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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]