerization products of aniline and ammonium persulfate using different reaction conditions in relation to the standard polyaniline synthesis (initial pH = 1). The reaction was performed in HCl medium (initial pH =3) and in buffered medium (pH = 3) In the first case it was observed the major formation of aniline oligomers with head-to-tail coupling, while in buffered medium macromolecules with 1,4 Michael-type segments between aniline and benzoquinone monoimine with different hydrolysis/oxidation ratio of iminic groups are formed, in addition to phenazine-like units. This characterization is in accordance to the distinct proposals of two groups (Surwade et al. e Stejskal et al.), which at first seemed contradictory. Again, relative to the products obtained in buffered solution (pH = 3), it is observed the formation of microspheres whose diameter range is 3-7 µm. The use of Raman microscopy and resonance Raman spectroscopy revealed a correlation between phenazine-like structure and the microspheres formation.

Protein engineering strategies, such as directed evolution, generate large libraries of enzyme variants, typically in the range of 106-108 variants. However, the availability of rapid, robust high-throughput screening methods has often limited the impact of directed evolution in discovering enzymes with enhanced catalyst performance. Raman spectroscopy is an established analytical technique, providing molecular specific information, permitting analysis in aqueous solutions and as such is an attractive, alternative screening method for biological systems. Although an inherently weak physical phenomenon, enhanced Raman scattering techniques, such as surface enhanced Raman scattering (SERS) and ultraviolet resonance Raman (UVRR) spectroscopy, can be used to overcome the associated sensitivity issues. Herein, we successfully monitored xanthine oxidase (XO) catalysed conversions of xanthine to uric acid, before extending to hypoxanthine, using two contrasting Raman scattering enhanced approaches. Firstly, a SERS-based assay was developed utilising silver nanoparticles to measure analytes directly and quantitatively on micromolar scale, in the absence of chromogenic substrates or lengthy chromatography. Secondly, a UVRR approach was developed enabling monitoring of the XO-mediated reaction in real-time and without the need to quench the system. Significantly, both methods demonstrated over &gt;30 fold reduction in acquisition times (when compared to conventional HPLC analysis), and offered excellent medium-term reproducibility and accuracy of results over significant time periods. Furthermore, investigations were made into developing this SERS-based assay into an enantiomeric screen using another vibrational spectroscopy approach, Raman optical activity (ROA), along with circular dichroism (CD). Successful chiral reduced nanoparticles were synthesised, with multiple characterisation techniques employed, affording enantiopure Au-cysteine and Ag-tyrosine colloids. However, it was not possible to generate consistent and reproducible SEROA responses, with these techniques ultimately being unsuccessful in analysing these chiral sensitive nanoprobes, and thus differentiating between the D- and L- forms. Finally, a novel SERS-based approach, in combination with the standard addition method (SAM), was developed for the routine analysis of uric acid (end product in XO catalysed reaction(s) and biomarker for various diseases), at clinically relevant levels in urine samples from patients. Results were highly comparable and in very good agreement with HPLC analyses, with an average < 9% difference in predictions between the two analytical approaches across all samples analysed, and a 60-fold reduction in acquisition time (when compared with HPLC). Together, the research presented in this thesis demonstrates the suitability of Raman enhanced techniques for quantitative analysis, measuring the analytes directly using a portable Raman instrument and, most importantly, offering significant reductions in acquisition times when compared to established analytical techniques.

Das Ziel der vorliegenden Arbeit ist die bessere Verarbeitung von Polyanilin (PANI), da dies bisher ein großer Nachteil unter leitfähigen Polymeren war. Dazu wird ein sperriges Tensid und Dotand, Dodecylbenzensulfonsäure (DBSA) verwendet. Zur Synthese der PANI kommen zwei verschiedene Methoden zur Anwendung, die in dieser Dissertation in zwei Kapiteln beschrieben werden. Im ersten Teil wurden in einem kleinen Reaktionsvolumen (250 mL) PANI-DBSA-Suspensionen synthetisiert, wobei mit einem binären Gemisch aus 2-Propanol und Wasser als Lösungsmittel gearbeitet wird um die Löslichkeit zu unterstützen. Die micellenunterstützte Synthese produziert grüne Dispersionen, welche nach länger als einem Jahr noch keine sichtbare Ausscheidung zeigen. Eine detaillierte spectroelektrochemische Untersuchung der PANI-DBSA-Nanokolloide wurde durchgeführt und gibt eine bessere Erklärung der Charge-Transfer-Prozesse zwischen PANI-Kolloiden und Elektrodenoberfläche. In einem alkalischen Medium ist das UV-Vis-Spektrum von der Beweglichkeit der Anionen und von einem elektrokinetischen Phänomen abhängig. Um den „metal-to-insulator”-Übergang zwischen PANI-Kolloiden, welcher durch pH-Wert-Änderung des Mediums geschehen kann, zu zeigen, wurden UV-Vis- und pre-resonanz-Raman-Spektroskopie verwendet. Im zweiten Teil der Dissertation wird zur Polymerisation von Anilin sowie seinen Copolymeren mit o-Toluidin eine neue Technik der Polymerisation beschrieben, welche durch inverse Emulsion erfolgt. Diese benutzt Benzoylperoxid, ein ungewöhnlicheres organisches Oxidationsmittel. Die erhaltenen PANI sind in gebräuchlichen organischen Lösungsmitteln, wie in Chloroform, vollständig löslich. Mit einer klar-transparenten, grünen Lösung von PANI können metallische Oberflächen oder Glas leicht tropfenbeschichtet werden. Zyklische Voltammetrie und spektroelektrochemische Verfahren kamen zum Einsatz, um die Elektroaktivität, das UV-Vis-Verhalten und die „metal-to-insulator”-Übergänge der chemisch synthetisierten PANI als Funktion des verwendeten Elektrodenpotentials zu untersuchen. Die elektrische Leitfähigkeit der Materialien ist relativ hoch (R = 10 ). SEM-Untersuchungen zeigen, dass die Menge des zugesetzten DBSA die Morphologie des Polymers stark beeinflusst. Aus in situ UV-Vis-spektroskopischen Messungen lässt sich eine gute elektrochromische Reversibilität des Polymers erkennen. DBSA kann Poly(o-toluidin) (POT) effektiv dotieren, auch wenn von der Methylgruppe eine sterische Hinderung ausgeht. Die spektroskopischen Untersuchungen, wie UV-Vis, FT-IR, Raman-Spektroskopie und zyklische Voltammetrie, zeigen deutlich, dass wirkliche Copolymere gebildet werden und die Möglichkeit von Kompositen nicht in Betracht kommt. Das entstandene Poly(anilin-co-o-toluidin) (PAT) ist in schwach polaren Lösungsmitteln wie Chloroform löslich. Wie erwartet, sind die elektrischen Leitfähigkeiten der Copolymere viel kleiner als die Leitfähigkeit von PANI-DBSA.

Ferrochelatase catalyzes the terminal step of heme biosynthesis by inserting ferrous iron into protoporphyrin IX. The current study is aimed at understanding the structural basis of porphyrin binding and distortion in ferrochelatase-catalyzed reaction by functional analysis of a highly conserved active site loop motif. The loop was shown to contact bound porphyrin based on crystallographic and molecular modelling observations, and its role in murine ferrochelatase was assessed by random mutagenesis and steady-state kinetic analysis. To overcome the limitations of conventional kinetic assay methods for ferrochelatase, a continuous assay was developed by monitoring porphyrin fluorescence decrease using natural substrates ferrous iron and protoporphyrin IX under anaerobic conditions. For wild-type murine ferrochelatase, the assay yielded KmPPIX of 1.4 uÌ?M, KmFe2+ of 1.9 uÌ?M and kcat of 4.0 min-1 at 30 °C. The results of random mutagenesis indicated that all the loop re sidues spanning Q248-L257 tolerated functional substitutions. While Q248, S249, G252, W256 and L257 possessed high informational content, the other five positions contained low informational content. Selected active loop variants exhibited kcat comparable to or higher than that of wild-type enzyme, while KmPPIX was increased in most variants. The kcat/KmPPIX remained largely unchanged, with the exception of a 10-fold reduction in variant K250M/V251L/W256Y. Molecular modeling of the active loop variants suggested that loop mutations resulted in alterations of the active site architecture. Distortion of porphyrin substrate, a crucial step in ferrochelatase-catalyzed metal chelation, was examined using resonance Raman spectroscopy. The results revealed that both wild-type enzyme and loop variants induced saddling of substrate protoporphyrin. Further, loop mutations generally interfered with porphyrin saddling, with the least deformation observed in variant K250M/V251L/W256Y.N-alkyl porphyrins are potent competitive inhibitors of mammalian ferrochelatase. The present study showed that while N-methyl protoporphyrin strongly inhibited the wild-type enzyme with an inhibition constant in the nanomolar range, it was less effective in inhibiting variants P255R and P255G. These results suggest that inhibitor binding may be associated with a protein conformational change mediated by P255. Wild-type ferrochelatase is a homodimeric [2Fe-2S] cluster-containing protein. Variants S249A/K250Q/V251C and S249A/K250R/G252W were found to retain enzymatic activity in the absence of FeS cluster and form active, higher order oligomers. These observations raise the possibility that FeS cluster and homodimeric organization are not essential to ferrochelatase catalysis.

The electronegativity of thiolate ligation in the hemeprotein nitric oxide synthase (NOS) proteins has been identified as an influence on autoinhibition in this enzyme. The mutation of a conserved tryptophan residue, which hydrogen bonds to the coordinating thiolate ligand and therefore influences its electronegativity, to either phenylalanine or tyrosine has had various effects including heme loss and dimer disruption in the inducible isoforms, while hyperactivity occurs in the neuronal isoforms. I have performed the analogous mutations in W180 of eNOSoxy, the endothelial isoform. UV/visible and resonance Raman spectroscopy have demonstrated that the mutants experienced increased basicity of the thiolate due to loss of the hydrogen bond between the mutated residue in the absence of the cofactor (6R)5,6,7,8-tetrahydrobiopterin (H4B). The mutants also displayed relative rates of NO2- production that were comparable to the nNOSoxy mutants, which is consistent with the nNOSoxy results. The presence of H4B alters porphyrin planarity, which enabled hydrogen bonding to occur in W180Y, thus restoring thiolate basicity to that of wild-type eNOSoxy. Reduced overall activities by the proteins suggest that H4B stabilizes the heme. The analogous W56 mutants of saNOS, a NOS oxygenase domain-like protein from Staphylococcus aureus (saNOS), have been previously characterized using resonance Raman spectroscopy. These mutants also exhibit increased thiolate electronegativity over wild-type. As the homodimers had already been investigated, saNOS was an ideal system in which to explore heterodimers. Heterodimers were generated through the co-expression of one wild-type and one mutated subunit, enabling the examination of each subunit individually through resonance Raman spectroscopy. The subunits of the resulting proteins were shown to have heme environments that resembled those of their corresponding homodimers. The activity of saNOS did not vary significantly for the various W56 mutants, suggesting that saNOS catalysis may be unaffected by thiolate electronegativity. / Thesis (Master, Chemistry) -- Queen's University, 2008-09-04 11:37:38.688

Dissertation (Ph.D.)--Auburn University, 2008. / Abstract. Vita. Includes bibliographic references.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 246-276).

Nesta tese são apresentados os resultados de síntese e caracterização dos nanocompósitos de poli(anilina) (PANI) e de poli(benzidina)(PBZ) com argilas. Através do emprego de diferentes técnicas espectroscópicas e de microscopia eletrônica foi possível confirmar a intercalação dos monômeros e sua polimerização. Quando a polimerização foi feita a partir do monômero intercalado na argila (polimerização in situ) são obtidos nanocompósitos no qual a maior parte das cadeias poliméricas está intercalada. Entretanto, quando a polimerização é feita sem a prévia intercalação do monômero (polimerização ex situ), ocorre a formação dos polímeros na superfície externa da argila. Usando a espectroscopia Raman ressonante e de Absorção de raios-X na borda K do nitrogênio (N K XANES) foi possível elucidar a estrutura dos segmentos cromofóricos da PANI e da PBZ intercaladas, e pela primeira vez foi mostrado a presença de um segmento cromofórico na PANI intercalada, não presente na PANI livre, que possui em sua estrutura ligações azo e anéis cíclicos do tipo fenazina. A síntese da PANI intercalada em diferentes argilas e em diferentes condições de síntese permitiu verificar a influência destes fatores na formação desses novos segmentos. / The present work shows the synthesis and characterization of the nanocomposites of poly(aniline) (PANI) and poly(benzidine) (PBZ) formed inside the montmorillonite (MMT)clay galleries. Through the use of a large number of spectroscopic and microscopic techniques was possible to confirm the monomer intercalation and polymerization between the clay layers. When the synthesis is carried out after previous monomer intercalation (in situ route) the formation of polymer occurs mainly between the clay layers. On the other hand, the formation of polymer in external clay surface (ex situ route) occurs when the polymerization is carried out without previous monomer intercalation. Using resonance Raman spectroscopy and X-ray near nitrogen K-edge absorption (N K XANES) was possible to elucidate the structure of chromophoric segments in intercalated PANI and PBZ. For the first time, we show the presence of a new chromophoric segment, having phenazinic and azo groups, in the PANI intercalated by in situ route, indicating that the confinement of monomer is one of the factors leading to the formation of these new segments. The synthesis of intercalated PANI in different clays and experimental reaction conditions show that these factors, not only the confinement, have played an important role in formation of the new chromophoric segments in the structure of intercalated PANI.

Neste trabalho foi realizado o estudo espectroscópico (UV-VIS-NIR, FTIR, Raman e RMN) dos produtos de polimerização da anilina com persulfato de amônio em condições de síntese diferentes da utilizada na síntese padrão da polianilina (pH inicial = 1). A reação foi realizada em meio de HCl (pH inicial = 3) e em meio tamponado (pH = 3). No primeiro caso foi observada a formação majoritária de oligômeros de anilina tipo cabeça-cauda, enquanto que em meio tamponado são formadas macromoléculas com segmentos do tipo 1,4 de Michael de anilina e benzoquinona monoimina com diferentes graus de hidrólise/oxidação, além de unidades tipo fenazina. Esta caracterização está de acordo com as propostas distintas de dois grupos (Surwade et al. e Stejskal et al.), que a princípio pareciam contraditórias. Ainda com relação aos produtos obtidos em meio tamponado (pH = 3), é observada a formação de microesferas com diâmetro entre 3-7 &#181;m. A microscopia Raman juntamente com a espectroscopia Raman ressonante revelou uma correlação entre estrutura tipo fenazina e a formação das microesferas. / In this work it was performed the spectroscopic study (UV-VIS-NIR, FTIR, Raman e NMR) of the polymerization products of aniline and ammonium persulfate using different reaction conditions in relation to the standard polyaniline synthesis (initial pH = 1). The reaction was performed in HCl medium (initial pH =3) and in buffered medium (pH = 3) In the first case it was observed the major formation of aniline oligomers with head-to-tail coupling, while in buffered medium macromolecules with 1,4 Michael-type segments between aniline and benzoquinone monoimine with different hydrolysis/oxidation ratio of iminic groups are formed, in addition to phenazine-like units. This characterization is in accordance to the distinct proposals of two groups (Surwade et al. e Stejskal et al.), which at first seemed contradictory. Again, relative to the products obtained in buffered solution (pH = 3), it is observed the formation of microspheres whose diameter range is 3-7 &#181;m. The use of Raman microscopy and resonance Raman spectroscopy revealed a correlation between phenazine-like structure and the microspheres formation.