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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.

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 HO 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.

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 -expressed recombinant HPL from (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 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.

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.

The simultaneous determination of particle size and density distributions by the so called density variation method via measurement of the same sample in and DO 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 CaCO 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.

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.

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 < M × 10(g/mol) < 65. Molecular mass relationships were obtained for intrinsic viscosity ([η]), translation diffusion coefficient () and velocity sedimentation coefficient (): [η] = 7.65 × 10 M,  = 2.41 × 10 M,  = 2.77 × 10 M. 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.

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.

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.

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.