RESUMO
The weakly basic antibiotic and anti-inflammatory drug, clofazimine (CFZ), was first described in 1957. It has been used therapeutically, most notably in the treatment of leprosy. However, the compound is extremely insoluble in aqueous media, and, indeed, there is poor consensus about what its intrinsic solubility is since the reported values range from 0.04 to 11 ng/mL. To understand the speciation and solubilization of CFZ as a function of pH, it is of paramount importance to know the true aqueous pKa. However, there is also poor consensus about the value of the pKa (reported measured values range from 6.08 to 9.11). In the present study, we report the determination of the CFZ ionization constant using two independent techniques. A state-of-the-art potentiometric analysis was performed, drawing on titration data in methanol-water solutions (46-75 wt % MeOH) of CFZ, using the bias-reducing consensus of two different procedures of extrapolating the apparent psKa values to zero cosolvent to approximate the true aqueous pKa as 9.43 ± 0.12 (25 °C, I = 0.15 M reference ionic strength). In parallel, spectrophotometric UV/vis titration data were acquired (250-600 nm at different pH) in 10 mM HEPES buffer solutions containing up to 54 wt % MeOH. The alternating least squares (ALS) method was used in the analysis of the absorbance-pH spectra. Uncharacteristically, the cosolvent UV/vis data in our study showed reverse cosolvent dependence (apparent pKa values increased with increasing cosolvent) which could be explained by a dimerization of the free base. The analysis of UV/vis data obtained from 54 wt % MeOH-water solution containing 20 µM CFZ yielded the apparent pKa 9.51 ± 0.17 (I ≈ 0.005 M). To assess whether self-assembly of CFZ was energetically feasible, density functional theory (DFT) calculations were used to study the putative CFZ dimers in aqueous and methanol media. The DFT-optimized geometries and infrared spectra of CFZ dimers using water and methanol as solvents were calculated and analyzed. Based on the lack of negative frequencies in calculated infrared spectra, it was confirmed that optimized geometries correspond to the true energetic minima. Visual analysis of optimized structures indicates the presence of stacking interactions between two CFZ molecules. The protonation site (the imine nitrogen atom) was determined by 1H NMR spectroscopy.
Assuntos
Clofazimina , Metanol , Potenciometria/métodos , Concentração de Íons de Hidrogênio , Água/química , Espectrofotometria/métodosRESUMO
A neutral polysaccharide (DIP-1) from Duchesnea indica (Andr.) Focke was obtained by hot water extraction, ethanol precipitation and chromatographic separation (DEAE-52 cellulose anion-exchange column and Sephadex G-100 gel column). The physicochemical properties of DIP-1 were elucidated by gel permeation chromatography, monosaccharide composition, Fourier transform infrared spectrometry, UV-visible spectrophotometry, scanning electron microscope and Congo red test. The results indicated that DIP-1 was consisted of mannose, glucosamine, glucose, galactose and arabinose in a ratio of 1.00:0.42:18.36:14.17:0.81, and its molecular weight was 218.3 kDa. Meanwhile, DIP-1 presented a straight hexahedron structure, but no triple-helical conformation. In antioxidant activity tests, DIP-1 exhibited powerful scavenging activities on hydroxyl, DPPH, ABTS radicals and reducing power in a dose-dependent manner. Especially, DIP-1 demonstrated high inhibitory activities against SKOV-3 and Hep-G2 cells in vitro, with IC50 values of 1.42 and 1.23 mg/ml, respectively. PRACTICAL APPLICATIONS: D. indica has been used for a long time as a Chinese medicine for therapy of many diseases, including cancer, inflammation, leprosy, fever, bleeding and so on. At present, polysaccharides have attracted comprehensive attention because of a large range of pharmacological and biological properties, including antitumor, antidiabetic, antioxidant and immunomodulatory activity. In the present study, we purified and characterized a neutral polysaccharide from D. indica for the first time. Moreover, the neutral polysaccharide exhibits significant antioxidant and antitumor activities. Therefore, the present study laid a foundation for the high-value application of D. indica polysaccharides in functional food and pharmaceutical industries.
Assuntos
Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Sequestradores de Radicais Livres/farmacologia , Polissacarídeos/farmacologia , Potentilla/química , Antineoplásicos/química , Antioxidantes/química , Fenômenos Químicos , Cromatografia em Gel , Sequestradores de Radicais Livres/química , Microscopia Eletrônica de Varredura , Peso Molecular , Monossacarídeos/química , Polissacarídeos/química , EspectrofotometriaRESUMO
The objective of this work was to determine the methemoglobinemia and correlate with dapsone levels in multibacillary leprosy patients under leprosy multi-drug therapy. Thirty patients with laboratory and clinical diagnosis of multibacillary leprosy were enrolled. Dapsone was analyzed by high performance liquid chromatography and methemoglobinemia by spectrophotometry. The mean dapsone concentrations in male was 1.42 g/mL and in female was 2.42 g/mL. The mean methemoglobin levels in male was 3.09 µg/mL; 191%, and in female was 2.84 ± 1.67%. No correlations were seen between dapsone levels and methemoglobin in male and female patients. Our results demonstrated that the dosage of dapsone in leprosy treatment does not promote a significant methemoglobinemia.
Assuntos
Dapsona/sangue , Hansenostáticos/administração & dosagem , Hanseníase Multibacilar/tratamento farmacológico , Metemoglobinemia/diagnóstico , Adolescente , Adulto , Cromatografia Líquida de Alta Pressão , Clofazimina/administração & dosagem , Dapsona/administração & dosagem , Dapsona/efeitos adversos , Feminino , Humanos , Hansenostáticos/efeitos adversos , Hanseníase Multibacilar/sangue , Masculino , Metemoglobinemia/induzido quimicamente , Rifampina/administração & dosagem , Espectrofotometria , Adulto JovemRESUMO
The objective of this work was to determine the methemoglobinemia and correlate with dapsone levels in multibacillary leprosy patients under leprosy multi-drug therapy. Thirty patients with laboratory and clinical diagnosis of multibacillary leprosy were enrolled. Dapsone was analyzed by high performance liquid chromatography and methemoglobinemia by spectrophotometry. The mean dapsone concentrations in male was 1.42 g/mL and in female was 2.42 g/mL. The mean methemoglobin levels in male was 3.09 µg/mL; 191 percent, and in female was 2.84 ± 1.67 percent. No correlations were seen between dapsone levels and methemoglobin in male and female patients. Our results demonstrated that the dosage of dapsone in leprosy treatment does not promote a significant methemoglobinemia.
Assuntos
Adolescente , Adulto , Feminino , Humanos , Masculino , Adulto Jovem , Dapsona/sangue , Hansenostáticos/administração & dosagem , Hanseníase Multibacilar/tratamento farmacológico , Metemoglobinemia/diagnóstico , Cromatografia Líquida de Alta Pressão , Clofazimina/administração & dosagem , Dapsona/administração & dosagem , Dapsona/efeitos adversos , Hansenostáticos/efeitos adversos , Hanseníase Multibacilar/sangue , Metemoglobinemia/induzido quimicamente , Rifampina/administração & dosagem , Espectrofotometria , Adulto JovemRESUMO
Dapsona (DDS) (4,4'diaminodifenilsulfona), fármaco de escolha para o tratamento da hanseníase, freqüentemente induz anemia hemolítica e metemoglobinemia. A N-hidroxilação, uma de suas principais vias de biotransformação, é constantemente relacionada com a metemoglobinemia observada com o uso do fármaco. Com o objetivo de prevenir a hemotoxicidade induzida pela DDS, N-acetilcisteína, fármaco precursor de glutationa, foi administrada em associação com DDS em ratos machos Wistar pesando 220-240 g. Os animais foram anestesiados e o sangue coletado da aorta para determinação da concentração plasmática de DDS por CLAE, determinação dos níveis de metemoglobina e de glutationa eritrocitária por espectrofotometria, e avaliação de parâmetros bioquímicos e hematológicos. Os resultados obtidos mostraram que a N-acetilcisteína potenciou o efeito metemoglobinizante da dapsona devido ao aumento de sua concentração plasmática e conseqüente aumento da formação da N-hidroxilamina. Concluímos que as interações medicamentosas com a dapsona exigem estudos individualizados a fim de evitar os efeitos adversos do fármaco.
Dapsone (DDS) (4,4'diaminodiphenylsulfone), the drug of choice for the treatment of leprosy, frequently induces hemolytic anemia and methemoglobinemia. N-hydroxylation, one of the major pathways of biotransformation, has been constantly related to the methemoglobinemia after the use of the drug. In order to prevent the dapsone-induced hemotoxicity, N-acetylcysteine, a drug precursor of glutathione, was administered in combination with DDS to male Wistar rats, weighting 220-240 g. The animals were then anaesthetized and blood was collected from the aorta for determination of plasma DDS concentration by HPLC, determination of methemoglobinemia and glutathione by spectrophotometry, and for biochemical and hematological parameters. Our results showed that N-acetylcysteine enhanced dapsone-induced methemoglobinemia due to increased dapsone plasmatic concentration and consequent increased N-hydroxylamine formation. We concluded that drug interactions with dapsone require individually studies in order to avoid undesirable effects of dapsone.
Assuntos
Animais , Ratos , Dapsona/farmacocinética , Hanseníase/metabolismo , Metemoglobinemia/complicações , Espectrofotometria/métodos , GlutationaRESUMO
Leprosy, a chronic infectious disease, is caused by a Mycobacterium leprae infection. After India, Brazil has the second greatest number of cases in the world. Increase of oxidative stress and antioxidant deficiency are present in infected subjects and can be related to infection progression. We studied alterations in serum levels of lipid peroxidation (LPO) and vitamin A in patients with different forms of leprosy. Four groups of leprosy patients and a control group (healthy subjects) were selected, and their vitamin A serum levels and LPO profile, measured as malonaldehyde (MDA) were measured by spectrophotometric assays. The mean MDA serum levels (micromol/L) were 3.80 +/- 0.5 for control group and 10.54 +/- 1.1 in the leprosy patients and this increase was gradual, being more accentuated in severe forms of the disease. Also, the vitamin A serum levels (microg/dL) were diminished in the infected subjects (38.51 +/- 4.2), mainly in lepromatous form, when compared with the control group (53.8 +/- 5.6). These results indicate that LPO can be an important factor in Mycobacterium leprae infection, which can be related to increases in phagocytic activity and the general breakdown of antioxidants, contributing to an increase of LPO during infection progression. The evaluation of oxidant/antioxidant status in these patients can be an important factor in the treatment, control, and/or prognosis of this disease.
Assuntos
Hanseníase/sangue , Peroxidação de Lipídeos , Malondialdeído/sangue , Vitamina A/sangue , Adulto , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , EspectrofotometriaRESUMO
Leprosy, a chronic infectious disease, is caused by a Mycobacterium leprae infection. After India, Brazil has the second greatest number of cases in the world. Increase of oxidative stress and antioxidant deficiency are present in infected subjects and can be related to infection progression. We studied alterations in serum levels of lipid peroxidation (LPO) and vitamin A in patients with different forms of leprosy. Four groups of leprosy patients and a control group (healthy subjects) were selected, and their vitamin A serum levels and LPO profile, measured as malonaldehyde (MDA) were measured by spectrophotometric assays. The mean MDA serum levels (μmol/L) were 3.80 ± 0.5 for control group and 10.54 ± 1.1 in the leprosy patients and this increase was gradual, being more accentuated in severe forms of the disease. Also, the vitamin A serum levels (μg/dL) were diminished in the infected subjects (38.51 ± 4.2), mainly in lepromatous form, when compared with the control group (53.8 ± 5.6). These results indicate that LPO can be an important factor in Mycobacterium leprae infection, which can be related to increases in phagocytic activity and the general breakdown of antioxidants, contributing to an increase of LPO during infection progression. The evaluation of oxidant/antioxidant status in these patients can be an important factor in the treatment, control, and/or prognosis of this disease.
A hanseníase, doença infecciosa crônica, é causada pelo Mycobacterium leprae. Depois da índia, o Brasil possui o segundo maior número de casos no mundo. O aumento do estresse oxidativo e da deficiência das defesas antioxidantes estão presentes em indivíduos infectados e podem associar-se à progressão da infecção. Foram estudadas alterações nos níveis séricos da peroxidação lipídica e vitamina A em pacientes com diferentes formas de hanseníase. Foram selecionados para o estudo quatro grupos de pacientes com hanseníase e um grupo controle (indivíduos saudáveis) e os níveis séricos de vitamina A e a peroxidação lipídica, medida através do malondialdeído (MDA), foram determinados por métodos espectrofotométricos. Os níveis séricos médios de MDA (μmol/L) foram 3,80 ± 0,5 no grupo controle e 10,54 ± 1,1 nos pacientes com hanseníase. Sendo este aumento gradual e exacerbado nas formas mais severas da doença. Quanto à vitamina A, os níveis séricos (μg/dL) encontraram-se diminuídos nos indivíduos infectados (38.51 ± 4.2), principalmente na forma lepromatosa, quando comparados com o grupo controle (53.8 ± 5.6). Estes resultados indicam que a peroxidação lipídica pode ser um fator importante na infecção mediada pelo Mycobacterium leprae podendo estar relacionada ao aumento da atividade fagocítica pelos macrófagos contribuindo para um aumento da LPO durante a progressão da infecção. A avaliação do perfil oxidante/antioxidante nestes pacientes pode ser um fator importante no tratamento, controle e/ou prognóstico desta doença.
Assuntos
Humanos , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Peroxidação de Lipídeos , Hanseníase/sangue , Malondialdeído/sangue , Vitamina A/sangue , Estudos de Casos e Controles , EspectrofotometriaRESUMO
Spectrophotometric determination of dapsone is described. The dapsone reacts with sodium 1,2-naphthoquinone-4-sulfonic in pH 6.98 buffer solution to form a salmon pink compound, and its maximum absorption wavelength is at 525 nm, epsilon525=3.68 x 10(4) l mol(-1) cm(-1). The absorbance of dapsone from 0.40 to 10 microg ml(-1) obeys Beer's law. The linear regression equation of the calibration graph is C=0.2334 A + 0.01288, with a linear regression correlation coefficient of 0.9998, the detection limit is 0.24 microg ml(-1), and recovery is from 99.2 to 102.4%. Effects of pH, surfactant, organic solvents, foreign ions, and standing time on the determination of dapsone have been examined. This method is simple and can be used for the determination of dapsone in injection solution of dapsone. The results obtained by this method agreed with those by the official method (dead-stop titration method [The Chinese Pharmacopoeia, Pharmacopoeia Commission, Ministry of Health, vol. 2, fifth ed., PRC Chemical Industry Press, Beijing, 2000, p.720]).
Assuntos
Dapsona/análise , Antimaláricos/análise , Compostos Cromogênicos , Composição de Medicamentos , Estabilidade de Medicamentos , Temperatura Alta , Concentração de Íons de Hidrogênio , Indicadores e Reagentes , Íons , Hansenostáticos/análise , Naftoquinonas , Solventes , Espectrofotometria/métodos , Espectrofotometria/estatística & dados numéricos , TensoativosRESUMO
Truncated hemoglobins (trHb's) form a family of low molecular weight O2 binding hemoproteins distributed in eubacteria, protozoa, and plants. TrHb's branch in a distinct clade within the hemoglobin (Hb) superfamily. A unique globin gene has recently been identified from the complete genome sequence of Mycobacterium leprae that is predicted to encode a trHb (M. leprae trHbO). Sequence comparison and modelling considerations indicate that monomeric M. leprae trHbO has structural features typical of trHb's, such as 20-40 fewer residues than conventional globin chains, Gly-based sequence consensus motifs, likely assembling into a 2-on-2 alpha-helical sandwich fold, and hydrophobic residues recognized to build up the protein matrix ligand diffusion tunnel. The ferrous heme iron atom of deoxygenated M. leprae trHbO appears to be hexacoordinated, like in Arabidopsis thaliana trHbO-3 (A. thaliana trHbO-3). Accordingly, the value of the second-order rate constant for M. leprae trHbO carbonylation (7.3 x 10(3) M(-1) s(-1)) is similar to that observed for A. thaliana trHbO-3 (1.4 x 10(4) M(-1) s(-1)) and turns out to be lower than that reported for carbon monoxide binding to pentacoordinated Mycobacterium tuberculosis trHbN (6.7 x 10(6) M(-1) s(-1)). The lower reactivity of M. leprae trHbO as compared to M. tuberculosis trHbN might be related to the higher susceptibility of the leprosy bacillus to toxic nitrogen and oxygen species produced by phagocytic cells.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Hemoglobinas/genética , Hemoglobinas/metabolismo , Mycobacterium leprae , Sequência de Aminoácidos , Proteínas de Bactérias/química , Sequência de Bases , Clonagem Molecular , Hemoglobinas/química , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Espectrofotometria , Hemoglobinas TruncadasRESUMO
As Mycobacterium leprae proliferate inside macrophages, it has been speculated that catalase encoded by katG may protect the bacilli from deleterious effects of peroxide generated from the macrophage and may also play a crucial role in the survival of M. leprae in vivo. However, unlike that of M. tuberculosis, the katG of M. leprae has been reported to be a pseudogene, implicating that isoniazid, which is activated to a potent tuberculocidal agent by catalase, is unlikely to be of therapeutic benefit to leprosy patients. These results raise a question as to how M. leprae avoids H202-mediated killing inside macrophages. To understand the survival of M. leprae in macrophages, the present study attempted to detect catalase-like activity in M. leprae. Catalase-like activity was found in M. leprae cell lysate by the diaminobenzidine (DAB) staining method with non-denaturing polyacrylamide gel electrophoresis. An ammonium sulphate precipitation study revealed that the catalase-like activity was precipitable with 80% ammonium sulphate. The effect of isoniazid (INH) on M. leprae growth was also tested by RT-PCR and radiorespirometric assay to examine catalase-like activity in M. leprae, because INH was activated by catalase. It was found that the viability of M. leprae was decreased at a concentration of 20 microg/ml by radiorespirometric assay and it was inhibited at higher concentrations as determined by RT-PCR. These data suggest that a catalase-like activity other than that encoded by katG is present in M. leprae.
Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias , Catalase/metabolismo , Isoniazida/farmacologia , Mycobacterium leprae/enzimologia , Peroxidases/metabolismo , Sulfato de Amônio , Animais , Sequência de Bases , Benzidinas , Catalase/genética , Primers do DNA , DNA Complementar/análise , Eletroforese em Gel de Ágar , Peróxido de Hidrogênio/metabolismo , Hanseníase/tratamento farmacológico , Hanseníase/enzimologia , Macrófagos Peritoneais/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium leprae/efeitos dos fármacos , Mycobacterium leprae/genética , Peroxidases/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Contagem de Cintilação , Homologia de Sequência do Ácido Nucleico , EspectrofotometriaRESUMO
A rapid, sensitive and selective spectrophotometric method has been developed for the quantitative determination of metoclopramide hydrochloride (MCP) and dapsone (DAP) in both pure and dosage forms. The method is based on the diazo coupling reaction of the drugs with a new coupling agent, dibenzoyl methane in an alkaline medium. The resulting coloured azo dyes exhibit maximum absorption at 440 nm for MCP and at 470 nm for DAP with a molar absorptivity of 2.85x10(4) and 1.71x10(4) l mol(-1) cm(-1) for MCP and DAP, respectively. All variables have been optimized. No interferences were observed from excipients and the validity of the method was tested against reference method.
Assuntos
Anti-Infecciosos/análise , Chalconas , Dapsona/análise , Hansenostáticos/análise , Metoclopramida/análise , Benzoatos/farmacologia , Concentração de Íons de Hidrogênio , Nitrito de Sódio/farmacologia , EspectrofotometriaRESUMO
A dapsona (4,4-diaminodifenilsulfona), quimioterápico bacteriostático utilizado no tratamento da hanseníase, vem sendo associada a intercorrências clínicas, principalmente devido à sua hemotoxicidade caracterizada por metemoglobinemia e anemia hemolítica. A N-hidroxilação, uma das principais vias de biotransformação da dapsona, vem sendo associada constantemente a quadros de metemoglobinemia decorrentes de sua utilização. Com o objetivo de verificar-se a inibição reversível da via de bioativação toxicológica, sem alterar as vias de destoxificação do composto, a acetilação citosólica, a cimetidina foi administrada concomitantemente à dapsona em ratos machos Wistar, com peso variando entre 200 e 220 g, divididos em 8 grupos (n=6 por grupo), em estudo de dose única...
Assuntos
Animais , Ratos , Anemia Hemolítica/terapia , Cimetidina/administração & dosagem , Dapsona/farmacocinética , Hanseníase , Metemoglobinemia/metabolismo , Cromatografia Líquida/métodos , Espectrofotometria , Interpretação Estatística de DadosRESUMO
Rifamycin S and rifabutin are clinical drugs used to treat tuberculosis and leprosy. The formation of reactive oxygen species during the redox-cycling of rifamycin S (quinone) and rifabutin (quinonimine) was evaluated. The semiquinone (or semiquinonimine) and hydroquinone (or hydroquinonimine) formed during the reduction of the parent molecules by microsomal electron transfer in the presence of nicotinamide-adenine dinucleotide phosphate, reduced (NADPH) or nicotinamide-adenine dinucleotide, reduced (NADH) reoxidizes in air to generate superoxide radical and hydrogen peroxide. In the presence of added iron, hydroxyl radicals, formed by the Fenton reaction, were detected using 5,5'-dimethyl-1-pyroline-N-oxide as the spin-trap. Rifamycin S, a quinone, redox cycles more efficiently than rifabutin, a quinonimine, as approximately five times the concentration of hydroxyl radical adduct of 5,5'-dimethyl-1-pyroline-N-oxide (DMPO) was detected, when compared with rifabutin. The NADPH-dependent microsomal production of hydroxyl radical in the presence of rifamycin S was somewhat higher than the NADH-rifamycin S system with most iron chelators. However, with rifabutin, NADH-dependent microsomal production of hydroxyl radical was higher than that found with the NADPH-rifabutin system. An exception was the iron chelator, diethylene-triamine-pentacetic acid (DTPA), in which NADPH-dependent rates exceeded the rates with NADH with both antibiotics. Rat liver sub-mitochondrial particles also generated hydroxyl radical in the presence of NADH and either rifamycin S or rifabutin. The electron transport chain inhibitors such as rotenone and antimycin A enhanced the signal intensity of DMPO-OH, suggesting NADH dehydrogenase (complex I) as the major component involved in the reduction of rifamycin S. Rifamycin S was shown to be readily reduced to rifamycin SV, the corresponding hydroquinone by Fe(II); under similar conditions Fe(II) did not reduce rifabutin. Using optical spectroscopy, we determined that rifamycin S forms a complex with Fe(II). The stoichiometry of the complex was Fe(rifamycin S)3 in phosphate buffer at pH 7.4. Rifabutin did not form a detectable complex with Fe(II). The redox cycling of rifamycin S and rifabutin did not cause microsomal lipid peroxidation. In fact, the Fe:ATP induced lipid peroxidation was completely inhibited by these two molecules. These results indicate that rifamycin S and rifabutin can interact with rat liver microsomes to undergo redox-cycling, with the subsequent production of hydroxyl radicals when iron complexes are present. Compared to NADPH, NADH is almost as effective (rifamycin S) or even more effective (rifabutin) in promoting these interactions. These interactions may play a role in the hepatotoxicity associated with the use of these antibiotics.
Assuntos
Antibacterianos/metabolismo , Microssomos Hepáticos/metabolismo , Rifabutina/metabolismo , Rifamicinas/metabolismo , Animais , Antibióticos Antituberculose/metabolismo , Peróxido de Hidrogênio/metabolismo , Radical Hidroxila/metabolismo , Masculino , NAD/farmacologia , NADP/farmacologia , Oxirredução , Ratos , Ratos Sprague-Dawley , Espectrofotometria , Superóxidos/metabolismoRESUMO
The binding of 9-aminoacridine and one bis-acridine compound to double helical poly(dA).poly-(dT) and poly[d(A-T)]2 and triple helical poly(dA).[poly(dT)]2 has been investigated using linear dichroism (LD) and circular dichroism (CD). A close examination of the negative reduced LD and the induced CD for the first pi-->pi* transition absorption region leads us to conclude that the acridine moiety of the 9-aminoacridine and bis-acridine molecule intercalates with both duplex and triplex DNA. Binding geometries of the acridine moieties in the examined polynucleotides are similar to those found for the ligands with DNA (Hansen et al. (1984) J. Chem. Soc., Chem. Commun., 509-511). It is also found that both 9-aminoacridine and bis-acridine effectively enhance the thermal stability of the triplex DNA. The corresponding spectra for the complexes of the minor groove binders DAPI and Hoechst with poly-(dA).[poly(dT)]2 were studied for comparison. They both show a positive LD and a mixing ratio dependent positive CD in the ligand absorption region, similar to those of their duplex complexes. This indicates that these ligands bind in the grooves of the triplex, probably to the one corresponding to the minor groove of the template duplex.
Assuntos
Acridinas/metabolismo , Aminacrina/metabolismo , DNA/metabolismo , Substâncias Intercalantes/metabolismo , Propilaminas/metabolismo , Aminacrina/análogos & derivados , Sítios de Ligação , Bisbenzimidazol/metabolismo , Dicroísmo Circular , Indóis/metabolismo , Ligantes , Desnaturação de Ácido Nucleico , Poli dA-dT/metabolismo , Polidesoxirribonucleotídeos/metabolismo , EspectrofotometriaRESUMO
Polarized visible reflection spectra of a chlorophyll a (Chl.a) Langmuir monolayer have been measured in situ at various surface pressures. By applying Hansen's optics to the three-phase plane-bounded system (air/Chl.a monolayer/water), the negative reflection absorbances observed were reproduced satisfactorily by the theoretical calculation. Molecular orientation of Chl.a in the monolayer was evaluated quantitatively as a function of surface pressure, from the reflection absorbance of p- and s-polarized spectra of the red (Qy) band. It has been proven that Chl.a molecules in the monolayer form aggregates (islands) even in the low surface pressure region and that during the monolayer compression the molecules are gradually reorganized from inhomogeneous islands to ordered structures, with the chromophores oriented on the average vertically to the water surface.
Assuntos
Clorofila/química , Clorofila A , Matemática , Modelos Teóricos , Conformação Molecular , Estrutura Molecular , Pressão , Espectrofotometria/instrumentação , Espectrofotometria/métodos , Spinacia oleracea , Propriedades de SuperfícieRESUMO
The R-state conformation of the Cu(II)-substituted insulin hexamer has been identified, and a number of its derivatives have been studied via 1H NMR, ESR, and UV-visible spectroscopy. This work establishes that the Cu(II)-substituted insulin hexamer undergoes an analogous T to R conformational transition in solution that has been identified previously for Zn(II)- and Co(II)-insulin hexamers [Roy, M., Brader, M.L., Lee, R. W.-K., Kaarsholm, N.C., Hansen, J., & Dunn, M.F. (1989) J. Biol. Chem. 264, 19081-19085]. The data indicate that each Cu(II) center of the R-state Cu(II)-insulin hexamer possesses a coordination site that is accessible to anions from solution. Both phenol and anionic ligands that coordinate to the Cu(II) ions are required to generate the necessary heterotropic interactions that stabilize the R-state structure. With phenylmethylthiolate (PMT), a Cu(II)-R6 adduct that displays the spectral features of blue (type 1) copper proteins is obtained. This complex is proposed to embody a pseudotetrahedral CuIIN3S(PMT) chromophore, in which N is HisB10 (imidazolyl). The remaining ligands examined gave rise to Cu(II)-R6 adducts that possessed the spectral characteristics of normal (type 2) Cu(II) proteins. Under reducing conditions, Cu(I)-T6 and Cu(I)-R6 hexamers have been identified.
Assuntos
Cobre/química , Insulina/química , Espectroscopia de Ressonância de Spin Eletrônica , Substâncias Macromoleculares , Espectroscopia de Ressonância Magnética , Compostos Organometálicos/química , Ligação Proteica , Conformação Proteica , Espectrofotometria , Relação Estrutura-AtividadeRESUMO
The phenol-induced conformational transition in the insulin hexamer is known to involve a large change in structure wherein residues 1-8 of the insulin B-chain are transformed from an extended coil (T-state) to a helix (R-state). This change in protein conformation both exposes a cryptic protein pocket on each subunit to which phenol binds and forces the HisB10 zinc sites to undergo a change in coordination geometry from octahedral to tetrahedral [Derewenda, U., Derewenda, Z., Dodson, E. J., Dodson, G. G., Reynolds, C. D., Smith, G. D., Sparks, C., & Swensen, D. (1989) Nature 338, 593-596]. Substitution of Co(II) for Zn(II) at the HisB10 sites introduces a sensitive chromophoric probe of the structural and chemical events that occur during this allosteric transition [Roy, M., Brader, M. L., Lee, R. W.-K., Kaarsholm, N. C., Hansen, J. F., & Dunn, M. F. (1989) J. Biol. Chem. 264, 19081-19085]. In this study, using rapid-scannig stopped-flow (RSSF) UV-visible spectroscopic studies, we demonstrate that a transient chemical intermediate is formed during the phenol-induced conversion of Co(II)-substituted hexamer from the T-state to the R-state. Decomposition of the RSSF spectra gave a spectrum for the intermediate with d-d transitions consistent with the assignment of the intermediate as either a distorted tetrahedral or a 5-coordinate Co(II) species. Possible structures for the intermediate and the implications of these findings to the allosteric mechanism are considered.
Assuntos
Sítio Alostérico , Cobalto/química , Insulina/química , Animais , Humanos , Cinética , Fenol , Fenóis , Ligação Proteica , Conformação Proteica/efeitos dos fármacos , Espectrofotometria , Relação Estrutura-Atividade , Suínos , Difração de Raios XRESUMO
Replacement of carbazole with meta-hydroxydiphenyl greatly improves the determination of uronic acids in the presence of neutral sugars by preventing substantially, but not completely, the browning that occurs during the heating of sugars in concentrated sulfuric acid and avoiding the formation of additional interference by the carbazole reagent (Blumenkrantz, N., and Asboe-Hansen, G. (1973) Anal. Biochem. 54, 484-489). However, interference is still substantial when uronic acids are determined in the presence of excess neutral sugar, particularly because of the browning that occurs during the first heating before addition of the diphenyl reagent. The browning can be essentially eliminated by addition of sulfamate to the reaction mixture (Galambos, J. T. (1967) Anal. Biochem. 19, 119-132). Although others have reported that sulfamate and the diphenyl reagent were incompatible, we find that a small amount of sulfamate suppresses color production by a 20-fold excess of some neutral sugars without substantial sacrifice of the sensitive detection of uronic acids by the diphenyl reagent. Sodium tetraborate is required for the detection of D-mannuronic acid and enhances color production by D-glucuronic acid. We propose this modified sulfamate/m-hydroxydiphenyl assay as a rapid and reliable means for the assay of uronic acids, particularly when present in much smaller amounts than neutral sugars.
Assuntos
Técnicas de Química Analítica/métodos , Ácidos Urônicos/análise , Compostos de Bifenilo , Carboidratos/análise , Cor , Temperatura Alta , Indicadores e Reagentes , Espectrofotometria , Ácidos SulfônicosRESUMO
Oxidation of palmitic acid by whole-cell suspensions of Mycobacterium leprae free from host tissues was investigated using manometric techniques. After a lag period of about 6-8 h, M. leprae suspensions catalysed an active oxidation of palmitic acid, and the oxidative process (oxygen uptake) was quite sensitive to rotenone, atabrine, amytal, antimycin A and cyanide. The spectrophotometric observations indicated that the M. leprae cytochrome system, under anaerobic conditions, was reduced in the presence of palmitic acid which was completely oxidized by oxygen. These data provide direct evidence that M. leprae cells are capable of oxidizing palmitic acid, and that oxidation is mediated by the electron transport system using oxygen as the terminal electron acceptor.
Assuntos
Mycobacterium leprae/metabolismo , Ácidos Palmíticos/metabolismo , Animais , Tatus , Citocromos/metabolismo , Concentração de Íons de Hidrogênio , Fígado/microbiologia , Manometria , Mycobacterium leprae/enzimologia , Nucleotidases/metabolismo , Oxirredução , Consumo de Oxigênio , Espectrofotometria , TemperaturaRESUMO
A comparative study on the microbiological and spectrophotometric methods for estimation of rifampicin in urine was carried out in 15 individuals. The urinary levels of rifampicin were determined on 2nd, 8th and 15th days at 3 hour, 6 hour and 24 hour samples by the above methods after administration of 600 mg rifampicin. The results suggest that the microbiological assay is more sensitive than spectrophotometric method. The difference was highly significant in all the cases by paired t-test. Incidentally it was also noticed that urinary excretion of rifampicin was comparatively more on 15th day.