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PubMed1979

Ultracentrifugation evidence for a somatostatin-binding protein in serum.

Using a technique of high speed centrifugation of serum and a well validated immunoassay for the measurement of serum somatostatin-like immunoreactivity, we have demonstrated that somatostatin, unlike other peptide hormones, appears to sediment with large molecular weight proteins. When synthetic somatostatin of increasing concentration was incubated with serum prior to ultracentrifugation, a linear plot of concentration of somatostatin added against concentration sedimenting (or apparently bound to protein) revealed an association curve. These data provide further evidence for the existence of a serum-binding protein for somatostatin.

Adult↗
PubMed1989

Comparison of the lipoprotein pattern of the horse, the pony and the lactating and non-lactating cow obtained by a combination of an ultracentrifugation and a precipitation technique.

1. The serum lipoprotein pattern was studied in four horses, four ponies and in three high producing lactating and three non-lactating cows. The lipoprotein pattern was estimated with a combination of the preparative ultracentrifugation and the heparin-manganese precipitation technique. 2. The lipid composition of the lipoproteins of horse, pony, lactating cow and non-lactating cow was determined. 3. In all three species more than 50% of serum total lipids was found in the HDL fraction. 4. The mean chylomicron fraction in horse and pony was 3.1%. In the cow it varied from 1.5 to 2.5%. 5. Between the lactating cow and the non-lactating cow there were substantial differences in the concentration of LDL and HDL. 6. The cholesterylester concentration in VLDL, LDL and HDL was clearly higher in the lactating cow than in the non-lactating cow.

Animals↗
PubMed2005

Fluorescence and analytical ultracentrifugation analyses of the interaction of the tyrosine kinase inhibitor, tyrphostin AG 1478-mesylate, with albumin.

Quantifying the interaction of drugs with carrier proteins in plasma is of importance for understanding effective drug delivery to disease-affected tissues. In this study, we employed analytical ultracentrifugation and steady-state fluorescence spectroscopy to characterize the interaction of a potential new anticancer drug, AG 1478-mesylate, with plasma proteins in a suspension of normal serum albumin (NSA). We found that mesylate salt of AG 1478, an epidermal growth factor receptor kinase inhibitor, sediments in 0.1%(w/v) NSA as a complex with a sedimentation coefficient of 3.8 S. This is consistent with the size of human serum albumin. This interaction was quantitated by meniscus depletion sedimentation and fluorescence titration analyses. AG 1478-mesylate binds to albumin with an apparent single-site affinity (K(d)) of 120 microM. In this article, we show that the cyclodextrin carrier molecule, Captisol, increases the apparent affinity of the hydrophobic AG 1478-mesylate for albumin (K(d)=4-6 microM), and we propose that the AG 1478-mesylate-Captisol (1:1) complex binds to albumin with at least 10-fold higher affinity than does AG 1478-mesylate ligand alone. A fluorenylmethoxycarbonyl-sulfonic acid (FMS) derivative of the 6-aminoquinazoline analog of AG 1478, which was designed to have improved serum-binding properties, was shown by fluorescence analysis to bind with approximately 100-fold greater affinity than the parent compound. This has significant implications in the effective delivery of therapeutic agents in vivo.

Protein Binding↗
PubMed2006

Sedimentation velocity analytical ultracentrifugation and SEDFIT/c(s): limits of quantitation for a monoclonal antibody system.

Sedimentation velocity analytical ultracentrifugation (SV-AUC) has emerged in the biopharmaceutical industry as a technique to detect small quantities of protein aggregates. However, the limits of detection and quantitation of these aggregates are not yet well understood. Although diverse factors (molecule, instrument, technique, and software dependent) preclude an all-encompassing measurement of these limits for the complete system, it is possible to use simulated data to determine the quantitation limits of the data analysis software aspect. The current study examines the performance of the SEDFIT/c(s) data analysis tool with simulated antibody monomer/dimer and monomer/aggregate systems. Under completely ideal conditions (zero noise, known meniscus, and shape factor homogeneity), the software limit of quantitation was 0.01% for the monomer/aggregate system and 0.03% for the less well-resolved monomer/dimer system. Under more realistic conditions (0.005 OD root mean square [RMS] noise, shape factor variability, and long solution column), the software limits of quantitation were 0.2 and 0.6% (0.002 and 0.006 OD) for the monomer/aggregate and monomer/dimer systems, respectively. Interestingly, diminished quantitation accuracy at very low levels of oligomer was not accompanied by deterioration of fit quality (as measured by root mean square deviation [RMSD] and residuals bitmap images).

Animals↗
PubMed2006

Monitoring the homogeneity of adenovirus preparations (a gene therapy delivery system) using analytical ultracentrifugation.

This study explores the capability of modern analytical ultracentrifugation (AUC) to characterize the homogeneity, under product formulation conditions, of preparations of adenovirus vectors used in gene therapy and to assess the lot-to-lot consistency of this unique drug product. We demonstrate that a single sedimentation velocity run on an adenovirus sample can detect and accurately quantify a number of different forms of virus particles and subvirus particles. These forms include (a) intact virus monomer particles, (b) virus aggregates, (c) empty capsids (ECs), and (d) smaller assembly intermediates or subparticles formed during normal or aberrant virus assembly (or as a result of damage to the intact adenovirus or EC material during all phases of virus production). This information, which is collected on adenovirus samples under the exact formulation conditions that exist in the adenovirus vial, is obtained by direct boundary modeling of the AUC data generated from refractometric and/or UV detection systems using the computer program SEDFIT developed by Peter Schuck. Although both detectors are useful, refractometric detection using the Rayleigh interferometer offers a key advantage for providing accurate concentration information due to the similar response factors for both protein and DNA and its insensitivity to light scattering effects. Additional AUC data obtained from analytical band sedimentation velocity and density gradient sedimentation equilibrium experiments in CsCl with UV detection were also generated. These results further support conclusions concerning the solution properties of adenovirus, the identity of the different virus species, and the overall capability of boundary sedimentation velocity analysis.

Adenoviridae↗
PubMed2005

Purification of infectious adenovirus in two hours by ultracentrifugation and tangential flow filtration.

Adenoviruses are excellent vectors for gene transfer and are used extensively for high-level expression of the products of transgenes in living cells. The development of simple and rapid methods for the purification of stable infectious recombinant adenoviruses (rAds) remains a challenge. We report here a method for the purification of infectious adenovirus type 5 (Ad5) that involves ultracentrifugation on a cesium chloride gradient at 604,000g for 15 min at 4 degrees C and tangential flow filtration. The entire procedure requires less than two hours and infectious Ad5 can be recovered at levels higher than 64% of the number of plaque-forming units (pfu) in the initial crude preparation of viruses. We have obtained titers of infectious purified Ad5 of 1.35x10(10) pfu/ml and a ratio of particle titer to infectious titer of seven. The method described here allows the rapid purification of rAds for studies of gene function in vivo and in vitro, as well as the rapid purification of Ad5.

Adenoviridae↗
PubMed2004

Spinning with Dave: David Yphantis's contributions to ultracentrifugation.

For nearly 50 years David Yphantis has helped advance analytical ultracentrifugation, promoted rigor in the thermodynamic analysis of biochemical data and encouraged students and colleagues to look for the deepest possible understanding of science. This article, written by five of Dave's students, presents some of the impressions he has made over the years.

Biochemistry↗
PubMed2006

Self-association of adrenodoxin studied by using analytical ultracentrifugation.

The mitochondrial steroid hydroxylase system of vertebrates utilizes adrenodoxin (Adx), a small iron-sulfur cluster protein of about 14 kDa as an electron carrier between a reductase and cytochrome P450. Although the crystal structure of this protein has been elucidated, the solution structure of Adx was discussed contrary in the literature [I.A. Pikuleva, K. Tesh, M.R. Waterman, Y. Kim, The tertiary structure of full-length bovine adrenodoxin suggests functional dimers, Arch. Biochem. Biophys. 373 (2000) 44-55; D. Beilke, R. Weiss, F. Löhr, P. Pristovsek, F. Hannemann, R. Bernhardt, H. Rüterjans, A new electron mechanism in mitochondrial steroid hydroxylase systems based on structural changes upon the reduction of adrenodoxin, Biochemistry 41 (2002) 7969-7978]. Therefore, it was necessary to study the self-association of this protein by using analytical ultracentrifugation over a larger concentration range. As could be demonstrated in sedimentation velocity experiments, as well as sedimentation equilibrium runs with explicit consideration of thermodynamic non-ideality, the full-length protein (residues 1-128) in the oxidized state resulted in a monomer-dimer equilibrium (K(a) approximately 3 x 10(2) M(-1)). For truncated Adx (1-108), as well as the reduced Adx, the association behavior was strongly reduced. The consequences of this behavior are discussed with respect to the physiological meaning for the Adx system.

Adrenodoxin↗
PubMed2005

Challenges for the modern analytical ultracentrifuge analysis of polysaccharides.

This article reviews some of the recent advances in analytical ultracentrifugation and how these advances have impacted--and can impact--on our understanding of the size, shape through conformation modelling, interactions and charge properties of polysaccharides in solution, particularly when used in combination with other solution techniques and also imaging techniques. Specifically we look at (1) polysaccharide polydispersity and simple shape analysis by sedimentation velocity, and in particular using new approaches such as SEDFIT analysis; (2) polysaccharide molecular-weight analysis by sedimentation equilibrium and MSTAR analysis and how this complements analysis of size exclusion chromatography coupled to multi-angle laser light scattering; (3) polysaccharide conformation analysis using traditional procedures such as the Wales-van Holde ratio, power law or 'scaling' relations, more specialised treatments for rigid cylindrical structures, semi-flexible chains and worm-like coils and complications through draining effects; (4) Analysis of polysaccharide interactions and in particular complex formation phenomena, focusing on interesting applications in the areas of mucoadhesion and sedimentation fingerprinting; and (5) the possibilities for macromolecular charge and charge screening measurement.

Amylopectin↗
PubMed2005

Damaged starch characterisation by ultracentrifugation.

The relative molecular size distributions of a selection of starches (waxy maize, pea and maize) that had received differing amounts of damage from ball milling (as quantified by susceptibility to alpha-amylase) were compared using analytical ultracentrifugation. Starch samples were solubilised in 90% dimethyl sulfoxide, and relative size distributions were determined in terms of the apparent distribution of sedimentation coefficients g*(s) versus s(20,w). For comparison purposes, the sedimentation coefficients were normalised to standard conditions of density and viscosity of water at 20 degrees C, and measurements were made with a standard starch loading concentration of 8 mg/mL. The modal molecular size of the native unmilled alpha-glucans were found to be approximately 50S, 51S and 79S for the waxy maize, pea and maize amylopectin molecules, respectively, whilst the pea and maize amylose modal molecular sizes were approximately 14S and approximately 12S, respectively. As the amount of damaged starch increased, the amylopectin molecules were eventually fragmented, and several components appeared, with the smallest fractions approaching the sedimentation coefficient values of amylose. For the waxy maize starch, the 50S material (amylopectin) was gradually converted to 14S, and the degradation process included the appearance of 24S material. For the pea starch, the situation was more complicated than the waxy maize due to the presence of amylose. As the amylopectin molecules (51S) were depolymerised by damage within this starch, low-molecular-weight fragments added to the proportion of the amylose fraction (14S)--although tending towards the high-molecular-weight region of this fraction. As normal maize starch was progressively damaged, a greater number of fragments appeared to be generated compared to the other two starches. Here, the 79S amylopectin peak (native starch) was gradually converted into 61 and 46S material and eventually to 11S material with a molecular size comparable to amylose. Amylose did not appear to be degraded, implying that all the damage was focused on the amylopectin fraction in all three cases. Specific differences in the damage profiles for the pea and maize starches may reflect the effect of lipid-complexed amylose in the maize starch.

Amylopectin↗
PubMed2006

Morphology of hybrid polystyrene-block-poly(ethylene oxide) micelles: analytical ultracentrifugation and SANS studies.

Morphology and structure of aqueous block copolymer solutions based on polystyrene-block-poly(ethylene oxide) (PS-b-PEO) of two different compositions, a cationic surfactant, cetyl pyridinium chloride (CPC), and either platinic acid (H2PtCl6.6H2O) or Pt nanoparticles were studied using a combination of analytical ultracentrifugation (AUC), transmission electron microscopy (TEM), and small angle neutron scattering (SANS). These studies combining methods contributing supplemental and analogous structural information allowed us to comprehensively characterize the complex hybrid systems and to discover an isotope effect when H2O was replaced with D2O. In particular, TEM shows formation of both micelles and larger aggregates after incorporation of platinic acid, yet the amount of aggregates depends on the H2PtCl6.6H2O concentration. AUC reveals the presence of micelles and micellar clusters in the PS-b-PEO block copolymers solution and even larger (supermicellar) aggregates in hybrids (with CPC). Conversely, SANS applied to D2O solutions of the similar species indicates that micelles are spherical and no other micellar species are found in block copolymer solutions. To reconcile the SANS and AUC data, we carried out AUC examination of the corresponding D2O block copolymer solutions. These measurements demonstrate a pronounced isotope effect on micelle aggregation and micelle size, i.e., no micelle aggregation in D2O solutions, revealing good agreement of AUC and SANS data.

Calorimetry, Differential Scanning↗
PubMed2003

A tetramer-octamer equilibrium in Mycobacterium leprae and Escherichia coli RuvA by analytical ultracentrifugation.

In the context of the bacterial RuvABC system, RuvA protein binds to and is involved in the subsequent processing of a four-way DNA structure called Holliday junction that is formed during homologous recombination. Four crystal structures of RuvA from Escherichia coli (EcoRuvA) showed that it was tetrameric, while neutron scattering and two other crystal structures for RuvA from Mycobacterium leprae (MleRuvA) and EcoRuvA showed that it was an octamer. To clarify this discrepancy, sedimentation equilibrium experiments by analytical ultracentrifugation were carried out and the results showed that MleRuvA existed as a tetramer-octamer equilibrium between 0.2-0.5 mg/ml in 0.1 M NaCl with a dissociation constant of 4 muM, and is octameric at higher concentrations. The same experiments in 0.3 M NaCl showed that MleRuvA is a tetramer up to 3.5 mg/ml, indicating that salt bridges are involved in octamer formation. Sedimentation equilibrium experiments with EcoRuvA showed that it was tetrameric at low concentration in both salt buffers but the protein was insoluble at high-protein concentrations in 0.1 M NaCl. It is concluded that free RuvA exists in an equilibrium between tetrameric and octameric forms in the typical concentration range and buffer found in bacterial cells.

Adenosine Triphosphatases↗
PubMed2004

Mutational analysis of the energetics of the GrpE.DnaK binding interface: equilibrium association constants by sedimentation velocity analytical ultracentrifugation.

DnaK, the prokaryotic Hsp70 molecular chaperone, requires the nucleotide exchange factor and heat shock protein GrpE to release ADP. GrpE and DnaK are tightly associated molecules with an extensive protein-protein interface, and in the absence of ADP, the dissociation constant for GrpE and DnaK is in the low nanomolar range. GrpE reduces the affinity of DnaK for ADP, and the reciprocal linkage is also true: ADP reduces the affinity of DnaK for GrpE. The energetic contributions of GrpE side-chains to GrpE-DnaK binding were probed by alanine-scanning mutagenesis. Sedimentation velocity (SV) analytical ultracentrifugation (AUC) was used to measure the equilibrium constants (Keq) for GrpE binding to the ATPase domain of DnaK in the presence of ADP. ADP-bound DnaK is the natural target of GrpE, and the addition of ADP (final concentration of 5 microM) to the preformed GrpE-DnaK(ATPase) complexes allowed the equilibrium association constants to be brought into an experimentally accessible range. Under these experimental conditions, the substitution of one single GrpE amino acid residue, arginine 183 with alanine, resulted in a GrpE-DnaK(ATPase) complex that was weakly associated (Keq =9.4 x 10(4) M). This residue has been previously shown to be part of a thermodynamic linkage between two structural domains of GrpE: the thermosensing long helices and the C-terminal beta-domains. Several other GrpE side-chains were found to have a significant change in the free energy of binding (DeltaDeltaG approximately 1.5 to 1.7 kcal mol(-1)), compared to wild-type GrpE.DnaK(ATPase) in the same experimental conditions. Overall, the strong interactions between GrpE and DnaK appear to be dominated by electrostatics, not unlike barnase and barstar, another well-characterized protein-protein interaction. GrpE, an inherent thermosensor, exhibits non-Arrhenius behavior with respect to its nucleotide exchange function at bacterial heat shock temperatures, and mutation of several solvent-exposed side-chains located along the thermosensing indicated that these residues are indeed important for GrpE-DnaK interactions.

Alanine↗
PubMed2005

Extended flexible linker structures in the complement chimaeric conjugate CR2-Ig by scattering, analytical ultracentrifugation and constrained modelling: implications for function and therapy.

Complement receptor 2 (CR2; CD21) is a membrane-bound regulator of complement activation, being comprised of 15 or 16 short complement repeat (SCR) domains. A recombinant glycosylated human CR2 SCR 1-2 domain pair was engineered with the Fc fragment of a mouse IgG1 antibody to create a chimaera CR2-Ig containing the major ligand binding domains. Such a chimaera has therapeutic potential as a complement inhibitor or immune modulator. X-ray and neutron scattering and analytical ultracentrifugation identified its domain structure in solution, and provided a comparison with controversial folded-back crystal structures for deglycosylated CR2 SCR 1-2. The radius of gyration R(G) of CR2-Ig was determined to be 5.39(+/-0.14) nm and 5.29(+/-0.01) nm by X-ray and neutron scattering, respectively. The maximum dimension of CR2-Ig was determined to be 17 nm. The molecular mass of CR2-Ig ranged between 101,000 Da and 107,000 Da as determined by neutron scattering and sedimentation equilibrium, in good agreement with the sequence-derived value of 106,600 Da. Sedimentation velocity gave a sedimentation coefficient of 4.49(+/-0.11) S. Stereochemically complete models for CR2-Ig were constructed from crystal structures for the CR2 SCR 1-2 and mouse IgG1 Fc fragments. The two SCR domains and the Fc fragment were joined by randomised conformational peptides. The analysis of 35,000 possible CR2-Ig models showed that only those models in which the two SCR domains were arranged in an open V-shape in random orientations about the Fc fragment accounted for the scattering and sedimentation data. It was not possible to define one single conformational family of Fab-like fragment relative to the Fc fragment. This flexibility is attributed to the relatively long linker sequence and the absence of the antibody light chain from CR2-Ig. The modelling also confirmed that the structure of CR2 SCR 1-2 is more extended in solution than in its crystal structure.

Amino Acid Sequence↗
PubMed2005

Purification, properties and extended solution structure of the complex formed between human immunoglobulin A1 and human serum albumin by scattering and ultracentrifugation.

Immunoglobulin A (IgA) is unique amongst antibodies in being able to form polymeric structures that may possess important functions in the pathology of specific diseases. IgA also forms complexes with other plasma proteins, the IgA1-human serum albumin (HSA) complex (IgA1-HSA) being typical. We have purified this complex using a novel two-step purification based on thiophilic chromatography and gel filtration, and characterised this. HSA is linked covalently to the tailpiece of IgA1 by a disulphide bond between Cys471 in IgA1 and Cys34 in HSA. IgA1-HSA binds to IgA receptors on neutrophils and monocytes, and elicits a respiratory burst that is comparable in magnitude to that of monomeric IgA1. The solution arrangement of IgA1-HSA was identified by X-ray scattering and ultracentrifugation. The radius of gyration R(G) of 7.5(+/-0.3) nm showed that IgA1-HSA is more extended in solution than IgA1 (R(G) of 6.1-6.2 nm). Its distance distribution function P(r) showed two peaks that indicated a well-separated solution structure similar to that for IgA1, and a maximum dimension of 25 nm, which is greater than that of 21 nm for IgA1. Sedimentation equilibrium showed that the IgA1:HSA stoichiometry is 1:1. Sedimentation velocity resulted in a sedimentation coefficient of 6.4S and a frictional ratio of 1.87, which is greater than that of 1.56 for IgA1. The constrained modelling of the IgA1-HSA structure using known structures for IgA1 and HSA generated 2432 conformationally randomised models of which 52 gave good scattering fits. The HSA structure was located at the base of the Fc fragment in IgA1 in an extended arrangement. Such a structure accounts for the functional activity of IgA1-HSA, and supports our previous modelling analysis of the IgA1 solution structure. The IgA1-HSA complex may suggest the potential for creating a new class of targeted therapeutic reagents based on the coupling of IgA1 to carrier proteins.

Humans↗
PubMed2006

Tetrameric structure of thermostable direct hemolysin from vibrio parahaemolyticus revealed by ultracentrifugation, small-angle X-ray scattering and electron microscopy.

The thermostable direct hemolysin (TDH) is a major virulence factor of Vibrio parahaemolyticus. We have characterized the conformational properties of TDH by small-angle X-ray scattering (SAXS), ultracentrifugation and transmission electron microscopy. Sedimentation equilibrium and velocity studies revealed that the protein is tetrameric in aqueous solvents. The Guinier plot derived from SAXS data provided a radius of gyration of 29.0 A. The elongated pattern with a shoulder of a pair distance distribution function derived from SAXS data suggested the presence of molecules with an anisotropic shape having a maximum diameter of 98 A. Electron microscopic image analysis of the negatively stained TDH oligomer showed the presence of C(4) symmetric particles with edge and diagonal lengths of 65 A and 80 A, respectively. Shape reconstruction was carried out by ab initio calculations using the SAXS data with a C(4) symmetric approximation. These results suggested that the tetrameric TDH assumes an oblate structure. The hydrodynamic parameters predicted from the ab initio model differed slightly from the experimental values, suggesting the presence of flexible segments.

Bacterial Toxins↗
PubMed2003

Effect of large refractive index gradients on the performance of absorption optics in the Beckman XL-A/I analytical ultracentrifuge: an experimental study.

An analytical centrifuge cell was modified to detect refraction of light transmitted through the cell caused by refractive index gradients formed by sedimenting solute during centrifugation. Sedimentation velocity and sedimentation equilibrium experiments were carried out in this cell on solutions containing high concentrations of protein and polysaccharide in a Beckman-Coulter XLA analytical ultracentrifuge. Analysis of the results indicates that in the absence of an optical artifact easily recognized as a "black band," the dependence of apparent absorbance upon radial position reported by the instrument may be considered a reliable measure of the solute concentration gradient.

Ovalbumin↗
PubMed2003

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.

Algorithms↗