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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 of any plant P450 is available so far because of difficulty in crystallising the protein. We employ analytical ultracentrifugation to characterise the oligomeric state of an E. coli-expressed recombinant HPL from Medicago truncatula (HPL-F) under different solution conditions. Sedimentation velocity analyses show that HPL-F (under detergent-free conditions) is largely a monomer with a sedimentation coefficient s20,w of ∼4.1 S (a value expected from the molar mass of the monomer). The effects of protein concentration, and detergent micelles on the oligomeric state of detergent-free HPL-F are reported for the first time. With increase in protein concentration only traces of dimers can be detected. However, HPL-F in association with detergent is a mixture of oligomers, which are not in reversible equilibrium with each other. These studies have important implications as they show that the oligomeric state of HPL-F changes with micellar association, both of which are related to the activity of the protein. They also show the virtue of combining sedimentation velocity with sedimentation equilibrium in the ultracentrifuge for the study of enzyme-detergent systems. [ABSTRACT FROM AUTHOR]

Hydrodynamic and optical investigations were performed on the binary dibutyl phthalate (DBP)-decalin liquid mixtures with the DBP content varying from 0 to 100%. This system may be suitable for modelling of the low-molecular-weight polymer behaviour in solution. Macro-characteristics of the system, such as medium viscosity and diffusivity, were evaluated in addition to the molar refraction and the flow birefringence of the system. A non-monotonous function of the molecular diffusivity of the mixture components was revealed. The low-speed ultracentrifugation data yield the dimensions of the mobile units varying with the DBP molar fraction in the mixture. The characteristics of the extreme composition are evaluated and compared with those known for other systems. [ABSTRACT FROM AUTHOR]

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 before aggregation and after re-dissolution. Moreover, absorbance concentration profiles identified a temperature dependent portion of polymer not participating in the aggregation process. Sedimentation equilibrium experiments allowed estimating the concentration influence on the molar mass. The study demonstrates for thermo-responsive polymers the suitability of analytical ultracentrifugation to analyze details of the association/aggregation process of stimuli-responsive polymers. [ABSTRACT FROM AUTHOR]

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 charge density/chemical composition. Combining synthetic boundary and sedimentation velocity experiments, and running at various speeds, allowed to identify the degree of homogeneity of the various distributions of the samples. It was illustrated that homogeneous batches of highly branched high molar mass polyelectrolytes can be synthesized with a negligible fraction of cross-linked molecules. Surprisingly, the sedimentation coefficient distribution of the main fraction was relatively narrow indicating a tight branching distribution. Overall, AUC delivers a variety of detail information, which cannot be obtained by other methods either due to lack of sensitivity or non-resolution of polydispersity. [ABSTRACT FROM AUTHOR]

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 in a case study, to test in a systematic manner the principles governing the evaluation strategies of the approaches applied. Therefore, emphasis was laid on the elaboration of modeling aspects rather than on biological details. Optimum results concerning the predictions of particle shapes and molecule properties have been obtained by utilizing tight constraints for modeling, such as symmetry and anisometry information. The use of pure ab initio conditions yields rather moderate shape and parameter predictions. Application of erroneous constraints generally leads to unrealistic particle shapes, although the parameter predictions may be satisfactory. The usage of the program DAMMIN turned out to be superior to application of the program GASBOR, whether the latter approach was used in the reciprocal- or real-space version. For hydrodynamic modeling, a modified version of the program HYDRO was adopted. By recourse to known crystallographic 3D structures for phosphorylases from other sources, SAXS profiles of anhydrous proteins can be modeled. Procedures for the addition of individual water molecules to anhydrous protein envelopes based on the atomic coordinates yield biologically relevant models for hydrated phosphorylases. This requires the usage of advanced surface calculation programs such as SIMS and of appropriate hydration algorithms such as those implemented in our programs HYDCRYST and HYDMODEL. The resulting SAXS profiles and structural and hydrodynamic parameters of the hydrated proteins can be compared with the data obtained by solution scattering. [ABSTRACT FROM AUTHOR]

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 have potential applications in other domains. [ABSTRACT FROM AUTHOR]

In this paper we study the solution properties of several metallo-supramolecular assemblies containing polystyrene (PS) and poly(ethylene oxide) (PEO) blocks linked via Ru2+ ions: PS20-[Ru2+]-PEO70, PS20-[Ru2+]-PS20, and PEO225-[Ru2+]-PEO225. Sedimentation equilibrium measurements were performed to determine the molar mass distribution and state of association, and the sedimentation velocity measurements were used to obtain the molar mass distribution, sedimentation coefficient distribution, their average values, and average diffusion coefficient. The results obtained from the two types of experiments are compared and discussed. [ABSTRACT FROM AUTHOR]

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 (D0) and velocity sedimentation coefficient (s0): [η] = 7.65 × 10−3 M0.8u0.1, D0 = 2.41 × 10−4 M−(0.59u0.05), s0 = 2.77 × 10−15 M0.41u0.05. Hydrodynamic data were interpreted by using the concept of electrostatic short- and long-range interactions. The equilibrium rigidity of poly(allylamine hydrochloride) chains in 0.1 M NaCl and structural and electrostatic contributions to it were quantitatively evaluated. It was shown that in pure water the conformation of poly(allylamine hydrochloride) chains is close to rod-like. [ABSTRACT FROM AUTHOR]

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 onto sedimentation are considered. This qualitative analysis allows an understanding of charge effects on the sedimentation behavior of colloids in a preformed pH-gradient and suggests ways for future quantitative evaluation of such data towards the determination of particle charge distributions. [ABSTRACT FROM AUTHOR]

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 of dispersions consists of particles, which have absorbed solvent, due to the fact, that they contain a few percent of soluble polymers. In the case of an aqueous dispersion then they are called hydrophilic. In these cases the density of these particles is unknown, it is between the density of the dry particle and that of the solvating medium. Applying the well-known ultracentrifugation method for determining particle size distributions from sedimentation velocity using the dry density results then in apparent diameters, which are smaller than the real diameters. To overcome this problem the degree of hydration has to be taken into account. So it is possible to calculate from the apparent diameter the diameter of the unswollen, compact particles as well as the diameter of the hydrated, swollen particles. The degree of hydration (or swelling) can be determined by preparative ultracentrifugation by pelleting the material and determining the weight of the wet material and after drying of the dry substance. To achieve equilibrium hydration pelleting is carried out at low concentrations and the particles are allowed to swell back at rest in the serum of the dispersion for several hours. Several examples are given. [ABSTRACT FROM AUTHOR]

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 represent over half of the thermodynamic data currently available for membrane proteins. Here, we consider the technical details of the EAUC methods used to study the stability of M2 in detergent solutions. Density-matching of detergent-buffer solutions yields precise values of the monomer-tetramer dissociation constant, and expressing these values in terms of peptide/detergent mole fraction units gives constant values over a modest range of experimentally relevant detergent concentrations. Furthermore, we have extended the EAUC method to calculate the number of detergent molecules bound to both monomer and tetramer species by employing a range of 2H2O buffer compositions. Determination of peptide-bound detergent not only reinforces the importance of density matching, but it opens the door to future analytical ultracentrifugation experiments involving membrane proteins in alternative detergents and lipid bicelles. [ABSTRACT FROM AUTHOR]

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 GC histograms of fixed-length sequence fragments (≈15-100 kb). This correspondence has been amply illustrated by genome sequences obtained over the past 5 years. Both AUC and sequencing have now generated large amounts of data that can be jointly mined. The dialogue between these two approaches should render tractable some tenacious problems of CsCl profile analysis, such as the correct treatment of concentration dependence for heterogeneous DNA. We focus on how absorbance profiles of a species' DNA vary as one changes the scale of one's observation (molecular weight), and dissect this scale-dependence into the contributions from its two main sources (diffusion, sequence effects). Our understanding of heterogeneous DNA in CsCl gradients can profit from the comparison of results from AUC and whole-genome sequencing, and the insights gained should prompt more strategic AUC analyses of DNA. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 of PhK induced by α and α . On the contrary, proline suppresses this process, probably, due to its specific interaction with PhK. We have also established that α-crystallin, a protein possessing chaperone-like activity, counteracts the self-association of PhK under molecular crowding conditions. Using dynamic light scattering we have shown that the increase in the light scattering intensity accompanying self-association of PhK is due to the formation of particles having hydrodynamic radius of hundreds of nanometers. The hydrodynamic radius of the start associates (seeds of association) was found to be approximately 80 nm. TMAO facilitates the formation of the associates of larger size whereas proline and α-crystallin suppress self-association of PhK. [ABSTRACT FROM AUTHOR]

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 coefficients that give aggregation numbers of 130 and 115 for DDM and C12E8, respectively, close to that given in the literature. The analysis of the number of interference fringes as a function of detergent concentration gave values for the critical micelle concentration and refractive index increments that are also in good agreement with published values. The values of the partial specific volumes of DDM and C12E8, obtained by sedimentation in H2O and D2O - where C12E8 floats - are identical to those obtained from density measurements. These results indicate that sedimentation velocity has good potential for proper characterisation of detergent-solubilized proteins. [ABSTRACT FROM AUTHOR]

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 and shape in the form of constraints or templates have been employed to improve the results. Templates for the subunit were preferably derived from SAXS-based models for the native hexagonal bilayer (HBL) complex that were biased by electron microscopic reconstructions. The obtained subunit models were carefully examined by variation of different selection and averaging methods and other checks such as surface renderings of the models. The findings were quantified by prediction of scattering profiles, I(h) and p(r), and structural and hydrodynamic parameters (V, RG, d max, s, D). The best matching models for the subunit were also scrutinized by comparing them to a model derived from currently available crystallographic data. The following results could be obtained: (i) The obtained parameter predictions for the dodecameric subunit are satisfactory, if compared to the SAXS data (consensus model, profiles and molecular parameters) or the results from hydrodynamic studies. (ii) The comparison between solution and crystal data of the dodecameric subunit, however, unequivocally proves a different behavior of the subunit in solution and the crystalline state. [ABSTRACT FROM AUTHOR]

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 are typically performed by a nonlinear least squares optimization algorithm. For models with many parameters, the error surface of this optimization often becomes very complex, the parameter solution tends to become trapped in a local minimum and the method may fail to converge. We introduce here a stochastic optimization approach for sedimentation velocity experiments utilizing genetic algorithms which is immune to such convergence traps and allows high-resolution fitting of nonlinear multi-component sedimentation models to yield distributions for sedimentation and diffusion coefficients, molecular weights, and partial concentrations. [ABSTRACT FROM AUTHOR]

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 coefficients (ks) and the estimated radius position at the cell base (rb). Deviation of the estimated rb value from the expected one is indicating the occurrence of ks that can be estimated by the program LammNum as a prerequisite for the calculation of reliable binding constants. [ABSTRACT FROM AUTHOR]

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 an effort to develop a fast UV/Vis detector, which is able to simultaneously detect the range from 200-800 nm. This is possible by the use of a modern CCD chip based generation of UV-Vis spectrometers, which translates the dispersed white light onto a CCD chip, where each pixel corresponds to a particular wavelength. In addition to the simultaneous detection of a large number of wavelengths in the range 200-800 nm, also with non integer values, these spectrometers are very fast. Current typical spectrum scan times with the necessary scan quality in the ultracentrifuge are in the range of 100 ms but this time can be significantly shortened down to 3 ms for higher light intensities and even down to 10 μs for a new generation of CCD chip based spectrometers. The introduction of a fiber based UV-Vis optics into a preparative XL-80K ultracentrifuge with the associated hardware developments will be described as a first generation prototype. In this study, we use a wavelength dependent optical lens system instead of the necessary but more complex wavelength independent mirror optical system for a first check on possibilities and limitations of the optical system. First examples for biopolymers and latexes will be presented and compared to those obtained in the commercial XL-A ultracentrifuge. Already the fast detection enables completely new possibilities like the determination of a particle size distribution in a few minutes. Multiwavelength detection at constant position in dependence of time will be demonstrated, which is an important mode for the use of speed profiles for very polydisperse samples. Also, the use of radial multiwavelength scans will be demonstrated producing a three dimensional data space for monitoring the sedimentation via radial scans with multiwavelength detection. However, despite the advantages, the current problems with the detector will also be discussed including the main problem that much intensity is lost in the important UV range as a result of fiber coupling and bending. [ABSTRACT FROM AUTHOR]

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 readings from three detectors (e.g. photomultipliers, avalanche photodiodes, etc.) simultaneously. For each detector, up to forty thousand intensity readings are acquired during each rotor revolution, for up to ten revolutions. Software synchronizes data acquisition with the spinning rotor. The use of continuous light sources allows simultaneous acquisition of dozens of intensity readings from all of the samples. Data acquisition is fast, allowing rapid radial scanning of samples. This data acquisition scheme is used in the Aviv Biomedical AU-FDS fluorescence detection retrofit system for the Beckman XLI analytical ultracentrifuge. It also will be used for the updated XLI absorbance system, as well as the next generation of analytical ultracentrifuge. [ABSTRACT FROM AUTHOR]

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 the density variation method appears of limited applicability. In addition, these systems are usually charged - a complication, which was so far not yet considered in the theory for the density variation method. In this work, we apply this method to the determination of the particle size and density of CaCO3 precursor particles, which form superstructures in order to elucidate their crystal modification. Interestingly, the particle densities can be determined rather reasonably, whereas the particle size is much more influenced by the nonideality. [ABSTRACT FROM AUTHOR]