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Light intensity measurements are required by absorbance, fluorescence, turbidity and low-angle light scattering detectors in order to determine the concentration distributions encountered in analytical ultracentrifugation. By using four fast analog-to-digital converters operating in parallel, a data acquisition system has been developed for the analytical ultracentrifuge that can acquire light intensity...
The advantages of simultaneously detecting multiple wavelengths in ultracentrifugation experiments are obvious, especially for interacting systems. In addition, the detection of the wavelength dependence of turbidity opens up the possibility to obtain independent information on the particle size in addition to the usual sedimentation coefficient distribution for colloidal systems. We therefore made...
We developed a system for acquisition and analysis of interference patterns from an analytical ultracentrifuge or a polarising diffusiometer. The system is the first of its kind to be based on highly sensitive Lebedev optical system. We also present a novel algorithm for automatic extraction of interference curves from photographs. The algorithm is fast, robust in the face of optical noise and may...
The binding constants of self-associating proteins can be determined from sedimentation velocity runs using the numerical solution of the Lamm equation. This procedure requires good starting values of sedimentation and diffusion coefficients or their possible concentration dependencies. We found, based on fits of synthetic data files, a connection between the concentration dependence of sedimentation...
Sedimentation experiments can provide a large amount of information about the composition of a sample, and the properties of each component contained in the sample. To extract the details of the composition and the component properties, experimental data can be described by a mathematical model, which can then be fitted to the data. If the model is nonlinear in the parameters, the parameter adjustments...
For modeling the low-resolution shape of the dodecameric subunit of Lumbricus terrestris hemoglobin, experimental small-angle X-ray scattering (SAXS) data and ab initio modeling approaches using a genetic algorithm or simulated annealing have been applied. In addition to the use of strict ab initio approaches, procedures which additionally include available structural information concerning symmetry...
The joint use of small-angle X-ray scattering (SAXS) and hydrodynamic data permits biologically useful reconstructions of protein structures to be determined. Low-resolution shapes of proteins can be obtained by SAXS-based modeling approaches, among them the ab initio approaches being the most recent and challenging ones. The programs DAMMIN and GASBOR have been applied to C. callunae starch phosphorylase...
We have characterized the hydrodynamic behaviour in H2O and D2O of two detergents, dodecyl-β-D-maltoside (DDM) and octaethylene glycol monododecyl ether (C12E8), by analysing sedimentation velocity profiles obtained with interference optics in terms of continuous particle distribution and non-interacting species. The analysis in H20 provides values for the sedimentation and diffusion...
Self-association of phosphorylase kinase (PhK) has been studied using analytical ultracentrifugation and dynamic light scattering under the conditions of molecular crowding arising from the presence of high concentrations of osmolyte. Sedimentation velocity analysis shows that in accordance with the predictions of molecular crowding theory, trimethylamine N-oxide (TMAO) greatly favours self-association...
This study considers the oligomeric of TBP1 from the extreme halophilic archaeaon Haloferax volcanii. This protein is in the first step of the cascade of binding events leading to transcription initiation. Sedimentation velocity shows that at 3M KCl, the protein appears to be in several oligomeric forms.
Since its introduction in the 1950's, analytical ultracentrifugation (AUC) of DNA in CsCl and other salt density gradients at sedimentation equilibrium has remained an elegant way to gain insight into the variation of base composition (GC, guanine + cytosine within animal and plant chromosomes, and into functional correlates of GC. Absorbance profiles of routine preparations of DNA in CsCl are essentially...
Though much progress has been made in the field of membrane protein folding, there is still much to learn about the association of transmembrane helices. Equilibrium analytical ultracentrifugation (EAUC) has been an important method of determining free energies of association for these types of systems. The M2 protein from the Influenza A virus, its transmembrane region and variants of that region...
The oligomeric state of fatty acid hydroperoxide lyase (HPL), of molar mass ∼55 kDa is uncertain and it has been reported as a trimer or tetramer in vivo. The enzyme has been found to be bi-functional and is active even in the absence of detergent. The association with detergent is known to stabilise the binding of the enzyme to its substrate and the enzyme is more active. No high resolution structure...
The classic idea of a particle is that of a hard particle for example a hard sphere. Deviations from this idea may refer to differences in shape. It may have the shape of an ellipsoid or a cylinder. These cases have in common, that the density of the particles is still well defined: It is the density of the solid material these particles consist of. In industrial practice however a great number...
The simultaneous determination of particle size and density distributions by the so called density variation method via measurement of the same sample in H2O and D2O proved to be of great value for latex systems. However, many colloids of practical interest are inorganic or inorganic-organic hybrid colloids. Their density is usually much higher than that of the solvents so that...
A new method is explored, where charged colloidal particles are sedimented in a pH-gradient, and an application in following the sedimentation velocity behavior of colloidal gold particles with cationic resp. anionic charge in a pH-gradient is described. A procedure to analyze this type of experiment is developed, where the effects of the changing pH and other parameters resulting from a pH-gradient...
The efficiency of two azoinitiators in the polymerization of allylamine salts in water and organic solvents was studied comparatively. Hydrodynamic and molecular properties of poly(allylamine hydrochloride) in 0.1 M NaCl were investigated in the molecular mass range 18 < MsD × 10−3(g/mol) < 65. Molecular mass relationships were obtained for intrinsic viscosity ([η]), translation diffusion coefficient...
High molar mass polyelectrolytes, which are polydisperse concerning the molar mass, the charge density, and the chain architecture were characterized by hydrodynamic methods, in particular analytical ultracentrifugation. The samples of interest were a series of copolymers differing in the degree of branching at constant chemical composition/charge density and highly branched polyelectrolytes of various...
Sedimentation velocity studies revealed concentration and temperature dependent aggregation of stimuli-responsive pre-associated polymers. The sedimentation coefficient monotonically increases with concentration and temperature while confirming pre-association prior to the phase separation. The aggregation process was proved as completely reversible by monitoring reproducible concentration signals...
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