The determination of density and molecular weight distributions of lipoproteins by sedimentation equilibrium.

A method is presented by which an experimental record of total concentration as a function of radial distance, obtained in a sedimentation equilibrium experiment conducted with a noninteracting mixture in the absence of a density gradient, may be analyzed to obtain the unimodal distributions of molecular weight and of partial molar volume when these vary concomitantly and continuously. Particular attention is given to the caracterization of classes of lipoproteins exhibiting Gaussian distributions of these quantities, although the analysis is applicable to other types of unimodal distribution. Equations are also formulated permitting the definition of the corresponding distributions of partial specific volume and of density. The analysis procedure is based on a method (employing Laplace transforms) developed previously, but differs from it in that it avoids the necessity of differentiating experimental results, which introduces error. The method offers certain advantages over other procedures used to characterize and compare lipoprotein samples (exhibiting unimodal distributions) with regard to the duration of the experiment, economy of the sample, and, particularly, the ability to define in principle all of the relevant distributions from one sedimentation equilibrium experiment and an external measurement of the weight average partial specific volume. These points and the steps in the analysis procedure are illustrated with experimental results obtained in the sedimentation equilibrium of a sample of human serum low density lipoprotein. The experimental parameters (such as solution density, column height, and angular velocity) used in the conduction of these experiments were selected on the basis of computer-simulated examples, which are also presented. These provide a guide for other workers interested in characterizing lipoproteins of this class.     

Determination of the sedimentation coefficient distribution by least-squares boundary modeling

A new method is presented for the calculation of apparent sedimentation coefficient distributions g*(s) for the size-distribution analysis of polymers in sedimentation velocity experiments. Direct linear least-squares boundary modeling by a superposition of sedimentation profiles of ideal nondiffusing particles is employed. It can be combined with algebraic noise decomposition techniques for the application to interference optical ultracentrifuge data at low loading concentrations with significant systematic noise components. Because of the use of direct boundary modeling, residuals are available for assessment of the quality of the fits and the consistency of the g*(s) distribution with the experimental data. The method can be combined with regularization techniques based on F statistics, such as used in the program CONTIN, or alternatively, the increment of s values can be adjusted empirically. The method is simple, has advantageous statistical properties, and reveals precise sedimentation coefficients. The new least-squares ls-g*(s) exhibits a very high robustness and resolution if data acquired over a large time interval are analyzed. This can result in a high resolution for large particles, and for samples with a high degree of heterogeneity. Because the method does not require a high frequency of scans, it can also be easily used in experiments with the absorbance optical scanning system. Published 2000 John Wiley & Sons, Inc.     

Non-ideality by sedimentation velocity of halophilic malate dehydrogenase in complex solvents

We have investigated the potential of sedimentation velocity analytical ultracentrifugation for the measurement of the second virial coefficients of proteins, with the goal of developing a method that allows efficient screening of different solvent conditions. This may be useful for the study of protein crystallization. Macromolecular concentration distributions were modeled using the Lamm equation with the approximation of linear concentration dependencies of the diffusion constant, D = D(o) (1 + k(D)c), and the reciprocal sedimentation coefficient s = s(o)/(1 + k(s)c). We have studied model distributions for their information content with respect to the particle and its non-ideal behavior, developed a strategy for their analysis by direct boundary modeling, and applied it to data from sedimentation velocity experiments on halophilic malate dehydrogenase in complex aqueous solvents containing sodium chloride and 2-methyl-2,4-pentanediol, including conditions near phase separation. Using global modeling for three sets of data obtained at three different protein concentrations, very good estimates for k(s) and s degrees and also for D degrees and the buoyant molar mass were obtained. It was also possible to obtain good estimates for k(D) and the second virial coefficients. Modeling of sedimentation velocity profiles with the non-ideal Lamm equation appears as a good technique to investigate weak inter-particle interactions in complex solvents and also to extrapolate the ideal behavior of the particle.     

A model for sedimentation in inhomogeneous media. II. Compressibility of aqueous and organic solvents

The effects of solvent compressibility on the sedimentation behavior of macromolecules as observed in analytical ultracentrifugation are examined. Expressions for the density and pressure distributions in the solution column are derived and combined with the finite element solution of the Lamm equation in inhomogeneous media to predict the macromolecular concentration distributions under different conditions. Independently, analytical expressions are derived for the sedimentation of non-diffusing particles in the limit of low compressibility. Both models are quantitatively consistent and predict solvent compressibility to result in a reduction of the sedimentation rate along the solution column and a continuous accumulation of solutes in the plateau region. For both organic and aqueous solvents, the calculated deviations from the sedimentation in incompressible media can be very large and substantially above the measurement error. Assuming conventional configurations used for sedimentation velocity experiments in analytical ultracentrifugation, neglect of the compressibility of water leads to systematic errors underestimating sedimentation coefficients by approximately 1% at a rotor speeds of 45000 rpm, but increasing to 2-5% with increasing rotor speeds and decreasing macromolecular size. The proposed finite element solution of the Lamm equation can be used to take solvent compressibility quantitatively into account in direct boundary models for discrete species, sedimentation coefficient distributions or molar mass distributions. Using the analytical expressions for the sedimentation of non-diffusing particles, the ls-g*(s) distribution of apparent sedimentation coefficients is extended to the analysis of sedimentation in compressible solvents. The consideration of solvent compressibility is highly relevant not only when using organic solvents, but also in aqueous solvents when precise sedimentation coefficients are needed, for example, for hydrodynamic modeling.     

On the analysis of protein self-association by sedimentation velocity analytical ultracentrifugation

Analytical ultracentrifugation is one of the classical techniques for the study of protein interactions and protein self-association. Recent instrumental and computational developments have significantly enhanced this methodology. In this paper, new tools for the analysis of protein self-association by sedimentation velocity are developed, their statistical properties are examined, and considerations for optimal experimental design are discussed. A traditional strategy is the analysis of the isotherm of weight-average sedimentation coefficients s(w) as a function of protein concentration. From theoretical considerations, it is shown that integration of any differential sedimentation coefficient distribution c(s), ls-g(*)(s), or g(s(*)) can give a thermodynamically well-defined isotherm, as long as it provides a good model for the sedimentation profiles. To test this condition for the g(s(*)) distribution, a back-transform into the original data space is proposed. Deconvoluting diffusion in the sedimentation coefficient distribution c(s) can be advantageous to identify species that do not participate in the association. Because of the large number of scans that can be analyzed in the c(s) approach, its s(w) values are very precise and allow extension of the isotherm to very low concentrations. For all differential sedimentation coefficients, corrections are derived for the slowing of the sedimentation boundaries caused by radial dilution. As an alternative to the interpretation of the isotherm of the weight-average s value, direct global modeling of several sedimentation experiments with Lamm equation solutions was studied. For this purpose, a new software SEDPHAT is introduced, allowing the global analysis of several sedimentation velocity and equilibrium experiments. In this approach, information from the shape of the sedimentation profiles is exploited, which permits the identification of the association scheme and requires fewer experiments to precisely characterize the association. Further, under suitable conditions, fractions of incompetent material that are not part of the reversible equilibrium can be detected.     

A method for directly fitting the time derivative of sedimentation velocity data and an alternative algorithm for calculating sedimentation coefficient distribution functions

The time-derivative method for deriving the sedimentation coefficient distribution, g(s*), from sedimentation velocity data that was developed by Walter Stafford has many advantages and is now widely used. By fitting Gaussian functions to the g(s*) distribution both sedimentation and diffusion coefficients (and therefore molecular masses) for individual species can be obtained. However, some of the approximations used in these procedures limit the accuracy of the results. An alternative approach is proposed in which the dc/dt data are fitted rather than g(s*). This new approach gives improved accuracy, extends the range to sedimentation coefficients below 1 S, and enhances resolution of multiple species. For both approaches the peaks from individual species are broadened when the data cover too wide a time span, and this effect is explored and quantified. An alternative algorithm for calculating ?(s*) from the dc/dt curves is presented and discussed. Rather than first averaging the dc/dt data for individual scan pairs and then calculating ?(s*) from that average, the ?(s*) distributions are calculated for every scan pair and then subsequently averaged. This alternative procedure yields smaller error bars for g(s*) and somewhat greater accuracy for fitted hydrodynamic properties when the time span becomes large.     

Macromolecular size-and-shape distributions by sedimentation velocity analytical ultracentrifugation

Sedimentation velocity analytical ultracentrifugation is an important tool in the characterization of macromolecules and nanoparticles in solution. The sedimentation coefficient distribution c(s) of Lamm equation solutions is based on the approximation of a single, weight-average frictional coefficient of all particles, determined from the experimental data, which scales the diffusion coefficient to the sedimentation coefficient consistent with the traditional s approximately M(2/3) power law. It provides a high hydrodynamic resolution, where diffusional broadening of the sedimentation boundaries is deconvoluted from the sedimentation coefficient distribution. The approximation of a single weight-average frictional ratio is favored by several experimental factors, and usually gives good results for chemically not too dissimilar macromolecules, such as mixtures of folded proteins. In this communication, we examine an extension to a two-dimensional distribution of sedimentation coefficient and frictional ratio, c(s,f(r)), which is representative of a more general set of size-and-shape distributions, including mass-Stokes radius distributions, c(M,R(S)), and sedimentation coefficient-molar mass distributions c(s,M). We show that this can be used to determine average molar masses of macromolecules and characterize macromolecular distributions, without the approximation of any scaling relationship between hydrodynamic and thermodynamic parameters.     

Conformational spectra--probing protein conformational changes

Stafford [Biophys. J. 17 (1996) MP452] has shown that it is possible, using the analytical ultracentrifuge in sedimentation velocity mode, to calculate the molecular weights of proteins with a precision of approximately 5%, by fitting Gaussian distributions to g(s*) profiles so long as partial specific volume and the radial position of the meniscus are known. This makes possible the analysis of systems containing several components by the fitting of multiple distributions to the total g(s*) profile. We have found the Stafford relationship to hold for a range of protein solutes, particularly good agreement being found when the g(s*) profiles are computed from Schlieren (dc/dr vs. r) data using the Bridgman equation [J. Am. Chem. Soc. 64 (1942) 2349] . On this basis, we have developed a new approach to the analysis of systems where two or more distinguishable conformations of a single species are present, either in the same sample cell or in different cells in the same rotor. In the former case, this allows us to analyse a given solution of pure protein (i.e. monodisperse with respect to M) to reveal the presence in that solution of two or more conformers under identical solvent conditions. In the latter case, we can detect with high sensitivity any conformational change occurring in the transition from one set of solvent conditions to another. Alternatively, in this case, we can analyse slightly different proteins (e.g. deletion mutants) for conformational changes under identical solvent conditions. Examples of these procedures using well-defined protein systems are given.     

Plant annexins form calcium-independent oligomers in solution

The oligomeric state in solution of four plant annexins, namely Anx23(Ca38), Anx24(Ca32), Anx(Gh1), and Anx(Gh2), was characterized by sedimentation equilibrium analysis and gel filtration. All proteins were expressed and purified as amino-terminal His(n) fusions. Sequencing of the Anx(Gh1) construct revealed distinct differences with the published sequence. Sedimentation equilibrium analysis of Anx23(Ca38), Anx24(Ca32), and Anx(Gh1) suggests monomer-trimer equilibria for each protein with association constants in the range of 0.9 x 10(10)-1.7 x 10(11) M(-2). All four proteins were subjected to analytical gel filtration under different buffer conditions. Observations from this experiment series agree quantitatively with the ultracentrifugation results, and strongly suggest calcium independence of the annexin oligomerization behavior; moreover, binding of calcium ions to the proteins seems to require disassembly of the oligomers. Anx(Gh2) showed a different elution profile than the other plant annexins; while having only a very small trimer content, this annexin seems to exist in a monomer-dimer equilibrium in solution.     

Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling

A new method for the size-distribution analysis of polymers by sedimentation velocity analytical ultracentrifugation is described. It exploits the ability of Lamm equation modeling to discriminate between the spreading of the sedimentation boundary arising from sample heterogeneity and from diffusion. Finite element solutions of the Lamm equation for a large number of discrete noninteracting species are combined with maximum entropy regularization to represent a continuous size-distribution. As in the program CONTIN, the parameter governing the regularization constraint is adjusted by variance analysis to a predefined confidence level. Estimates of the partial specific volume and the frictional ratio of the macromolecules are used to calculate the diffusion coefficients, resulting in relatively high-resolution sedimentation coefficient distributions c(s) or molar mass distributions c(M). It can be applied to interference optical data that exhibit systematic noise components, and it does not require solution or solvent plateaus to be established. More details on the size-distribution can be obtained than from van Holde-Weischet analysis. The sensitivity to the values of the regularization parameter and to the shape parameters is explored with the help of simulated sedimentation data of discrete and continuous model size distributions, and by applications to experimental data of continuous and discrete protein mixtures.     

Boundary analysis in sedimentation transport experiments: a procedure for obtaining sedimentation coefficient distributions using the time derivative of the concentration profile.

A procedure is described for computing sedimentation coefficient distributions from the time derivative of the sedimentation velocity concentration profile. Use of the time derivative, (delta c/delta t)r, instead of the radial derivative, (delta c/delta r)t, is desirable because it is independent of time-invariant contributions to the optical baseline. Slowly varying baseline changes also are significantly reduced. An apparent sedimentation coefficient distribution (i.e., uncorrected for the effects of diffusion), g*(s), can be calculated from (delta c/delta t)r as [formula: see text] where s is the sedimentation coefficient, omega is the angular velocity of the rotor, c0 is the initial concentration, r is the radius, rm is the radius of the meniscus, and t is time. An iterative procedure is presented for computing g*(s)t by taking into account the contribution to (delta c/delta t)r from the plateau region to give (delta c/delta t)corr. Values of g*(s)t obtained this way are identical to those of g*(s) calculated from the radial derivative to within the roundoff error of the computations. Use of (delta c/delta t)r, instead of (delta c/delta r)t, results in a significant increase (greater than 10-fold) in the signal-to-noise ratio of data obtained from both the uv photoelectric scanner and Rayleigh optical systems of the analytical ultracentrifuge. The use of (delta c/delta t)r to compute apparent sedimentation coefficient distributions for purposes of boundary analysis is exemplified with an antigen-antibody system.     

Cell-cycle analysis of perturbed cell populations: computer simulation of sequential DNA distributions.

A mathematical model is presented that permits simulation of a time sequence of DNA distributions with a single set of cell-cycle parameters. The method is particularly suited to the quantitative analysis of sets of sequential DNA distributions from perturbed cell populations. The model permits determination of the durations and associated dispersions of the phases of the cell cycle as well as the point in the cell cycle at which the perturbing agent exerts its effect. The mathematical details of the simulation technique are presented, and the technique is applied to the analysis of DNA distributions from perturbed cell populations. Three cell populations are modeled: CHO-line cells released from a block at the interface of the G1-and S-phases, 3T3 cells released from a G1-phase block produced by serum starvation, and S49 mouse lymphoma cells responding to a block in the G1-phage produced by N6,02'-dibutyryl adenosine 3':5'-cyclic monophosphate (Bt2cAMP).     

Cumulative sedimentation analysis of Escherichia coli debris size

A new method to measure Escherichia coli cell debris size after homogenization is presented. It is based on cumulative sedimentation analysis under centrifugal force, coupled with Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) analysis of sedimented proteins. The effects that fermentation and homogenization conditions have on the resulting debris distributions were investigated using this method. Median debris size decreased significantly from approximately 0.5 mum to 0.3 mum as the number of homogenization passes increased from 2 to 10. Under identical homogenization conditions, uninduced host cells in stationary phase had a larger debris size than exponential cells after 5 homogenizer passes. This difference was not evident after 2 or 10 passes, possibly because of confounding intact cells and the existence of a minimum debris size for the conditions investigated. Recombinant cells containing protein inclusion bodies had the smallest debris size following homogenization. The method was also used to measure the size distribution of inclusion bodies. This result compared extremely well with an independent determination using centrifugal disc photosedimentation (CDS), thus validating the method. This is the first method that provides accurate size distributions of E. coli debris without the need for sample pretreatment, theoretical approximations (e.g. extinction coefficients), or the separation of debris and inclusion bodies prior to analysis. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioang 55: 556-564, 1997.     

Sedimentation velocity, multi-speed method for analyzing polydisperse solutions

A method is described for the sedimentation velocity analysis of solutions composed of macromolecular solutes of widely disparate size. In sedimentation velocity experiments, usually a single rotor speed is chosen for the entire run, and consequently, the range of observable sedimentation coefficients can be severely limited. This limitation can be removed if the speed is varied during the run, starting with a relatively low speed so that the largest particles can be easily observed. The speed is increased during the run until full speed is attained and the run continued at full speed until the smallest species of interest have cleared the solution. The method, called wide distribution analysis, is based on the method developed originally by Yphantis and co-workers (Proc. Natl. Acad. Sci. USA 78 (1981) 1431) and on the time derivative method of Stafford (Anal. Biochem. 203 (1992) 295), essentially eliminating both the time-independent and radially-independent noise thereby improving the precision, especially for interference optics. An algorithm for analysis of data from both absorbance and interference optics and experimental protocols compatible with the Beckman XL-I Analytical Ultracentrifuge are presented. With these protocols an extremely wide range of sedimentation coefficients from approximately 1.0 to 250000 S can be accommodated in a single multi-speed run.     

Sedimentation velocity analysis of heterogeneous protein-protein interactions: sedimentation coefficient distributions c(s) and asymptotic boundary profiles from Gilbert-Jenkins theory

Interacting proteins in rapid association equilibrium exhibit coupled migration under the influence of an external force. In sedimentation, two-component systems can exhibit bimodal boundaries, consisting of the undisturbed sedimentation of a fraction of the population of one component, and the coupled sedimentation of a mixture of both free and complex species in the reaction boundary. For the theoretical limit of diffusion-free sedimentation after infinite time, the shapes of the reaction boundaries and the sedimentation velocity gradients have been predicted by Gilbert and Jenkins. We compare these asymptotic gradients with sedimentation coefficient distributions, c(s), extracted from experimental sedimentation profiles by direct modeling with superpositions of Lamm equation solutions. The overall shapes are qualitatively consistent and the amplitudes and weight-average s-values of the different boundary components are quantitatively in good agreement. We propose that the concentration dependence of the area and weight-average s-value of the c(s) peaks can be modeled by isotherms based on Gilbert-Jenkins theory, providing a robust approach to exploit the bimodal structure of the reaction boundary for the analysis of experimental data. This can significantly improve the estimates for the determination of binding constants and hydrodynamic parameters of the complexes.     

Genetic stability of strains preserved on LENTICULE discs and by freeze-drying: a comparison using fluorescent amplified fragment length polymorphism analysis

Fluorescent amplified fragment length polymorphism (FAFLP) analysis, a high-resolution genome fingerprinting method, was used to ascertain the DNA integrity of bacterial strains during preservation by lenticulation and by traditional freeze-drying into glass ampoules. This was achieved by comparing FAFLP genotypes of a range of paired bacterial isolates recovered from LENTICULE discs (preserved between 1995 and 2004) and from freeze-dried (FD) cultures in glass ampoules (preserved between 1966 and 2000). A choice of two endonuclease combinations EcoRI/MseI or HindIII/HhaI was used for FAFLP analysis of the five different bacterial genera comprising of 10 strains. Each of these 10 strains exhibited unique FAFLP profiles. However, there were no detectable differences between the FAFLP profiles for each of the individual strains, irrespective of their preservation format or their year of preservation. Thus, the FAFLP data suggests that LENTICULE production does not result in any detectable genetic changes during drying onto LENTICULE discs and storage for at least 5 years. The provision of such FD reference cultures on LENTICULE discs rather than FD glass ampoules will provide a cost-effective format that is easier to use.     

Size-distribution analysis of proteins by analytical ultracentrifugation: strategies and application to model systems.

Strategies for the deconvolution of diffusion in the determination of size-distributions from sedimentation velocity experiments were examined and developed. On the basis of four different model systems, we studied the differential apparent sedimentation coefficient distributions by the time-derivative method, g(s*), and by least-squares direct boundary modeling, ls-g*(s), the integral sedimentation coefficient distribution by the van Holde-Weischet method, G(s), and the previously introduced differential distribution of Lamm equation solutions, c(s). It is shown that the least-squares approach ls-g*(s) can be extrapolated to infinite time by considering area divisions analogous to boundary divisions in the van Holde-Weischet method, thus allowing the transformation of interference optical data into an integral sedimentation coefficient distribution G(s). However, despite the model-free approach of G(s), for the systems considered, the direct boundary modeling with a distribution of Lamm equation solutions c(s) exhibited the highest resolution and sensitivity. The c(s) approach requires an estimate for the size-dependent diffusion coefficients D(s), which is usually incorporated in the form of a weight-average frictional ratio of all species, or in the form of prior knowledge of the molar mass of the main species. We studied the influence of the weight-average frictional ratio on the quality of the fit, and found that it is well-determined by the data. As a direct boundary model, the calculated c(s) distribution can be combined with a nonlinear regression to optimize distribution parameters, such as the exact meniscus position, and the weight-average frictional ratio. Although c(s) is computationally the most complex, it has the potential for the highest resolution and sensitivity of the methods described.     

Effect of temperature on the self-assembly of the Escherichia coli ClpA molecular chaperone

Protein quality control pathways rely upon ATP-dependent proteases, such as Escherichia coli ClpAP, to perform maintenance roles in the cytoplasm of the cell. ATP-dependent proteases remove misfolded and partially synthesized proteins. This action is particularly important in situations where an unregulated accumulation of such proteins will have a deleterious effect on the cell. ClpAP is composed of a tetradecameric serine protease, ClpP (21.6 kDa monomer), and the ATPase/protein unfoldase ClpA (84.2 kDa monomer). ClpA also uses its protein unfolding activity to remodel proteins and protein complexes; thus, in the absence of the proteolytic component, ClpA is considered a molecular chaperone. Previous reports, by others, suggested that ClpA exists in a monomer-dimer equilibrium at 4 °C. In contrast, using a combination of sedimentation velocity, sedimentation equilibrium, and dynamic light scattering, we recently reported that ClpA exists in a monomer-tetramer equilibrium at 25 °C. Here we report an investigation of the effect of temperature on the self-association of the E. coli ClpA protein unfoldase using analytical ultracentrifugation techniques. The results of sedimentation velocity and sedimentation equilibrium experiments performed at multiple loading concentrations of ClpA over a range of temperatures from 3.9 to 38.2 °C are discussed. Sedimentation velocity experiments show a decrease in weight average s(20,w) at the extremes of temperature. This result, along with extensive sedimentation equilibrium data and analysis, suggests the presence of a dimeric intermediate of ClpA that is differentially populated as a function of temperature. Further, analysis of sedimentation equilibrium data as a function of temperature led us to propose a monomer-dimer-tetramer equilibrium to describe the temperature dependence of ClpA self-assembly in the absence of nucleotide.     

New Alicornops (Rhinocerotidae) remains from Lower and Middle Siwaliks,Pakistan

Unpublished rhinocerotid remains from the Lower and Middle Siwaliks of Pakistan are described in this paper and recognized as two species of Alicornops. Alicornops complanatum (Heissig) is identified in the Dhok Pathan Formation of the Middle Siwaliks and Alicornops laogouense Deng in the Kamlial Formation of the Lower Siwaliks. The Dhok Pathan Formation levels with A. complanatum are roughly correlated with the late Miocene-Pliocene European mammal zones MN10-15. In turn, levels with A. laogouense of the Kamlial Formation would correlate with the middle–late Aragonian (middle Miocene) European MN5. The recognition of the Chinese species A. laogouense in the Potwar Plateau represents the first discovery of this taxon in Pakistan and increases the geographical and stratigraphic distributions of this species, and adds to the rhinocerotid association from the Siwaliks. In turn, the presence of A. complanatum in the Siwaliks of Potwar Plateau also enlarges its geographic distribution in Pakistan, as it was previously known from the Bugti Hills of Balouchistan. The absence of Alicornops from the Siwaliks in the Chinji and Nagri formations (between late MN5 and MN9 zones) might be due to an inadequate fossil record, as other rhinocerotid species are known from Kamlial to Dhok Pathan formations. However, the two recorded species of Alicornops could also reveal two independent migration waves as supported by the appearance of other taxa in different formations. A summary of fossil Cenozoic rhinocerotids from different areas of Pakistan is also presented.     

Sedimentation equilibrium analysis of interference optical data by systematic noise decomposition.

An analytical method is described for removal of systematic signal offsets from interference optical data of sedimentation equilibrium gradients. It is demonstrated that the time-invariant signal contributions can be extracted from hydrodynamic modeling of interference profiles acquired during the approach to sedimentation equilibrium. This method is based on a technique for the explicit algebraic calculation of time-invariant noise components from sedimentation data, recently described for the direct modeling of sedimentation velocity experiments (P. Schuck and B. Demeler, Biophys. J. 76, 2288-2296, 1999). The calculated systematic signal offset is very well defined by the experimental data, stable over time, and its calculation is robust and to a large extent independent of the hydrodynamic model. The calculated time-invariant signal can be used to reduce the systematic errors in the measured sedimentation equilibrium profiles by more than an order of magnitude. It is shown that the resulting net equilibrium fringe profiles after subtraction of the time-invariant noise component allow equilibrium analyses consistent with those obtained from absorbance profiles. However, due to a higher dynamic range and the higher number of data points, the parameters derived from the net interference analysis can exhibit significantly improved precision. The presented study demonstrates the feasibility and potential of this analytical method for full exploitation of the remarkable precision of the interference optical data acquisition system, allowing sedimentation equilibrium experiments at loading concentrations below 0.05 mg/ml.