Devido a baixa concentração de metais pesados em amostras ambientais. há quase sempre uma necessidade de uma etapa de pré-concentração Agentes complexantes, em especial azo-compostos, quando adsorvidos numa resina trocadora de ânions e contendo grupos ácidos suIfônicos, são particularmente úteis na separação de metais, agindo como uma resina quelante capaz de sequestrar íons metálicos da solução aquosa. Neste trabalho estudamos as propriedades da resina quelante obtida por modificação do trocador aniônico macroporoso Amberlyst A-26 çom Eriochrome Red B e o Eriochrome Blue Black R. Estes reagentes proporcionam separações de íons metálicos que diferem suficientemente em afinidade pelos átomos de oxigênio doadores do ligante. Estudamos o equilíbrio de distribuição de Zn (II), Ni(II) e Co (II) sob diferentes condições experimentais. Resultados preliminares indicaram o Eriochrome Red B como o mais satisfatório para concentrar o zinco em solução aquosa, quando imobilizado na Amberlyst A-26, com subsequente determinação por espectrometria de absorção atômica com chama. Foi obtida uma recuperação maior que 99 % de um volume de um amostra em solução aquosa de 650,0 mL, atestando um alto fator de concentração de 65 e permitindo a determinação de zinco em águas numa concentração de 5 ng mL . O procedimento foi aplicado na análise de águas naturais e de efluentes industriais tratados...
A screening using several fungi (Phanerochaete chrysosporium, Pleurotus ostreatus, Trametes versicolor and Aureobasidium
pullulans) was performed on the degradation of syringol derivatives of azo dyes possessing either carboxylic or
sulphonic groups, under optimized conditions previously established by us. T. versicolor showed the best biodegradation
performance and its potential was confirmed by the degradation of differently substituted fungal bioaccessible
dyes. Enzymatic assays (lignin peroxidase, manganese peroxidase, laccase, proteases and glyoxal oxidase) and GC-MS
analysis were performed upon the assay obtained using the most degraded dye. The identification of hydroxylated
metabolites allowed us to propose a possible metabolic pathway. Biodegradation assays using mixtures of these bioaccessible
dyes were performed to evaluate the possibility of a fungal wastewater treatment for textile industries.; Instituto de Biotecnologia e Química Fina (IBQF).
Fundação para a Ciência e a Tecnologia (FCT) - Praxis XXI/BD/15878/98.
Tese de Doutoramento em Ciências (área de conhecimento Química); O objectivo deste trabalho consistiu na síntese de N-glicopéptidos
fluorescentes com potencial actividade biológica.
Inicialmente preparou-se uma glucosilamina a qual foi ligada a corantes,
aminoácidos e a péptidos pela posição anomérica, originando uma ligação amida entre
o açúcar e o grupo carboxílico do composto a ligar.
Sintetizou-se o péptido Gly-Phe-Pro por síntese sequencial e foram ainda feitas
tentativas de preparação de um glicopéptido fluorescente por síntese convergente.
Como unidades fluorescentes utilizaram-se os corantes ácido 4-(nafto[1,2-d]
[1,2,3]triazol-2-il)benzóico e o ácido cumarina-3-carboxílico, anteriormente estudados
pelo nosso grupo de investigação, que foram ligados à glucosilamina, à glicina ou à
extremidade glicina do péptido Gly-Phe-Pro.
Foi realizada a desprotecção do açúcar ligado aos fluoróforos e também
quando ligado a péptidos fluorescentes por um método catalítico em condições
A formação da ligação amida foi realizada em solução por métodos
convencionais usados em síntese peptídica.
Na pesquisa de novos fluoróforos foram feitos estudos com corantes
vinilsulfónicos tendo sido sintetizado um composto pela ligação de um corante deste
tipo à cisteína.
Foram realizadas reacções de aza-Michael com a metilvinilsulfona comercial e
com um precursor de um corante vinilsulfónico.
Os compostos foram purificados por cromatografia em coluna e/ou
recristalização e caracterizados pelas técnicas analíticas habituais.; The aim of this work was the synthesis of fluorescent N-glycopeptides with
potential biological activity.
The project began with the synthesis of a glucosylamine that was used to
perform amide linkage by the anomeric position with a carboxylic group from dyes...
Eight fluorescent compounds containing a naphthotriazole moiety substituted at position 2 by a (vinylsulfonyl)aryl group or its precursors, containing hydroxyl or sulphonic groups or N-methylglycine, were prepared and characterized. The products were recovered in moderate yields after column chromatography or recrystallization and identified by proton and carbon nuclear magnetic resonance spectroscopy. Double resonance, heteronuclear multiple quantum coherence and heteronuclear multiple bond correlation experiments were carried out for complete assignment of proton and carbon signals. Absorption and emission spectra were obtained, in acetonitrile, for all the compounds and the fluorescence quantum yields determined. All compounds are promising fluorescent probes due to their high fluorescence quantum yields.; Fundação para a Ciência e a Tecnologia (FCT) - REEQ/ 630/QUI/2005; REDE/1517/RMN/2005; FEDER - REEQ/ 630/QUI/2005; REDE/1517/RMN/2005; Research grant VZ MSMT-0021627501, Czech Republic
Dissertação de Mestrado em Engenharia do Ambiente; Neste trabalho pretendeu avaliar-se a viabilidade da utilização da macroalga castanha, Sargassum muticum, no tratamento de efluentes contendo crómio hexavalente e trivalente pelo processo de biossorção.
A escolha do biossorvente usado baseou-se nos seguintes parâmetros: uso de um material natural, custo e abundância na costa portuguesa.
Numa primeira fase submeteu-se o biossorvente a um pré-tratamento (protonação), de modo a melhorar o seu desempenho no processo de biossorção. Procedeu-se também à caraterização do biossorvente, tendo sido identificados, por espectroscopia de infravermelho, os principais grupos funcionais presentes na superfície da alga – grupos hidroxilo, carboxilo, amina e sulfónicos. Realizaram-se ainda análises SEM/EDS à alga protonada verificando-se uma alteração na composição química da parede da alga.
Numa segunda fase estudou-se a cinética de redução do Cr (VI) a Cr (III). Realizaram-se diversos ensaios onde, para diferentes condições experimentais, se avaliou a influência de parâmetros como o pH, a temperatura, a massa de alga utilizada e a concentração inicial de metal. Tendo em conta, igualmente, ensaios realizados numa fase preliminar deduziu-se que as condições operacionais ótimas para aplicação da alga protonada como biossorvente seriam: pH = 2...
Results are presented for Pt deposited on 4-aminobenzenesulphonic acid-functionalized carbon, Pt/C_ABSA. Vulcan XC-72R was functionalized in order to influence catalyst dispersion and decrease the resistance of three phase boundary, by introducing sulphonic groups and lessening the amount of Nafion® added to the catalyst layers. Electrochemical characterization of the supported catalyst was done in a 0.5 M H2SO4 solution with added methanol or chloride demonstrating well defined features and stable voltammograms after cycling, with apparent higher currents when compared with commercial catalyst. The structure sensitive adsorption of anions on platinum is confirmed by either the blocking effect of chloride ions or methanol, evident in both anodic and cathodic features of the voltammograms. Optimal performance was found for a Nafion® concentration of 15-20 wt %.
The development of renin inhibitors for the treatment of hypertension requires highly sensitive substrates to evaluate potency and to characterize the mechanism of tight-binding inhibitors. A series of intramolecularly quenched fluorogenic renin substrates, based on the N-terminal tetradecapeptide sequence of human angiotensinogen (hTDP), was synthesized using a solid-phase technique. Incorporation of the fluorescent amino acid L-Amp [L-2-amino-3-(7-methoxy-4-coumaryl)propionic acid] and the DNP (2,4-dinitrophenyl) group at various positions resulted in >90% quenching efficiency and strong product fluorescence. Shortening the hTDP sequence to an octapeptide from histidine in P5 to histidine in P3′ (substrate 3) resulted in an acceptable kcat/Km (41000 M−1·s−1) and further systematic variation gave substrate 9, DNP-Lys-His-Pro-Phe-His-Leu-Val-Ile-His-L-Amp, with a kcat/Km value of 350000 M−1·s−1 and 94% quenching efficiency. The free side chain of lysine, replacing the isoleucine residue at P6 position in the angiotensinogen sequence, contributed to the increased value for kcat. The pH dependence of kcat/Km for renin and substrate 9 showed that the optimal pH is at pH 6–7. It also showed two titrating groups on the acidic side of the pH optimum...
1. Modification of potato (Solanum tuberosum) lectin with acetic anhydride blocked 5.1 amino and 2.7 tyrosyl groups per molecule of lectin and decreased the haemagglutinating activity of the lectin. De-O-acetylation regenerated 2.0 of the tyrosyl groups and resulted in a recovery of activity. 2. Modification with citraconic anhydride or cyclohexane-1,2-dione did not greatly affect activity, although modification of amino and arginyl groups could be demonstrated. 3. Treatment with tetranitromethane nitrated 3.7 tyrosine residues per molecule of lectin with concomitant loss of activity. The presence of 0.1m-NN′N″-triacetylchitotriose (a potent inhibitor of the lectin) in the reaction medium protected all the tyrosyl residues from nitration and the lectin was fully active. 4. Modification of tryptophyl groups with 2-hydroxy-5-nitrobenzyl bromide and 2,3-dioxoindoline-5-sulphonic acid modified 0.9 and 2.6 residues per molecule of lectin respectively with a loss of activity in each case. Reaction of potato lectin with 2,3-dioxoindoline-5-sulphonic acid in the presence of inhibitor protected 2.4 residues of tryptophan from the reagent. Loss of haemagglutination activity was prevented under these conditions. 5. Reaction of carboxy groups...
The effects of p-chloromercuribenzoic acid and chloromercuribenzene-p-sulphonic acid on pancreatic islets were studied in vitro. Obese–hyperglycaemic mice were used as the source of microdissected islets containing more than 90% β-cells. p-Chloromercuribenzoic acid and chloromercuribenzene-p-sulphonic acid stimulated insulin release at concentrations of 0.01mm or above. This stimulation was significantly inhibited by the omission of Ca2+ or the addition of adrenaline, diazoxide or 2,4-dinitrophenol. p-Chloromercuribenzoic acid or chloromercuribenzene-p-sulphonic acid did not interfere with the insulin-releasing ability of glucose. Micro-perifusion experiments revealed that the release of insulin in response to organic mercurial occurred almost instantaneously, was reversible, and was biphasic. The two mercurials inhibited glucose transport as well as glucose oxidation, and increased the mannitol and sucrose spaces of isolated islets. Compared with the effects on insulin release, those on glucose transport and membrane permeability were characterized by a longer latency and/or required higher concentrations of organic mercurial. Apart from a seemingly higher proportion of β-cells exhibiting certain degenerative features, in islets exposed to 0.1mm-chloromercuribenzene-p-sulphonic acid for 60min...
Using thiol blocking agents, we examined the role of sulphydryl groups for function of the lysosomal sulphate transport system. Monothiol binding reagents, p-hydroxymercuribenzoic acid (p-HMB) and p-chloromercuribenzene sulphonic acid (p-CMBS), dithiol binding reagents such as CuCl2, the alkylating agent, N-ethylmaleimide (NEM), and NADH all inhibited lysosomal sulphate transport. The inhibitory effects of NEM and Cu2+ were not additive, suggesting that they both act upon the same critical sulphydryl group(s). Unlike the case for NEM, the inhibitory effects of Cu2+ were reversed by the reducing agent, dithiothreitol. Exposure to NEM resulted in a seven-fold increase in Km to 867 microM versus a control value of 126 microM and a modest decrease in Vmax to 99 pmolperunit beta-hexosaminidase per 30 s versus a control value of 129 pmolperunit beta-hexosaminidase per 30 s. Similar although somewhat less dramatic results were obtained using Cu2+ with an increase of Km to 448 microM and a Vmax of 77 pmolperunit beta-hexosaminidase per 30 s. The sulphate transport activity of detergent solubilized lysosomal membranes could be bound to a p-chloromercuribenzoic acid (p-CMB)-Sepharose sulphydryl affinity resin and eluted with mercaptoethanol. Sulphydryl groups thus appear to play a role in sulphate transport through effects on substrate affinity. Sulphydryl-binding appears to be a strategy that may be useful for purification of the transporter.
1. We have investigated the effects of externally applied trinitrobenzene sulphonic acid (TNBS) on sodium currents of voltage-clamped frog skeletal muscle fibres. TNBS is a membrane-impermeant reagent which reacts specifically with amino groups under physiological conditions. 2. TNBS shifts the voltage dependence of steady-state sodium current inactivation (h infinity) by approximately 18 mV to more hyperpolarized potentials when measured at pH 9. This effect of TNBS is irreversible, suggesting that the reagent permanently modifies some membrane component(s). 3. Time constants for the development of and recovery from inactivation of sodium current are similarly shifted to more hyperpolarized potentials by TNBS treatment. External TNBS did not affect the completeness of sodium current inactivation during depolarizing pulses. 4. The activation of sodium current is less affected by TNBS than is inactivation. The voltage dependence of peak sodium current activation was shifted by only approximately 4 mV by TNBS treatment at pH 9. The kinetics of sodium current activation during depolarization were little affected by TNBS. 5. The TNBS-induced shift in h infinity and sodium current activation are independent of pH when measured at pHs between 7.4 and 11. 6. The results are consistent with the hypothesis that TNBS increases the negative surface charge of the membrane by reacting with membrane amino groups having a high pK.
BACKGROUND: Growing evidence suggests that intestinal recovery from injury induced by radiation, endotoxin, and protein deficiency is improved by the ingestion of nucleosides and nucleotides. AIM: This study examined the effect of dietary nucleosides and nucleotides supplementation on trinitrobenzene sulphonic acid induced colonic damage in experimental colitis. METHODS: Sprague-Dawley rats were randomised into two groups and fed nucleic acid free 20% casein diet (control) or this diet supplemented with 0.5% nucleoside-nucleotide mixture for four weeks. On the second week, colonic inflammation was induced in rats by intracolonic administration of 0.25 ml of 50% ethanol containing 25 mg of trinitrobenzene sulphonic acid. Additionally, other sets of rats were treated with 0.25 ml of 50% ethanol, 25 mg of trinitrobenzene sulphonic acid in 0.25 ml saline, or 0.25 ml of 0.9% saline. RESULTS: After two weeks, colon weight, macroscopic and microscopic damage scores, were significantly greater (p < 0.05) in the nucleoside-nucleotide supplemented group compared with the non-supplemented control groups. The same variables seen in the trinitrobenzene sulphonic acid-ethanol group fed nucleoside-nucleotide free diet were greater (p < 0.05) than in the rest of the groups fed nucleoside-nucleotide free diet and treated with ethanol...
Cellular localization of 2,4-dinitrophenyl (DNP) groups in the peripheral lymphoid system of guinea-pigs was studied at various times after painting the skin with 2,4-dinitrochlorobenzene (DNCB) by the immunofluorescent method using anti-DNP antibody. The cells taking up the stain (DNP cells) were shown to be mainly lymphocytes. At 1-6 h after painting the majority of DNP cells were found in the peripheral blood and the spleen but the maximum number was reached in the lymph node draining the site of DNCB application 12 h after painting. Injecting cyclophosphamide (CY) 3 days before painting with DNCB, heightened the number of DNP cells residing in the draining node. The animals treated with the tolerogen, 2,4-dinitrobenzene sulphonic acid sodium salt (DNBSO3Na), and then painted with DNCB, had fewer DNP cells than those animals which had simply been painted once with DNCB. The culture supernatants prepared from the draining nodes of both normal and tolerant animals partially blocked the anti-DNP antibody binding with DNP cells. It is suggested that the cells associated with DNP groups residing in the draining lymph node act as immunogens in the immunizing process of contact sensitivity.
Background—It is well established that glutamine
supplemented elemental diets result in less severe intestinal damage in
experimental colitis. However, few studies have examined the mode of
action of glutamine in reducing intestinal damage. Aims—To examine the effects of glutamine
supplemented elemental diets on the potent inflammatory cytokines
interleukin 8 (IL-8) and tumour necrosis factor α (TNF-α) in
trinitrobenzene sulphonic acid (TNBS) induced colitis which presents
with both acute and chronic features of ulcerative colitis. Methods—Sprague-Dawley rats were randomised into
three dietary groups and fed 20% casein (controls), or 20% casein
supplemented with either 2% glutamine (2% Gln) or 4% glutamine (4%
Gln). After two weeks they received intracolonic TNBS to induce
colitis. Results—Both Gln groups of rats gained more weight than
the control group (p<0.05) which had progressive weight loss. Colon weight, macroscopic, and microscopic damage scores for the Gln groups
were lower than in the control group (p<0.05). IL-8 and TNF-α
concentrations in inflamed colonic tissues were lower in the Gln groups
than in the control group (p<0.05), and correlated well with disease
severity. Bacterial translocation was lower both in incidence (p<0.05)
and in the number of colony forming units (p<0.05) for the Gln groups...
Aluminium phthalocyanines substituted to different degrees with hydrophilic sulphonic acid and hydrophobic phthalimidomethyl groups were investigated in vivo as new agents for the photodynamic therapy of malignant tumours. Parameters studied included the photodynamic action on EMT-6 mammary tumours in BALB/c mice, the therapeutic window and the potential for direct cell killing, assayed via an in vivo/in vitro test. Although the efficiency of photoinactivation of the EMT-6 tumour increases by a factor of ten with reduction of the number of sulphonic acid groups from four to two, no further effect was seen with the addition of the hydrophobic phthalimidomethyl groups. Addition of the latter groups however increased the potential for direct cell killing by a factor of two and expanded the therapeutic window by a factor of four, thus improving the usefulness of the dye as a photosensitiser for the photodynamic therapy of cancer.
Desde sua introdução no mercado por volta de 1950, polímeros iônicos vem despertando um grande interesse em diversas áreas, tecnológicas e acadêmicas, devido às acentuadas modificações das propriedades dos polímeros devido à introdução de grupos iônicos em suas cadeias. Neste trabalho, foram sintetizados copolímeros estatísticos de estirenoisopreno com diferentes composições e massas molares via polimerização aniônica. As unidades de isopreno foram hidrogenadas com diimida. A seguir, foi realizada a sulfonação das unidades de estireno. Os grupos sulfonato resultantes foram, posterionnente, neutralizados com trimetilsilanolato de sódio. A análise telmogravimétrica das amostras mostra que ocorre um aumento na estabilidade télmica do polímero após a hidrogenação. Uma grande perda de massa ocorre nas amostras do polímero sufonado no intervalo de temperatura entre 60 e 210ºC. O copolímero sulfonado é muito higroscópico e esta perda de massa pode ser atribuída pelo menos parcialmente à eliminação de água pela amostra. No copolímero neutralizado, esta perda de massa não é observada. A temperatura de transição vítrea dos copolímeros, determinada através de DSC, mostra que todos os copolímeros...
Succinic anhydride (SA) dissolved in Ringer solution buffered with N-tris (hydroxymethyl) methyl-2-aminoethane sulphonic acid (SA-TES solution) potentiates the depolarizing action of acetylcholine (ACh, 10-40 microM) on frog muscle and the tension induced by bath application of this agonist. Applied from one side of a double-barrelled micropipette, SA-TES increases the amplitude of iontophoretically elicited ACh potentials. The potentiation of the effects of ACh by SA-TES does not involve changes in either the activity of the ACh esterase or the input resistance of the muscle membrane. For depolarizations of frog sartorius muscle, dose-response relationships obtained for ACh concentrations from 0.5 to 20 microM indicate that SA-TES increases the apparent affinity of ACh by a factor of 3. SA-TES exerts an "accelerating' effect on the responses elicited by bath-applied ACh; i.e., it increases the rate of depolarization when ACh is added to the bath and the rate of repolarization upon washing out. These effects are particularly marked in preparations treated with neostigmine (3 microM). SA-TES does not potentiate the depolarizing action of agonists which do not contain an ester group. Moreover, the time course of the responses elicited by these compounds is not influenced by SA-TES. SA-TES fails to influence significantly the effects of the neurally released transmitter. Only a 10% increase in the average amplitude of the endplate potentials was observed. SA hydrolyzes in about 30 min at room temperature; however the SA-TES solution retains its activity for several weeks. Succinate is inactive...
A human liver 3 alpha-hydroxysteroid dehydrogenase isoenzyme, a member of the aldo-keto reductase family, shows a marked preference for NADP(H) over NAD(H), and is activated by sulphobromophthalein, which increases the Km values for both NADP(H) and substrates. Here we report kinetic alterations in binding of the coenzymes and the activator to the enzyme caused by site-directed mutagenesis of Lys-270 and Arg-276, which are strictly conserved among the aldo-keto reductase family of enzymes. The mutated enzymes, K270M and R276M, showed increases in the Km for NADP+ of 22- and 290-fold respectively; the Km for alcohol substrate and the kca1 of the NADP(-)-linked reaction were also elevated, by 9- and 5-fold respectively. No kinetic constant of the NAD(+)-linked reaction was altered by more than 3-fold. Calculation of the free-energy changes showed that the 2'-phosphate group of NADP+ contributes 16.3 kJ/mol (3.9 kcal/mol) of binding energy to its interaction with the wildtype enzyme, and the mutagenesis to K270M and R276M destabilized the binding energy of NADP+ by 6.3 and 13.0 kJ/mol (1.5 and 3.1 kcal/mol) respectively. In addition, the mutations attenuated enzyme activation by sulphobromophthalein, which bound to the mutant enzymes as an inhibitor. The inhibition for the R276M mutant was competitive with respect to NADP+ and non-competitive with respect to the substrate...
Papain was labelled by attachment of the fluorescent groups 2-(4'-acetamidoanilino)naphthalene-6-sulphonic acid (AANS) or N-(acetylaminoethyl)-8-naphthylamine-1-sulphonic acid (AEDANS) to the active-site cysteine residue, with the aim of using the labelled papains as probes in competitive titrations of unlabelled cysteine proteinases with their inhibitors. The interaction between the labelled papains and cystatins was accompanied by an increase in fluorescence emission of up to 38-fold for AANS-papain and approximately 3.5-fold for AEDANS-papain. Fluorescence titrations gave dissociation equilibrium constants of 3.1 and 0.6 microM for the binding of chicken cystatin and recombinant human cystatin C respectively to AANS-papain and of 11.9 microM for the binding of chicken cystatin to AEDANS-papain. The kinetics of interaction of chicken cystatin with AANS-papain showed an unusual biphasic dependence of the observed pseudo-first-order rate constant on inhibitor concentration, consistent with the reaction occurring via both pathways of a general two-step binding mechanism. AANS-papain was selected as the most suitable probe for competitive titrations of unlabelled active or inactivated cysteine proteinases with inhibitors. This technique...
Preliminary results are presented for Pt deposited on 4-aminobenzenesulphonic acid-functionalized carbon, Pt/C_ABSA. Vulcan XC-72R was functionalized with the objective of influencing the dispersion of catalyst nanoparticles and to decrease the resistance of three phase boundaries, by introducing sulphonic groups. Electrochemical characterization of the supported catalyst was done in a 0.5 M sulphuric acid solution with added chloride and also using methanol demonstrating well defined features and stable voltammograms after 30 cycles, with apparent higher currents when compared with commercial catalyst. The structure sensitive adsorption of anions on platinum is confirmed by the blocking effect of chloride ions in the hydrogen adsorption-desorption region as well as by the consequent dissolution of platinum, evident in both anodic and cathodic features of the voltammograms regarding surface oxide formation and reduction. The partial reversibility of the effect of chloride ions
is discussed. Research is in progress in order to accomplish a comprenhensive characterization of the synthesized catalyst and to ascertain the effect of the sulphonic