RESUMO
AIMS: To establish the role of cardiolipin (CL) of the membrane in response to the presence of tetradecyltrimethylammonium in Pseudomonas putida A (ATCC 12633). METHODS AND RESULTS: Two ORFs of Ps. putida A (ATCC 12633), which in Ps. putida KT2440 encode the putative CL synthase genes cls and cls2, were cloned, sequenced and mutated. Only the double mutant lacking cls and cls2 showed a reduction of the CL content, 83% lower than the amount produced by the wild-type. Accompanying this change was a 40% decrease in the content of unsaturated fatty acid. Consequently, the membrane of the mutant was more rigid than the one of the parental strain, as observed using fluorescence polarization techniques. The mutant strain showed reduced viability in the presence of tetradecyltrimethylammonium. The incorporation of exogenous CL into its membrane relieved sensitivity to the cationic detergent. CONCLUSIONS: Pseudomonas Putida cells with low levels of CL die in the presence of tetradecyltrimethylammonium, because they cannot counter the fluidizing effect of the cationic surfactant. SIGNIFICANCE AND IMPACT OF THE STUDY: The modification in the membrane phospholipids composition allows knowing the adaptation strategy of Ps. putida when these bacteria are exposed to cationic surfactant.
Assuntos
Antibacterianos/farmacologia , Pseudomonas putida/efeitos dos fármacos , Tensoativos/farmacologia , Compostos de Trimetil Amônio/farmacologia , Cardiolipinas/análise , Cardiolipinas/metabolismo , Clonagem Molecular , Polarização de Fluorescência , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Fases de Leitura Aberta , Fosfolipídeos/metabolismo , Pseudomonas putida/química , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/genéticaRESUMO
Pseudomonas putida A (ATCC 12633), a degrader of cationic surfactants, releases outer membrane vesicles (OMVs) when grown with tetradecyltrimethylammonium bromide (TTAB) as the sole carbon, nitrogen and energy source. The OMVs exhibit a bilayer structure and were found to be composed of lipopolysaccharides, proteins and phospholipids (PLs) such as cardiolipin, phosphatidylcholine, phosphatidic acid and phosphatidylglycerol (PG). The OMVs showed a marked increase in the PG content, approximately 43 % higher than the amount registered in the parent cells from which the vesicles were derived. After growth of P. putida with TTAB, the amount of lipoprotein covalently cross-linked to the peptidoglycan showed a twofold decrease when compared with values found after growth without the surfactant [16 ± 2 and 28 ± 3 µg (mg cell envelope protein)- 1, respectively]. This decrease in the amount of lipoprotein can be related to areas of loss of contact between the outer membrane and the peptidoglycan and, therefore, to OMV production. In addition, due to its amphiphilic nature, TTAB can contribute to OMV biogenesis, through a physical mechanism, by induction of the curvature of the membrane. Taking into account that OVMs were produced when the cells were grown under external stress, caused by the surfactant, and that TTAB was detected in the vesicles [48ânmol TTAB (nmol PL)- 1], we concluded that this system of TTAB elimination is a mechanism that P. putida A (ATCC 12633) would utilize for alleviating stress caused by cationic surfactants.
Assuntos
Membrana Celular/metabolismo , Vesículas Extracelulares/metabolismo , Pseudomonas putida/metabolismo , Estresse Fisiológico/fisiologia , Tensoativos/farmacologia , Compostos de Trimetil Amônio/farmacologia , Animais , Cardiolipinas/metabolismo , Lipoproteínas/metabolismo , Peptidoglicano/metabolismo , Ácidos Fosfatídicos/metabolismo , Fosfatidilcolinas/metabolismo , Fosfatidilgliceróis/metabolismo , CoelhosRESUMO
The present study assessed the role of membrane components of Pseudomonas putida A (ATCC 12633) under chemical stress conditions originated by treatment with tetradecyltrimethylammonium bromide (TTAB), a cationic surfactant. We examined changes in fatty acid composition and in the fluidity of the membranes of cells exposed to TTAB at a specific point of growth as well as of cells growing with TTAB. The addition of 10-50 mg TTAB l(-1) promoted an increase in the saturated/unsaturated fatty acid ratio. By using fluorescence polarization techniques, we found that TTAB exerted a fluidizing effect on P. putida A (ATCC 12633) membranes. However, a complete reversal of induced membrane fluidification was detected after 15 min of incubation with TTAB. Consistently, the proportion of unsaturated fatty acids was lower in TTAB-treated cells as compared with non-treated cells. In the presence of TTAB, the content of phosphatidylglycerol increased (120â%), whilst that of cardiolipin decreased (60â%). Analysis of the fatty acid composition of P. putida A (ATCC 12633) showed that phosphatidylglycerol carried the major proportion of saturated fatty acids (89â%), whilst cardiolipin carried an elevated proportion of unsaturated fatty acids (18â%). The increase in phosphatidylglycerol and consequently in saturated fatty acids, together with a decrease in cardiolipin content, enabled greater membrane resistance, reversing the fluidizing effect of TTAB. Therefore, results obtained in the present study point to changes in the fatty acid profile as an adaptive response of P. putida A (ATCC 12633) cells to stress caused by a cationic surfactant.
Assuntos
Membrana Celular/química , Lipídeos de Membrana/análise , Fosfolipídeos/análise , Pseudomonas putida/efeitos dos fármacos , Estresse Fisiológico , Tensoativos/metabolismo , Compostos de Trimetil Amônio/metabolismo , Cátions/metabolismo , Cátions/toxicidade , Membrana Celular/fisiologia , Fluidez de Membrana/efeitos dos fármacos , Pseudomonas putida/fisiologia , Tensoativos/toxicidade , Compostos de Trimetil Amônio/toxicidadeRESUMO
This study presents the first report of the purification and characterization of a monooxygenase enzyme from Pseudomonas putida A (ATCC 12633) that is responsible for the oxidation of physiologically relevant quaternary ammonium compounds, the tetradecyltrimethylammonium bromide. The degradation of tetradecyltrimethylammonium bromide by P. putida A (ATCC 12633) is initiated by N-dealkylation and catalysed by tetradecyltrimethylammonium monooxygenase (TTABMO), resulting in the formation of tetradecylalkanal and trimethylamine. Based on sequence analysis, the gene for TTABMO (ttbmo) corresponded to an ORF named PP2033 in the genome of P. putida KT2440. Mutation in ttabmo blocked the utilization of tetradecyltrimethylammonium bromide by Pseudomonas putida A (ATCC 12633) as carbon and nitrogen sources. The enzyme can be highly overexpressed in P. putida Δttabmo-T7 in active form and purified as a hexahistidine fusion protein. Like the native enzyme, the his-TTABMO was found to be a monomer with molecular mass of 40 kDa, the isoelectric point 7.3, that catalyses the breakdown of tetradecyltrimethylammonium bromide and utilized NADPH and FAD as cofactor. The biochemical properties and the analysis of the respective protein sequence revealed that TTABMO represents a typical flavoprotein monooxygenase, which is member of a flavoprotein family that is distinct from Baeyer-Villiger monooxygenases.
Assuntos
Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Pseudomonas putida/enzimologia , Pseudomonas putida/genética , Compostos de Trimetil Amônio/metabolismo , Alquilação , Sequência de Aminoácidos , Biocatálise , Clonagem Molecular , Metilaminas/metabolismo , Oxigenases de Função Mista/química , Oxigenases de Função Mista/isolamento & purificação , Dados de Sequência Molecular , Filogenia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
In this study, the degradation of tetradecyltrimethylammonium bromide (TTAB) by freely suspended and alginate-entrapped cells from the bacteria Pseudomonas putida (P. putida) A ATCC 12633 was investigated in batch cultures. The optimal conditions to prepare beads for achieving a higher TTAB degradation rate were investigated by changing the concentration of sodium alginate, pH, temperature, agitation rate and initial concentration of TTAB. The results show that the optimal embedding conditions of calcium alginate beads are 4 % w/v of sodium alginate content and 2 × 10(8) cfu ml(-1) of P. putida A ATCC 12633 cells that had been previously grown in rich medium. The optimal degradation process was carried out in pH 7.4 buffered medium at 30 °C on a rotary shaker at 100 rpm. After 48 h of incubation, the free cells degraded 26 mg l(-1) of TTAB from an initial concentration of 50 mg l(-1) TTAB. When the initial TTAB concentration was increased to 100 mg l(-1), the free cells lost their degrading activity and were no longer viable. In contrast, when the cells were immobilized on alginate, they degraded 75 % of the TTAB after 24 h of incubation from an initial concentration of 330 mg l(-1) of TTAB. The immobilized cells can be stored at 4 °C for 25 days without loss of viability and can be reused without losing degrading capacity for three cycles.
Assuntos
Alginatos , Pseudomonas putida/metabolismo , Tensoativos/metabolismo , Compostos de Trimetil Amônio/metabolismo , Cátions , Ácido Glucurônico , Ácidos Hexurônicos , Microscopia Eletrônica de VarreduraRESUMO
The present study examined the aerobic metabolism of trimethylamine in Pseudomonas putida A ATCC 12633 grown on tetradecyltrimethylammonium bromide or trimethylamine. In both conditions, the trimethylamine was used as a nitrogen source and also accumulated in the cell, slowing the bacterial growth. Decreased bacterial growth was counteracted by the addition of AlCl(3). Cell-free extracts prepared from cells grown aerobically on tetradecyltrimethylammonium bromide exhibited trimethylamine monooxygenase activity that produced trimethylamine N-oxide and trimethylamine N-oxide demethylase activity that produced dimethylamine. Cell-free extracts from cells grown on trimethylamine exhibited trimethylamine dehydrogenase activity that produced dimethylamine, which was oxidized to methanal and methylamine by dimethylamine dehydrogenase. These results show that this bacterial strain uses two enzymes to initiate the oxidation of trimethylamine in aerobic conditions. The apparent K(m) for trimethylamine was 0.7 mM for trimethylamine monooxygenase and 4.0 mM for trimethylamine dehydrogenase, but both enzymes maintain similar catalytic efficiency (0.5 and 0.4, respectively). Trimethylamine dehydrogenase was inhibited by trimethylamine from 1 mM. Therefore, the accumulation of trimethylamine inside Pseudomonas putida A ATCC 12633 grown on tetradecyltrimethylammonium bromide or trimethylamine may be due to the low catalytic efficiency of trimethylamine monooxygenase and trimethylamine dehydrogenase.
Assuntos
Metilaminas/metabolismo , Oxirredutases N-Desmetilantes/metabolismo , Oxigenases/metabolismo , Pseudomonas putida/metabolismo , Aerobiose , Dimetilaminas/metabolismo , Cinética , Oxirredução , Oxirredutases N-Desmetilantes/antagonistas & inibidores , Pseudomonas putida/crescimento & desenvolvimento , Compostos de Trimetil Amônio/metabolismoRESUMO
AIMS: To evaluate the effect of tetradecyltrimethylammonium bromide (TTAB) and aluminium stresses on the phospholipid (PL) composition of Pseudomonas putida A ATCC 12633. METHODS AND RESULTS: Pseudomonas putida were grown with TTAB in the presence or absence of AlCl(3), and the PL composition was analysed. The presence of TTAB resulted in an increase in phosphatidylglycerol and phosphatidic acid levels (6- and 20-fold, respectively) with respect to the levels in cells grown without the surfactant. With AlCl(3), phosphatidylcholine (PC) increased (threefold) and cell-free extracts contained approximately threefold more phosphatidylcholine synthase activities than extracts without AlCl(3), indicating that the PC level is dependent upon activation of this enzyme. CONCLUSIONS: The negative charges of the headgroups of PL are the primary membrane-associated factors for the response to TTAB. PC are involved in cellular responses to binding Al(3+) and should be viewed as a temporary reservoir of available Al(3+) to allow a more efficient utilization of TTAB by Ps. putida. SIGNIFICANCE AND IMPACT OF THE STUDY: The changes in the PL of Ps. putida in the presence of TTAB and AlCl(3) indicate that different responses are utilized by bacteria to maintain optimal PL composition in the presence of such environmental pollutants.
Assuntos
Compostos de Alumínio/farmacologia , Fosfatidilcolinas/metabolismo , Pseudomonas putida/efeitos dos fármacos , Pseudomonas putida/metabolismo , Compostos de Trimetil Amônio/farmacologia , Proteínas de Bactérias/metabolismo , Fosfatidilcolinas/química , Pseudomonas putida/química , Pseudomonas putida/enzimologia , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismoRESUMO
This article describes a simple fluorescence method for the determination of tetradecyltrimethylammonium mono-oxygenase (TTAB mono-oxygenase) activity involving N-dealkylation of tetradecyltrimethylammonium bromide with concomitant production of trimethylamine (TMA). Activity was determined by measuring the formation of TMA using the morin reagent and aluminum (Al). Morin reacts with Al to form a fluorescent complex, Al-morin. In the presence of TMA, Al is tightly associated with TMA and cannot be sequestered by morin, thus providing evidence for formation of the Al-TMA complex. The concentration of TMA is estimated by calibration graphs constructed by plotting the fluorescence intensity of the Al-morin complex versus TMA concentration. The fluorescence intensities of the Al-morin complexes quenched by TMA are linearly dependent on both the time of the TTAB mono-oxygenase reaction and the amount of protein used in the reaction. The kinetic behavior is characterized by K0.5=4.26x10(-4) M, and the apparent Hill coefficient (napp)=2.24. These values are both comparable to those determined by GC-MS (K0.5=4.41x10(-4) M and napp=2.35). The advantages of this assay include rapid and efficient implementation and potential employment for routine accurate determinations of TTAB mono-oxygenase activity over a wide range of substrate concentrations.
Assuntos
Metilaminas/metabolismo , Oxigenases de Função Mista/química , Oxigenases de Função Mista/metabolismo , Espectrometria de Fluorescência/métodos , Tensoativos/análise , Tensoativos/química , Compostos de Trimetil Amônio/análise , Compostos de Trimetil Amônio/química , Alumínio/química , Carbono/química , Catálise , Flavonoides/química , Metilaminas/química , Nitrogênio/química , Tensoativos/metabolismo , Compostos de Trimetil Amônio/metabolismoRESUMO
AIMS: To establish if tetradecyltrimethylammonium (TDTMA) might be degraded by pure culture of Pseudomonas strains, and how the presence of a Lewis' acid in the medium influences its biodegradability. METHODS AND RESULTS: From different strains of Pseudomonas screened, only Pseudomonas putida A ATCC 12633 grows with 50 mg l(-1) of TDTMA as the sole carbon and nitrogen source. A monooxygenase activity catalyzed the initial step of the biodegradation. The trimethylamine (TMA) produced was used as nitrogen source or accumulated inside the cell. To decrease the intracellular TMA, the culture was divided, and 0.1 mmol l(-1) AlCl(3) added. In this way, the growth and TDTMA consumption increased. The internal concentration of TMA, determined using the fluorochrome Morin, decreased by the formation of Al(3+) : TMA complex. CONCLUSIONS: Pseudomonas putida utilized TDTMA as its sole carbon and nitrogen source. The TMA produced in the initial step of the biodegradation by a monooxygenase activity was used as nitrogen source or accumulated inside the cell, affecting the bacterial growth. This effect was alleviated by the addition of AlCl(3). SIGNIFICANCE AND IMPACT OF THE STUDY: The use of Lewis' acids to sequester intracellular amines offers an alternative to achieve an efficient utilization of TDTMA by Ps. putida.
Assuntos
Microbiologia Ambiental , Pseudomonas putida/metabolismo , Tensoativos , Compostos de Trimetil Amônio , Alumínio/farmacologia , Técnicas Bacteriológicas , Biodegradação Ambiental , Cátions , Concentração de Íons de Hidrogênio , Metilaminas/análise , Metilaminas/metabolismo , Especificidade da Espécie , Tensoativos/análise , Compostos de Trimetil Amônio/análiseRESUMO
Pseudomonas aeruginosa expresses hemolytic phospholipase C (PlcH) with choline or under phosphate-limiting conditions. PlcH from these conditions were differently eluted from the Celite-545 column after application of an ammonium sulfate linear reverse gradient. The PlcH from supernatants of bacteria grown in the presence of choline was eluted with 30% ammonium sulfate and was more than 85% inhibited by tetradecyltrimethylammonium. PlcH from supernatants of bacteria grown with succinate and ammonium ions in a low-phosphate medium was eluted as a peak with 10% of salt and was less than 10% inhibited by tetradecyltrimethylammonium. PlcH from low phosphate was purified associated with a protein of 17 kDa. This complex was dissociated and separated on a Sephacryl S-200 column with 1% (w/v) sodium dodecyl sulfate. After this dissociation, the resulting protein of 70 kDa, corresponding to PlcH, was inhibited by tetradecyltrimethylammonium, showing a protection effect of the accompanying protein. RT-PCR analyses showed that in choline media, the plcH gene was expressed independently of plcR. In low-phosphate medium, the plcH gene was expressed as a plcHR operon. Because plcR encodes for chaperone proteins, this result correlates with the observation that PlcH from supernatants of bacteria grown in the presence of choline was purified without an accompanying protein. The consequence of the absence of this chaperone was that tetradecyltrimethylammonium inhibited the PlcH activity.
Assuntos
Colina/farmacologia , Pseudomonas aeruginosa/enzimologia , Tensoativos/farmacologia , Compostos de Trimetil Amônio/farmacologia , Fosfolipases Tipo C/antagonistas & inibidores , Proteínas de Bactérias , Meios de Cultura , Indução Enzimática , Regulação Bacteriana da Expressão Gênica , Hemólise , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crescimento & desenvolvimento , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/metabolismoRESUMO
AIMS: The aim of this work was to establish if the response to tetradecyltrimethylammonium (TDTMA), a representative quaternary ammonium compound (QAC), involves changes in the phospholipid (PL) composition of Pseudomonas putida A ATCC 12633. METHODS AND RESULTS: Pseudomonas putida was exposed to 50 mg l(-1) of TDTMA for 15 min, and PL composition was analysed. With respect to control values, phosphatidic acid and phosphatidylglycerol increased by 140% and 120%, respectively; cardiolipin decreased about 60%. In TDTMA-adapted bacteria, the most significant change was a 380% increase in phosphatidic acid. Accompanying this change was a 130% increase in phosphatidylglycerol and a 70% decrease in cardiolipin. The changes in adapted cells were reverted after two subcultures without biocide. CONCLUSIONS: Pseudomonas putida responded to TDTMA through quantitative changes in PLs with specific variations in the content of phosphatidic acid, phosphatidylglycerol and cardiolipin. These modifications indicated that these PLs are involved in cellular responses to QACs, utilizing phosphatidic acid principally to neutralize the high positive charge density given for the ammonium quaternary moiety from TDTMA. SIGNIFICANCE AND IMPACT OF THE STUDY: The changes in PL composition give a new insight about the response inflicted by Ps. putida when these bacteria are exposed to QACs.
Assuntos
Desinfetantes/farmacologia , Fosfolipídeos/metabolismo , Pseudomonas putida/efeitos dos fármacos , Compostos de Trimetil Amônio/farmacologia , Cardiolipinas/metabolismo , Meios de Cultura , Relação Dose-Resposta a Droga , Farmacorresistência Bacteriana , Ácidos Fosfatídicos/metabolismo , Fosfatidilgliceróis/metabolismo , Pseudomonas putida/crescimento & desenvolvimento , Pseudomonas putida/metabolismoRESUMO
The effect of three cationic surfactants bearing the same polar head group and different chain length (cetyltrimethyl ammonium bromide (CTAB); tetradecyltrimethylammonium bromide (TTAB); dodecyltrimethylammonium bromide (DTAB)) on the conformation and function of the sea anemone pore-forming toxins sticholysins I and II (St I and St II) was studied by fluorescence and circular dichroism spectroscopy and evaluation of hemolytic activity (HA). Preincubation of the toxins with the longer chain surfactants CTAB and TTAB at concentrations slightly above their critical micelle concentration (CMC) leads to an enhancement of their HA. Significant increases in the fluorescence intensity with a slightly red shift in lambda(max) were observed at concentrations close to the surfactants' CMC, suggesting changes in the environment of the tryptophan residues. The changes in the fluorescence intensity are more noticeable and take place at lower surfactant concentrations for St I, irrespective of the surfactant alkyl chain length, although the differences between St I and St II increase as the surfactant alkyl chain length increases. This is evinced not only by the higher fluorescence intensity values and the lower surfactant concentrations required to reach them, but also by the higher acrylamide-quenching constant values (Ksv) for St I. However, the surfactant's effects on the toxins' HA were not found to be directly related to the observed changes in fluorescence intensity, as well as near- and far-UV-CD spectra. In particular, the latter spectra indicate that changes in HA and in fluorescence behavior take place without noticeable modifications in St I and St II secondary and tertiary structures. The results suggest that the interaction with the surfactants induces only subtle conformational changes in the toxins that favor the formation of lytic competent structures.
Assuntos
Venenos de Cnidários/farmacologia , Hemólise/efeitos dos fármacos , Proteínas Citotóxicas Formadoras de Poros/farmacologia , Compostos de Amônio Quaternário/farmacologia , Anêmonas-do-Mar , Tensoativos/farmacologia , Animais , Cátions , Cetrimônio , Compostos de Cetrimônio/química , Dicroísmo Circular , Venenos de Cnidários/química , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Sinergismo Farmacológico , Eritrócitos/efeitos dos fármacos , Humanos , Técnicas In Vitro , Micelas , Compostos Orgânicos/química , Compostos Orgânicos/farmacologia , Proteínas Citotóxicas Formadoras de Poros/química , Conformação Proteica , Compostos de Amônio Quaternário/química , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Tensoativos/química , Compostos de Trimetil Amônio/químicaRESUMO
Counterion and composition effects on the size and interface dynamics of discotic nematic lyotropic liquid crystals made of tetradecyltrimethylammonium halide (TTAX)-decanol (DeOH)-water-NaX, with X = Cl(-) and Br(-), were investigated using NMR and fluorescence spectroscopies. The dynamics of the interface was examined by measuring deuterium quadrupole splittings from HDO (0.1% D(2)O in H(2)O) and 1,1-dideuterodecanol (20% 1,1-dideuterodecanol in DeOH) in 27 samples of each liquid crystal. Aggregation numbers, N(D), from 15 samples of each mesophase were obtained using the fluorescence of pyrene quenched by hexadecylpyridinium chloride. N(D) of TTAB and TTAC are about 230+/-30 and 300+/-20, respectively. N(D) of TTAC increases with increasing concentration of all mesophase components, whereas TTAB shows no correlation between size and composition. The dimension of these aggregates prevents the occurrence of undulations, previously observed in lamellar phases. The quadrupole splitting of decanol-d(2) in TTAC is about 5 kHz smaller than in TTAB, and the splitting of HDO is observed only in TTAB. All results are consistent with a more dynamic TTAC interface. The TTAC aggregate should be more dissociated from counterions and the excess ammonium-ammonium electrostatic repulsions contribute to increase the mobility of the interface components.
Assuntos
Cristais Líquidos/química , Espectroscopia de Ressonância Magnética , Micelas , Pirenos/química , Compostos de Trimetil Amônio/químicaRESUMO
A capillary zone electrophoretic (CZE) method has been developed for the determination of impurities (phosphyte and phosphate) in technical-grade ibandronate, which is a potent nitrogen-containing bisphosphonate. Successful separation of the drug from the impurities was achieved using 1mM tetradecyl-trimethyl-ammonium bromide (TTAB) and 5mM potassium chromate (pH 10.0) as background electrolyte with an indirect detection at 254 nm. The optimised method was validated for specificity, precision, linearity and accuracy. The limit of detection (LOD) was 2 microg/mL and the limit of quantification (LOQ) was 7 microg/mL for both phosphyte and phosphate. The developed CZE method used to determine phosphyte and phosphate as bisphosphonates impurities can be used to evaluate the quality of regular production samples of ibandronate.
Assuntos
Conservadores da Densidade Óssea/análise , Difosfonatos/análise , Contaminação de Medicamentos , Eletroforese Capilar/métodos , Cromatos/química , Eletroforese Capilar/instrumentação , Concentração de Íons de Hidrogênio , Ácido Ibandrônico , Fosfatos/análise , Fosfitos/análise , Compostos de Potássio/química , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrofotometria Ultravioleta , Compostos de Trimetil Amônio/químicaRESUMO
Most drugs have to cross cell membranes to reach their final target. A better understanding of the distribution, interactions, and dynamics of biologically active molecules in model bilayers is of fundamental importance in understanding drug functioning and design. 2H NMR quadrupole splittings (delta nu(Q)) and longitudinal relaxation times (T1) from the aromatic ring of benzyl alcohol-d5 (C0), a commonly used anesthetic, and a series of linear alkyl benzyl-d5 ethers with chain lengths from 1 to 12 carbon atoms (C1-C12), were measured. The molecules were dissolved in a nematic discotic lyotropic liquid crystal solution made of tetradecyltrimethylammonium chloride (TTAC)/decanol (DeOH)/NaCl/H2O. Values of delta nu(Q) and T1 from 1,1-dideuteriodecanol (15% enriched) and DHO (H2O with 0.2% D2O) were also measured. Delta nu(Q) of DeOH and DHO remained constant throughout the series. The value of delta nu(Q) of the para position of the ring (delta nu(p)) in C1 is 30% smaller than the delta nu(p) of C0. This is attributed to the existence of an H-bond between the alcohol hydroxyl proton and the solvent, which influences the average orientation of the ring. The relaxation data show that T1o,m is always longer than T1p and both decrease with the increase in alkyl chain length. Molecular dynamics simulations of the experimentally studied systems were performed. The aggregate was represented as a bilayer. The distribution, average orientation, and order parameters of the aromatic ring of the guest molecules in the bilayer were examined. Rotational correlation functions of all the C-D bonds and the OH bond from H2O were evaluated, allowing an estimate of the correlation times and T1. According to these results all spins relax in extreme narrowing conditions, except DeOH. Experimental and calculated T1 values differ at most by a factor of 3. However, the order of magnitude and the observed trends are well reproduced by the calculations. The aromatic ring of C0 possesses a unique average orientation in the bilayer. For the ether series, the orientation is modified and the C2 symmetry axis of the aromatic ring is exchanging between two orientations averaging the quadrupole splittings from the ortho and meta positions. The simulation supports the existence of an H-bond between C0 and the solvent not found in the ethers, which should be responsible for the observed differences.