RESUMEN
In this contribution the use of Analytical Ultracentrifugation (AUC) for the modern analysis of colloids is reviewed. Since AUC is a fractionation technique, distributions of the sedimentation coefficient, particle size and shape, molar mass and density can be obtained for particle sizes spanning the entire colloidal range. The Ångström resolution and the reliable statistics with which particle size distributions can be obtained from analytical ultracentrifugation makes this a high resolution analysis technique for the characterization of nanoparticles in solution or suspension. Several examples showing successful applications of AUC to complex problems in colloid science are given to illustrate the broad range and versatility of questions that can be answered by AUC experiments.
Asunto(s)
Coloides/química , Biofisica/métodos , Técnicas de Química Analítica , Cromatografía/métodos , Computadores , ADN/química , Sustancias Macromoleculares/química , Modelos Estadísticos , Óptica y Fotónica , Tamaño de la Partícula , Programas Informáticos , Factores de Tiempo , UltracentrifugaciónRESUMEN
We report an analytical ultracentrifugation study on sedimentation in dilute stable dispersions of uniform, magnetic iron oxide (Fe3O4) colloids. On increase of the dipolar coupling constant, tuned by the average particle size, the linear concentration dependence of the sedimentation velocity shows an abrupt transition from the hindered sedimentation expected for hard spheres to a marked acceleration already for weak dipolar interactions. This transition is not reproduced by sedimentation theory derived from an effective, isotropic pair correlation function of the type proposed by De Gennes and Pincus, for reasons which are made clear. Accelerated settling, instead, follows a scaling based on the mass action law for dimer formation with dipoles in head-to-tail configuration. Our work illustrates that orientational averaging of dipole interactions of ferromagnetic colloids in the dilute regime is inapplicable, with an obvious implication for the possible existence of isotropic gas-liquid criticality for such colloids.
RESUMEN
Activation of the complement system generates potent chemoattractants and leads to the opsonization of cells for immune clearance. Short-lived protease complexes cleave complement component C3 into anaphylatoxin C3a and opsonin C3b. Here we report the crystal structure of the C3 convertase formed by C3b and the protease fragment Bb, which was stabilized by the bacterial immune-evasion protein SCIN. The data suggest that the proteolytic specificity and activity depend on the formation of dimers of C3 with C3b of the convertase. SCIN blocked the formation of a productive enzyme-substrate complex. Irreversible dissociation of the complex of C3b and Bb is crucial to complement regulation and was determined by slow binding kinetics of the Mg(2+)-adhesion site in Bb. Understanding the mechanistic basis of the central complement-activation step and microbial immune evasion strategies targeting this step will aid in the development of complement therapeutics.
Asunto(s)
Proteínas Bacterianas/química , C3 Convertasa de la Vía Alternativa del Complemento/química , Proteínas Inactivadoras de Complemento/química , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Sitios de Unión , Catálisis , Dominio Catalítico , Complemento C3/química , Complemento C3/metabolismo , C3 Convertasa de la Vía Alternativa del Complemento/metabolismo , Convertasas de Complemento C3-C5/química , Convertasas de Complemento C3-C5/metabolismo , Complemento C3b/química , Complemento C3b/metabolismo , Proteínas Inactivadoras de Complemento/inmunología , Proteínas Inactivadoras de Complemento/metabolismo , Vía Alternativa del Complemento/inmunología , Cristalografía por Rayos X , Humanos , Cinética , Modelos Moleculares , Unión Proteica , Multimerización de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Staphylococcus aureus/química , Staphylococcus aureus/inmunología , Staphylococcus aureus/metabolismo , Especificidad por Sustrato , Resonancia por Plasmón de SuperficieRESUMEN
We report a method to determine the particle size distribution of small colloidal silica spheres via analytical ultracentrifugation and show that the average particle size, variance, standard deviation, and relative polydispersity can be obtained from a single sedimentation velocity (SV) analytical ultracentrifugation (AUC) experiment. The particle size distribution (psd) from the enhanced van Holde-Weischet (vHW) analysis accounts for the dynamic light scattering results quite well. In addition, the vHW psd equals the psd from a continuous distribution of sedimentation coefficients analysis where whole sedimentation velocity boundaries are fitted. The SV AUC interference optical data also yield the specific particle volume such that distributions of sedimentation coefficients for colloidal spheres can be converted directly to particle size distributions. Our results show that SV AUC experiments may yield a quantitative particle size distribution without a priori knowledge of the particle size and the shape of the size distribution.
RESUMEN
We demonstrate the existence of discrete single molecular [Mo(132)] Keplerate-type clusters in aqueous solution. Starting from a discrete spherical [Mo(132)] cluster, the formation of an open-basket-type [Mo(116)] defect structure is shown for the first time in solution using analytical ultracentrifugation sedimentation velocity experiments.
RESUMEN
We present a convenient and low-cost method to prepare milligram amounts of completely monodisperse DNA restriction fragments in a physico-chemical laboratory setting to study (in part II) the effect of limited flexibility on the concentration dependent sedimentation velocity. Four fragments of 200, 400, 800, and 1600 bp were designed to span a range of 1-11 persistence lengths. The fragments were synthesized by cloning fragments of controlled lengths obtained by PCR into bacterial plasmid DNA. The constructs were amplified in large-scale bacterial cultures from which the fragments were obtained by a modified alkaline lysis procedure and subsequent digestion with EcoRV. A method is presented to isolate the DNA from the digestion mixture using horizontal agarose-slab gels and agarose columns in a home-built preparative gel electrophoresis set-up. We show that a combination of optical absorbance readings, ethidium bromide fluorescence, and hyperchromicity measurements allows assessment of both the purity of the DNA solutions and the fraction of double-stranded DNA.
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ADN/química , Desoxirribonucleasas de Localización Especificada Tipo II/química , Clonación Molecular , ADN/biosíntesis , Electroforesis en Gel de Agar , Escherichia coli/genética , Escherichia coli/metabolismo , Etidio , Colorantes Fluorescentes , Plásmidos , Reacción en Cadena de la Polimerasa , Soluciones , Espectrofotometría UltravioletaRESUMEN
We report sedimentation velocity and equilibrium measurements performed with an analytical ultracentrifuge to elucidate the effects of limited flexibility on the transport properties of semiflexible, monodisperse, double-stranded, blunt-ended DNA restriction fragments. We study a homologous series of fragments with 400, 800, and 1600 base pairs (3 to 11 persistence lengths), which are specifically designed and synthesized for this purpose (Part I). The molecular weights following from the sedimentation measurements agree well with the values expected on the basis of the number of base pairs. The sedimentation coefficients at infinite dilution are in good agreement with theoretical predictions for wormlike cylinders. The first order in volume fraction (varphi) coefficient K of the varphi-dependent sedimentation coefficient s(varphi)=1-Kvarphi decreases from 1178 for the shortest fragment to 882 for the longest fragment. These values are much larger than predicted for uncharged rigid rods, indicating the presence of associates with an enhanced aspect ratio and excluded volume. The precise match of the molecular weights obtained from exponential sedimentation-diffusion equilibrium distributions with weights calculated from the number of base pairs shows that any association is reversible and disappears at sufficiently low DNA concentration.
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ADN/química , Desoxirribonucleasas de Localización Especificada Tipo II/química , Coloides , Peso Molecular , Plásmidos , Espectrofotometría Ultravioleta , UltracentrifugaciónRESUMEN
UNLABELLED: The authors examined the serum copper concentrations in a group of 40 patients with chronic heart failure and significant systolic left ventricular dysfunction with an ejection fraction of less than 35%. In 21 patients the examination was made on admission to hospital on account of deterioration of cardiac insufficiency (group A), in 19 patients with stable chronic cardiac failure the examination was made at the out-patient department (group B). Assessment of serum copper concentrations was made by the method of absorption spectrophotometry under standard conditions. Assessment of the ejection fraction of the left ventricle and dimensions of the cardiac compartments was made by two-dimensional echocardiography. The heart rate was assessed from the electrocardiogram at rest. RESULTS: The authors found statistically significantly higher copper concentrations in patients of group A (19.78 +/- 1.67 mumol/l) as compared with group B (14.80 +/- 1.54 mumol/l, p < 0.001). The correlation between copper concentrations, the left ventricular ejection fraction, dimensions of the cardiac compartments and heart rate are not significant. CONCLUSION: Serum copper concentrations in patients with chronic heart failure and significant systolic left ventricular dysfunction are related to the severity of the disease and are higher in patients admitted on account of cardiac insufficiency than in patients with stable cardiac failure.
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Cobre/sangre , Insuficiencia Cardíaca/sangre , Disfunción Ventricular Izquierda/sangre , Anciano , Anciano de 80 o más Años , Enfermedad Crónica , Femenino , Insuficiencia Cardíaca/complicaciones , Insuficiencia Cardíaca/fisiopatología , Humanos , Masculino , Persona de Mediana Edad , Volumen Sistólico , Sístole , Disfunción Ventricular Izquierda/complicacionesRESUMEN
Vasculitis is accepted to be the basis of Behçet's disease (BD) which is a multisystem disease, and the arachidonic acid(AA) metabolites acting as balancing mediators in the organism are accepted to be responsible for the vasculitis. In this study, we examined the prostaglandin E2 (PGE2) and leukotriene C4 (LTC4) levels of the patients with BD before and after colchicine therapy. We found a statistical decrease in the PGE2 and LTC4 levels after colchicine therapy compared to the previous levels, concluding that colchicine inhibits the inflammation and the polymorphonuclear leukocyte (PML) chemotaxis by inhibiting the cyclooxygenase and lipoxygenase pathways.