RESUMO
This article presents a novel application of small and wide angle X-ray scattering (SWAXS) in the assessment of aspirin and lactose content in a binary pharmaceutical powder formulation. It is shown that the content correlates with the intensity of the SAXS signal and the intensity of polymorph fingerprints in the WAXS spectra that are collected from the same samples. Because the polymorph WAXS fingerprints and the SAXS signal are two independent characteristics of the same sample, simultaneous SWAXS analysis provides the basis for a dual independent assessment of the same contents.
Assuntos
Aspirina/química , Pós/química , Espalhamento a Baixo Ângulo , Difração de Raios X/métodos , Lactose/química , Tamanho da Partícula , Raios XRESUMO
We report on the characterization of mesostructured aerosol silica particles in the gas phase using in situ synchrotron small-angle X-ray scattering (SAXS) in order to unveil the influence of the basic production parameters. The investigated system was based on tetraethylorthosilicate (TEOS) as the inorganic precursor and on cetyltrimethyl-ammonium bromide (CTAB) as the surfactant. The heating temperature, surfactant to silicate ratio, and particle flow rate were thoroughly investigated, and for this purpose, an in-house-built aerosol reactor equipped with a special X-ray observation chamber was used. Complementary fine structural analysis was applied on dried deposits of the silica aerosols comprising direct Fourier transforms as well as simple two-phase model fits. This resulted in robust estimates for the silica wall thickness and surfactant core radius of the hexagonally ordered mesostructure. The particle shape and size distribution were examined by scanning electron microscopy (SEM). The quality of the inner nanostructure was revealed from an analysis of the peak width. The comparison of data from the gas phase and powder deposit shows that, in general, slower drying conditions (heating temperature about 80 °C) and a medium surfactant to Si ratio (about 0.14) lead to nanostructures of the best quality in terms of well-defined long-range organization.
Assuntos
Gases/química , Dióxido de Silício/química , Difração de Raios X , Aerossóis , Elétrons , Tamanho da Partícula , Pós , Espalhamento a Baixo Ângulo , TemperaturaRESUMO
Valsartan is a marketed drug with high affinity to the type 1 angiotensin (AT1) receptor. It has been reported that AT1 antagonists may reach the receptor site by diffusion through the plasma membrane. For this reason we have applied a combination of differential scanning calorimetry (DSC), Raman spectroscopy and small and wide angle X-ray scattering (SAXS and WAXS) to investigate the interactions of valsartan with the model membrane of dipalmitoyl-phosphatidylcholine (DPPC). Hence, the thermal, dynamic and structural effects in bulk as well as local dynamic properties in the bilayers were studied with different valsartan concentrations ranging from 0 to 20 mol%. The DSC experimental results showed that valsartan causes a lowering and broadening of the phase transition. A splitting of the main transition is observed at high drug concentrations. In addition, valsartan causes an increase in enthalpy change of the main transition, which can be related to the induction of interdigitation of the lipid bilayers in the gel phase. Raman spectroscopy revealed distinct interactions between valsartan with the lipid interface localizing it in the polar head group region and in the upper part of the hydrophobic core. This localization of the drug molecule in the lipid bilayers supports the interdigitation view. SAXS measurements confirm a monotonous bilayer thinning in the fluid phase, associated with a steady increase of the root mean square fluctuation of the bilayers as the valsartan concentration is increased. At high drug concentrations these fluctuations are mainly governed by the electrostatic repulsion of neighboring membranes. Finally, valsartans' complex thermal and structural effects on DPPC bilayers are illustrated and discussed on a molecular level.
Assuntos
1,2-Dipalmitoilfosfatidilcolina/química , Bicamadas Lipídicas/química , Tetrazóis/química , Valina/análogos & derivados , 1,2-Dipalmitoilfosfatidilcolina/metabolismo , Algoritmos , Bloqueadores do Receptor Tipo 1 de Angiotensina II/química , Bloqueadores do Receptor Tipo 1 de Angiotensina II/metabolismo , Ligação Competitiva , Varredura Diferencial de Calorimetria , Cinética , Bicamadas Lipídicas/metabolismo , Modelos Químicos , Modelos Moleculares , Estrutura Molecular , Espalhamento a Baixo Ângulo , Análise Espectral Raman , Temperatura , Tetrazóis/metabolismo , Termodinâmica , Valina/química , Valina/metabolismo , Valsartana , Difração de Raios XRESUMO
An in-house built aerosol generator setup for in situ gas phase studies of aerosol and nanoparticles is described. The aerosol generator with an ultrasonic ceramic disk mist maker provides high enough particle concentrations for structural gas phase analysis by synchrotron small angle x-ray scattering (for water approximately 4 x 10(8) droplets/s with a droplet size of approximately 2.5 microm). The working principle was proved by scattering of gold nanoparticles. For evaporation induced self-assembly studies of nanostructured particles, an additional thermal treatment chamber was included in the setup. The first on-line gas phase data with our setup for mesostructured silica particles are presented for different thermal treatments. Scanning electron microscope imaging revealed the average particle size to be approximately 1 microm. Furthermore, to quantify their internal nanostructure, diffraction experiments of deposited silica aerosols were carried out and the corresponding electron density map indicates a silica wall thickness of about 1 nm.
Assuntos
Aerossóis/química , Gases/química , Microfluídica/instrumentação , Nanopartículas/química , Nanopartículas/ultraestrutura , Sonicação/instrumentação , Difração de Raios X/instrumentação , Desenho de Equipamento , Análise de Falha de Equipamento , Microfluídica/métodos , Transição de Fase , Espalhamento a Baixo Ângulo , Difração de Raios X/métodosRESUMO
The collagen diffraction patterns of human aortas under uniaxial tensile test conditions have been investigated by synchrotron small-angle X-ray scattering. Using a recently designed tensile testing device the orientation and d-spacing of the collagen fibers in the adventitial layer have been measured in situ with the macroscopic force and sample stretching under physiological conditions. The results show a direct relation between the orientation and extension of the collagen fibers on the nanoscopic level and the macroscopic stress and strain. This is attributed first to a straightening, second to a reorientation of the collagen fibers, and third to an uptake of the increasing loads by the collagen fibers.
Assuntos
Aorta/fisiologia , Colágeno/fisiologia , Síncrotrons , Difração de Raios X/métodos , Aorta/química , Fenômenos Biomecânicos , Colágeno/química , Elasticidade , Humanos , Espalhamento de Radiação , Resistência à Tração , Difração de Raios X/instrumentaçãoRESUMO
N-alkyl-N,N-dimethylamine-N-oxides (CnNO, n = 10-20 is the number of alkyl carbon atoms) stimulate the skeletal sarcoplasmic reticulum (SR) Ca(2+)-transporting ATPase activity at low concentrations and inhibit it at high concentrations. The minimum concentration (cmin), at which CnNO inhibits the ATPase, continuously decreases up to n = 16-18 and then increases. The values of Cmin are smaller than the CnNO critical micelle concentration (cmc) for C10NO-C14NO homologs, but larger than cmc for C18NO-C20NO homologs. The ATPase inhibition is caused by the CnNO-induced lipid bilayer structural perturbation in the ATPase annular region, modulated by the partition equilibria of the CnNO molecules between the bilayer and aqueous phase for short alkyl chain (n = 10-16) CnNO homologs, and between the bilayer, micelles and aqueous phase for long alkyl chain (n = 18-20) CnNO homologs.
Assuntos
ATPases Transportadoras de Cálcio/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Retículo Sarcoplasmático/enzimologia , Animais , Dimetilaminas/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Feminino , Técnicas In Vitro , Cinética , Bicamadas Lipídicas , Óxidos/farmacologia , Ligação Proteica , Coelhos , Retículo Sarcoplasmático/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
We have determined the structural properties and bending fluctuations of fully hydrated phosphatidylcholine multibilayers in the fluid (Lalpha) phase, as well as the structure of the ripple (Pbeta') phase near the main phase transition temperature (TM) by x-ray diffraction. The number of carbons, nHC, per acyl chain of the studied disaturated lipids varied from 14 to 22. All lipids exhibit a nonlinear increase of the lamellar repeat distance d in the Lalpha phase upon approaching TM, known as "anomalous swelling." The nonlinear increase reduces with chain length, but levels off at a constant value of about 0.5 A for lipids with more than 18 hydrocarbons per chain. A detailed analysis shows that anomalous swelling has two components. One is due to an expansion of the water layer, which decreases with chain length and finally vanishes for nHC >18. The second component is due to a bilayer thickness increase, which remains unchanged in its temperature dependence, including a nonlinear component of about 0.5 A in the vicinity of TM. Thus, anomalous swelling above 18 hydrocarbons per chain is due to the pretransitional effects on the membrane only. These results are supported by a bending fluctuation analysis revealing increased undulations close to TM only for the short chain lipids. We have further calculated the electron density maps in the ripple phase and find no coupling of the magnitude of the ripple amplitude to the chain length effects observed in the Lalpha phase. Hence, in agreement with an earlier report by Mason et al. [Phys. Rev. E 63, 030902 (2001)] there is no connection between the formation of the ripple phase and anomalous swelling.
Assuntos
Biofísica/métodos , Fosfatidilcolinas/química , Carbono/química , Hidrocarbonetos/química , Bicamadas Lipídicas/química , Lipídeos/química , Modelos Estatísticos , Temperatura , Água , Difração de Raios XRESUMO
Biological membranes play an essential role in the drug action. They constitute the first barrier for drugs to exert their biological action. AT1 antagonists are amphiphilic molecules and are hypothesized to act on AT1 receptor through incorporation (first step) and lateral diffusion through membrane bilayers (second step). Various biophysical methods along with Molecular Modelling were applied in order to explore the plausible two step proposed mechanism of action for this class of antihypertensive drugs.
Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II , Anti-Hipertensivos/química , Anti-Hipertensivos/farmacologia , Membrana Celular/efeitos dos fármacos , Aminoácidos/química , Aminoácidos/metabolismo , Anti-Hipertensivos/uso terapêutico , Sítios de Ligação , Compostos de Bifenilo/química , Compostos de Bifenilo/farmacologia , Varredura Diferencial de Calorimetria , Membrana Celular/química , Membrana Celular/metabolismo , Humanos , Hipertensão/tratamento farmacológico , Hipertensão/etiologia , Imidazóis/química , Imidazóis/farmacologia , Irbesartana , Losartan/química , Losartan/farmacologia , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Receptor Tipo 1 de Angiotensina/química , Tetrazóis/química , Tetrazóis/farmacologia , Difração de Raios XRESUMO
In an attempt to understand the multifunctional involvement of beta(2)-glycoprotein I (beta(2)GPI) in autoimmune diseases, thrombosis, atherosclerosis, and inflammatory processes, substantial interest is focused on the interaction of beta(2)GPI with negatively charged ligands, in particular, with acidic phospholipids. In this study, unilamellar vesicles composed of cardiolipin were used as in vitro membrane system to test and further refine a model of interaction based on the crystal structure of beta(2)GPI. The data suggest that beta(2)GPI anchors to the membrane surface with its hydrophobic loop adjacent to the positively charged lysine rich region in domain V. Subsequently, beta(2)GPI penetrates the membrane interfacial headgroup region as indicated by a restriction of the lipid side chain mobility, but without formation of a nonbilayer lipid phase. A structural rearrangement of beta(2)GPI upon lipid binding was detected by microcalorimetry and may result in the exposure of cryptic epitopes located in the complement control protein domains. This lipid-dependent conformational change may induce oligomerization of beta(2)GPI and promote intermolecular associations. Thus, the aggregation tendency of beta(2)GPI may serve as the basis for the formation of a molecular link between cells but may also be an essential feature for binding of autoantibodies and hence determine the role of beta(2)GPI in autoimmune diseases.
Assuntos
Apolipoproteínas/metabolismo , Glicoproteínas/metabolismo , Lipossomos/metabolismo , Fosfolipídeos/metabolismo , Dimiristoilfosfatidilcolina , Polarização de Fluorescência , Temperatura Alta , Humanos , Modelos Teóricos , Desnaturação Proteica , Espalhamento de Radiação , Espectrometria de Fluorescência , Raios X , beta 2-Glicoproteína IRESUMO
The effects of the two cannabinomimetic drugs (-)-2-(6a,7,10,10a-tetrahydro-6,6,9-trimethyl-1-hydroxy-6H-dibenzo[b,d]pyranyl-2-(hexyl)-1,3-dithiolane (AMG-3) and its pharmacologically less active 1-methoxy analogue (AMG-18) on the thermotropic and structural properties of dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC) liposomes have been studied by X-ray diffraction and differential scanning calorimetry (DSC). DSC data revealed that the incorporation of the drugs affect differently the thermotropic properties of DPPC. The presence of the more active drug distinctly broadened and attenuated both the pretransition and main phase transition of DPPC bilayers, while the inactive analogue had only minor effects. Small and wide angle X-ray diffraction data showed that the two cannabinoids have different effects on the lipid phase structures and on the hydrocarbon chain packing. The pharmacologically active analogue, AMG-3, was found to efficiently fluidize domains of the lipids in the L(beta)' gel phase, and to perturb the regular multibilayer lattice. In the liquid crystalline L(alpha) phase, AMG-3 was also found to cause irregularities in packing, suggesting that the drug induces local curvature. At the same concentration, the inactive AMG-18 had only minor structural effects on the lipids. At about 10-fold or higher concentrations, AMG-18 was found to produce similar but still less pronounced effects in comparison to those observed by AMG-3. The dose-dependent, different thermotropic and structural effects by the two cannabinoid analogues suggest that these may be related to their biological activity.
Assuntos
1,2-Dipalmitoilfosfatidilcolina/química , Canabinoides/química , Bicamadas Lipídicas , Fluidez de Membrana , Varredura Diferencial de Calorimetria , Canabinoides/farmacologia , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Relação Estrutura-Atividade , Difração de Raios XRESUMO
We present a method for analyzing small angle x-ray scattering data on multilamellar phospholipid bilayer systems at full hydration. The method utilizes a modified Caillé theory structure factor in combination with a Gaussian model representation of the electron density profile such that it accounts also for the diffuse scattering between Bragg peaks. Thus the method can retrieve structural information even if only a few orders of diffraction are observed. We further introduce a procedure to derive fundamental parameters, such as area per lipid, membrane thickness, and number of water molecules per lipid, directly from the electron density profile without the need of additional volumetric measurements. The theoretical apparatus is applied to experimental data on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, and 1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine liposome preparations.
Assuntos
Lipossomos/química , Lipossomos/metabolismo , Água/metabolismo , Modelos Moleculares , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/metabolismo , Espalhamento de Radiação , Água/química , Difração de Raios XRESUMO
Many biological membranes contain lipids that do not form a lamellar phase but the roles of these lipids are not well understood. An artificial membrane assembled from the main non-bilayer lipid and the major integral protein of pea thylakoids revealed that the protein spatially inhibits the formation of non-bilayer structures in the lamellae. Without this inhibition, excess lipids are secreted, creating lipid reservoirs for metabolism and/or later uptake. This determines the protein:lipid ratio in the membrane and hence the balance between structural flexibility and the stability of the key constituents that participate in cooperative interactions.
Assuntos
Lipídeos de Membrana/fisiologia , Bicamadas Lipídicas , Proteínas de Plantas/metabolismo , Plantas/metabolismoRESUMO
Experimental evidence supporting the hypothesis of gel-liquid crystalline phase coexistence in the stable ripple phase of diacylphosphatidylcholines has been obtained from time-resolved X-ray small- (SAXS) and wide-angle diffraction (WAXS) in the millisecond to second time domain. The pretransition of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) exhibits a thin lamellar liquid crystalline intermediate phase (designated Lalpha) if driven far away from equilibrium by an infrared temperature jump (T-jump) technique. The findings can be described by a two-step model. (1) Instantaneously with the T-jump, an anomalously thin lamellar liquid crystalline intermediate phase (d = 5.6-5.8 nm) forms, coexisting with the original gel-phase Lbeta'. Within the first seconds, the lamellar repeat distance of the intermediate increases to a value of about 6.7 nm. A closer examination of these kinetics reveals two relaxation components: a fast process, proceeding within tenths of a second, and a slow process, on the time scale of a few seconds. (2) Finally, both the liquid crystalline and the gel-phase relax into the stable ripple phase Pbeta'. The total process time of the transition is nearly independent of the addition of NaCl, but varies strongly with the chain length of the lecithin species.
Assuntos
1,2-Dipalmitoilfosfatidilcolina/química , Cristalização , Géis , Cinética , Modelos Moleculares , Conformação Molecular , Termodinâmica , Difração de Raios XRESUMO
In many biological membranes, the major lipids are "non-bilayer lipids," which in purified form cannot be arranged in a lamellar structure. The structural and functional roles of these lipids are poorly understood. This work demonstrates that the in vitro association of the two main components of a membrane, the non-bilayer lipid monogalactosyldiacylglycerol (MGDG) and the chlorophyll-a/b light-harvesting antenna protein of photosystem II (LHCII) of pea thylakoids, leads to the formation of large, ordered lamellar structures: (i) thin-section electron microscopy and circular dichroism spectroscopy reveal that the addition of MGDG induces the transformation of isolated, disordered macroaggregates of LHCII into stacked lamellar aggregates with a long-range chiral order of the complexes; (ii) small-angle x-ray scattering discloses that LHCII perturbs the structure of the pure lipid and destroys the inverted hexagonal phase; and (iii) an analysis of electron micrographs of negatively stained 2D crystals indicates that in MGDG-LHCII the complexes are found in an ordered macroarray. It is proposed that, by limiting the space available for MGDG in the macroaggregate, LHCII inhibits formation of the inverted hexagonal phase of lipids; in thylakoids, a spatial limitation is likely to be imposed by the high concentration of membrane-associated proteins.
Assuntos
Diglicerídeos/química , Galactolipídeos , Glicolipídeos/química , Lipídeos/química , Complexo de Proteínas do Centro de Reação Fotossintética/química , Dicroísmo Circular , Complexos de Proteínas Captadores de Luz , Substâncias Macromoleculares , Proteínas de Membrana/química , Proteínas de Membrana/ultraestrutura , Microscopia Eletrônica , Pisum sativum , Complexo de Proteínas do Centro de Reação Fotossintética/ultraestrutura , Complexo de Proteína do Fotossistema II , Proteínas de Plantas/química , Espalhamento de Radiação , Tilacoides/química , Tilacoides/ultraestruturaRESUMO
The high affinity of human plasma beta2-glycoprotein I (beta(2)GPI), also known as apolipoprotein-H (ApoH), for negatively charged phospholipids determines its implication in a variety of physiological pathways, including blood coagulation and the immune response. beta(2)GPI is considered to be a cofactor for the binding of serum autoantibodies from antiphospholipid syndrome (APS) and correlated with thrombosis, lupus erythematosus and recurrent fetal loss. We solved the beta(2)GPI structure from a crystal form with 84% solvent and present a model containing all 326 amino acid residues and four glycans. The structure reveals four complement control protein modules and a distinctly folding fifth C-terminal domain arranged like beads on a string to form an elongated J-shaped molecule. Domain V folds into a central beta-spiral of four antiparallel beta-sheets with two small helices and an extended C-terminal loop region. It carries a distinct positive charge and the sequence motif CKNKEKKC close to the hydrophobic loop composed of residues LAFW (313-316), resulting in an excellent counterpart for interactions with negatively charged amphiphilic substances. The beta(2)GPI structure reveals potential autoantibody-binding sites and supports mutagenesis studies where Trp316 and CKNKEKKC have been found to be essential for the phospholipid-binding capacity of beta(2)GPI.
Assuntos
Glicoproteínas/química , Sequência de Aminoácidos , Animais , Apolipoproteínas/química , Sítios de Ligação , Gráficos por Computador , Cristalografia por Raios X , Drosophila , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , beta 2-Glicoproteína IRESUMO
The structural organization of the neutral lipid core in human low density lipoproteins (LDL) was investigated in physicochemically defined, distinct human LDL subspecies in the density range of 1. 0244-1.0435 g/ml by evaluation of the core lipid transition temperature, chemical composition, and the behavior of spin-labeled core lipids. Calorimetric studies were performed on more than 60 LDL preparations, and the transition temperature, which varied between 19 and 32 degreesC, was correlated to the chemical composition and revealed a discontinuity at a critical cholesteryl ester to triglyceride ratio of approximately 7:1. For electron spin resonance studies, several LDL preparations were probed with spin-labeled cholesteryl esters and triglycerides, respectively. In LDL with a high triglyceride content, both labels exhibited similar mobility behavior. In contrast, in LDL with only small concentrations of triglycerides, the behavior of labeled cholesteryl esters and labeled triglycerides differed distinctly. The cholesteryl esters were strongly immobilized below the transition temperature, whereas the triglycerides remained fluid throughout the measured temperatures. These results suggest that the critical cholesteryl ester to triglyceride mass ratio of 7:1 corresponds to two concentric compartments with a radial ratio of 2:1, where the liquid triglycerides occupy the core, and the cholesteryl esters form the frozen shell. At higher triglyceride contents, the triglyceride molecules insert into the cholesteryl ester shell and depress the peak transition temperature of the LDL core, whereas at lower triglyceride contents, excess cholesteryl esters are dissolved in the core.
Assuntos
Lipoproteínas LDL/química , Triglicerídeos/química , Varredura Diferencial de Calorimetria , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Nêutrons , Espalhamento de Radiação , Temperatura , Raios XRESUMO
The effects of alkali chlorides on phosphatidylcholine-water bilayer systems in the Lalpha-phase were investigated by using small- and wide-angle X-ray scattering. The ternary system LiCl-POPC-H2O under isothermal conditions has shown that above Li+/POPC molar ratios of 0.1 and a lipid concentration above 5% (w/w), a splitting of the lamellar Bragg diffraction peaks into discrete components indicates a phase separation into different lamellar liquid crystalline (smectic A) phases. It is also shown that in saturated distearoyl phosphatidylcholine and in egg phosphatidylcholine, alkali chlorides induce Lalpha-phase separation. The number and repeat distance of the coexisting lamellar phases depend on the nature and concentration of the alkali chloride, the concentration of the phosphatidylcholine, and the degree of the acyl chain unsaturation.
Assuntos
Cloretos/química , Fosfatidilcolinas/química , Água/química , Fenômenos Químicos , Físico-Química , Cristalização , Concentração de Íons de Hidrogênio , Cloreto de Lítio/química , Espalhamento de Radiação , Raios XRESUMO
Small-angle x-ray scattering was used to investigate structural changes upon binding of individual substrates or a transition state analog complex (TSAC; Mg-ADP, creatine, and KNO3) to creatine kinase (CK) isoenzymes (dimeric muscle-type (M)-CK and octameric mitochondrial (Mi)-CK) and monomeric arginine kinase (AK). Considerable changes in the shape and the size of the molecules occurred upon binding of Mg-nucleotide or TSAC. The radius of gyration of Mi-CK was reduced from 55.6 A (free enzyme) to 48.9 A (enzyme plus Mg-ATP) and to 48.2 A (enzyme plus TSAC). M-CK showed similar changes from 28.0 A (free enzyme) to 25.6 A (enzyme plus Mg-ATP) and to 25.5 A (enzyme plus TSAC). Creatine alone did not lead to significant changes in the radii of gyration, nor did free ATP or ADP. AK also showed a change of the radius of gyration from 21.5 A (free enzyme) to 19.7 A (enzyme plus Mg-ATP), whereas with arginine alone only a minor change could be observed. The primary change in structure as seen with monomeric AK seems to be a Mg-nucleotide-induced domain movement relative to each other, whereas the effect of substrate may be of local order only. In CK, however, additional movements have to be involved.
Assuntos
Trifosfato de Adenosina/metabolismo , Arginina Quinase/química , Arginina Quinase/metabolismo , Creatina Quinase/química , Creatina Quinase/metabolismo , Conformação Proteica , Algoritmos , Animais , Galinhas , Clonagem Molecular , Simulação por Computador , Cristalografia por Raios X/métodos , Dimerização , Escherichia coli , Isoenzimas , Mitocôndrias/enzimologia , Mitocôndrias Musculares/enzimologia , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sarcômeros/enzimologia , Espalhamento de Radiação , Raios XRESUMO
The radical scavenging capacity of active nitroxide spin label radicals located at different depths in the surface monolayer of native and alpha-tocopherol enriched low density lipoprotein (LDL) has been evaluated at early stages of copper-mediated lipid peroxidation. Spin labels induced a concentration-dependent prolongation in lag time and a pronounced decrease in the initial rate of conjugated diene (CD) formation. These effects strongly argue for a protective, antioxidative action of spin labels, which in turn become destroyed with the extent of oxidation by radical recombination reactions. The results revealed that the decrease in spectral intensity proceeds at a higher rate for nitroxide radicals located in a more hydrophobic environment. The loss in spin label activity is accompanied by simultaneous alpha-tocopherol consumption and progresses rather independently of initial alpha-tocopherol content. The data provided no evidence that spin labels either save alpha-tocopherol or compete with it for radicals. The authors, therefore, deduce that due to enhanced accessibility and mobility, spin labels located in the interior of LDL eliminate lipid-derived radicals, which otherwise would promote lipid peroxidation. Lowering of temperature clearly below the core-lipid phase transition temperature of LDL exerts a significant effect on the kinetics of copper-induced LDL oxidation, whereas the characteristics of the radical scavenging mechanisms of the spin label molecules located in the surrounding phospholipid monolayer are conserved. Taken together, the susceptibility of LDL to primary oxidative stress conditions was efficiently retarded by small amounts of radical scavengers. This effect was more pronounced for nitroxide radicals embedded deeper in the phospholipid monolayer and was rather independent of alpha-tocopherol enrichment.
Assuntos
Sequestradores de Radicais Livres/farmacologia , Lipoproteínas LDL/química , Lipoproteínas LDL/efeitos dos fármacos , Cobre/farmacologia , Óxidos N-Cíclicos , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Técnicas In Vitro , Cinética , Peroxidação de Lipídeos/efeitos dos fármacos , Lipoproteínas LDL/sangue , Estresse Oxidativo/efeitos dos fármacos , Marcadores de Spin , Temperatura , Vitamina E/farmacologiaRESUMO
The development of surface roughness of two synthetic goethites with a high, 176 m2/g, and a low, 7 m2/g, initial specific surface area by acid attack is described in terms of fractal geometry. The dissolution kinetics were influenced by the presence of micropores and small mesopores created during dissolution and leading to an initial increase in surface area causing S-shaped dissolution curves. The fractal dimensions were calculated from the adsorption isotherms obtained with N2 as adsorbate. These values of surface fractal dimension agree quite well with the values of the mass and surface fractal dimension obtained with small angle X-ray scattering (SAXS). Copyright 1998 Academic Press. Copyright 1998Academic Press