scholarly journals Sedimentation coefficient distributions of large particles

The Analyst ◽  
2016 ◽  
Vol 141 (14) ◽  
pp. 4400-4409 ◽  
Author(s):  
Peter Schuck
Keyword(s):  
Analytical Ultracentrifugation ◽  
Sedimentation Coefficient ◽  
Mathematical Framework ◽  
Analysis Methods ◽  
Large Particles

A uniform mathematical framework for sedimentation coefficient distributions in analytical ultracentrifugation establishes new relationships and resolves differences in analysis methods.

scholarly journals Measurement of length distribution of beta-lactoglobulin fibrils by multiwavelength analytical ultracentrifugation

2020 ◽  
Vol 49 (8) ◽  
pp. 745-760 ◽  
Author(s):  
Maximilian J. Uttinger ◽  
Timon R. Heyn ◽  
Uwe Jandt ◽  
Simon E. Wawra ◽  
Bettina Winzer ◽  
...  
Keyword(s):  
Scaling Laws ◽  
Amyloid Fibrils ◽  
Analytical Ultracentrifugation ◽  
Length Distribution ◽  
Sedimentation Coefficient ◽  
Size Reduction ◽  
Diameter Distribution ◽  
Cylindrical Shape ◽  
Modal Values ◽  
Beta Lactoglobulin

AbstractThe whey protein beta-lactoglobulin is the building block of amyloid fibrils which exhibit a great potential in various applications. These include stabilization of gels or emulsions. During biotechnological processing, high shear forces lead to fragmentation of fibrils and therefore to smaller fibril lengths. To provide insight into such processes, pure straight amyloid fibril dispersions (prepared at pH 2) were produced and sheared using the rotor stator setup of an Ultra Turrax. In the first part of this work, the sedimentation properties of fragmented amyloid fibrils sheared at different stress levels were analyzed with mulitwavelength analytical ultracentrifugation (AUC). Sedimentation data analysis was carried out with the boundary condition that fragmented fibrils were of cylindrical shape, for which frictional properties are known. These results were compared with complementary atomic force microscopy (AFM) measurements. We demonstrate how the sedimentation coefficient distribution from AUC experiments is influenced by the underlying length and diameter distribution of amyloid fibrils.In the second part of this work, we show how to correlate the fibril size reduction kinetics with the applied rotor revolution and the resulting energy density, respectively, using modal values of the sedimentation coefficients obtained from AUC. Remarkably, the determined scaling laws for the size reduction are in agreement with the results for other material systems, such as emulsification processes or the size reduction of graphene oxide sheets.

scholarly journals Structure, Subunit Topology, and Actin-binding Activity of the Arp2/3 Complex from Acanthamoeba

1997 ◽  
Vol 136 (2) ◽  
pp. 331-343 ◽  
Author(s):  
R. Dyche Mullins ◽  
Walter F. Stafford ◽  
Thomas D. Pollard
Keyword(s):  
Electron Micrographs ◽  
Actin Filaments ◽  
Analytical Ultracentrifugation ◽  
Fluorescent Antibody ◽  
Complex Molecule ◽  
Gel Filtration ◽  
Sedimentation Coefficient ◽  
Leading Edge ◽  
Actin Binding ◽  
Filament Bundles

The Arp2/3 complex, first isolated from Acanthamoeba castellani by affinity chromatography on profilin, consists of seven polypeptides; two actinrelated proteins, Arp2 and Arp3; and five apparently novel proteins, p40, p35, p19, p18, and p14 (Machesky et al., 1994). The complex is homogeneous by hydrodynamic criteria with a Stokes' radius of 5.3 nm by gel filtration, sedimentation coefficient of 8.7 S, and molecular mass of 197 kD by analytical ultracentrifugation. The stoichiometry of the subunits is 1:1:1:1:1:1:1, indicating the purified complex contains one copy each of seven polypeptides. In electron micrographs, the complex has a bilobed or horseshoe shape with outer dimensions of ∼13 × 10 nm, and mathematical models of such a shape and size are consistent with the measured hydrodynamic properties. Chemical cross-linking with a battery of cross-linkers of different spacer arm lengths and chemical reactivities identify the following nearest neighbors within the complex: Arp2 and p40; Arp2 and p35; Arp3 and p35; Arp3 and either p18 or p19; and p19 and p14. By fluorescent antibody staining with anti-p40 and -p35, the complex is concentrated in the cortex of the ameba, especially in linear structures, possibly actin filament bundles, that lie perpendicular to the leading edge. Purified Arp2/3 complex binds actin filaments with a Kd of 2.3 μM and a stoichiometry of approximately one complex molecule per actin monomer. In electron micrographs of negatively stained samples, Arp2/3 complex decorates the sides of actin filaments. EDC/NHS cross-links actin to Arp3, p35, and a low molecular weight subunit, p19, p18, or p14. We propose structural and topological models for the Arp2/3 complex and suggest that affinity for actin filaments accounts for the localization of complex subunits to actinrich regions of Acanthamoeba.

Characterization of Factor VIII Immune Complexes By Analytical Ultracentrifugation

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3787-3787
Author(s):  
Pete Lollar ◽  
Ernest T. Parker ◽  
John F. Healey ◽  
Christopher B. Doering
Keyword(s):  
Immune Response ◽  
Factor Viii ◽  
Immune Complexes ◽  
Hemophilia A ◽  
Analytical Ultracentrifugation ◽  
Conflicts Of Interest ◽  
Sedimentation Coefficient ◽  
Coagulation Factor ◽  
Velocity Sedimentation ◽  
Molar Excess

Abstract Inhibitory polyclonal IgG antibodies (inhibitors) to factor VIII (fVIII) represent the most significant complication in patients with congenital hemophilia A. FVIII also is the most frequently targeted coagulation factor in autoimmunity. Antibodies recognizing epitopes in the fVIII A2 and C2 domains are present in most inhibitor patients. In the current study, we characterized the hydrodynamic properties of fVIII immune complexes formed by murine anti-human anti-A2 and anti-C2 fVIII monoclonal antibodies (MAbs) 4A4 and 3D12. 4A4 is representative of the most frequently identified group of anti-A2 MAbs identified in the murine hemophilia A immune response to human fVIII. 3D12 is a classical anti-C2 MAb that inhibits the binding of fVIII to von Willebrand factor (VWF) and phospholipid membranes. Velocity sedimentation of immune complexes formed by varying ratios of 4A4 and 3D12 with a high-expression fVIII construct designated ET3 was conducted at 55,000g and 20 °C by measuring protein absorbance at 280 nm in a Beckman XL-I analytical ultracentrifuge. Sedimentation coefficient (s20,w) distributions of fVIII, MAbs and immune complexes were determined using SEDFIT. The sedimentation coefficients of fVIII in the absence of MAbs and of the MAbs in the absence of fVIII were 7.7 S and 6.4 S, respectively. Under conditions of excess MAb (equimolar 4A4 and 3D12 each in five-fold molar excess over fVIII), a 10.3 S immune complex was observed, representing singly-ligated MAbs (Figure, red trace). Under conditions of excess fVIII (fVIII in four-fold molar excess over equimolar 4A4 and 3D12), 11.9 S doubly-ligated MAb complexes were observed (Figure, green trace). A mixture containing equimolar fVIII and 4A4/3D12 MAb binding sites produced a dominant 14.0 S species and a minor 18.8 S species, indicative of cross-linked 3D12-fVIII-4A4 immune complexes (Figure, blue trace). Indefinite association or immunoprecipitation was not observed. These results demonstrate that a biclonal, bivalent anti-fVIII antibody population can form higher-order immune complexes. These complexes may be a driving factor in the immune response to fVIII by promoting B cell activation and/or antigen presentation. Additionally, these results indicate that analytical ultracentrifugation is a useful tool to characterize fVIII immune complexes. Figure Figure. Disclosures No relevant conflicts of interest to declare.

Conceptual Examination of Gas Phase Particulate Formation in Gas Turbine Combustors

1980 ◽  
Vol 102 (3) ◽  
pp. 613-618 ◽  
Author(s):  
A. S. Kesten ◽  
J. J. Sangiovanni ◽  
P. Goldberg
Keyword(s):  
Gas Turbine ◽  
Gas Phase ◽  
Mathematical Framework ◽  
Droplet Combustion ◽  
Laboratory Studies ◽  
Gas Turbine Combustors ◽  
Large Particles ◽  
Fuel Pyrolysis ◽  
Insight Into ◽  
Recent Laboratory

Recent laboratory studies of droplet combustion indicate the potential for substantial gas phase particulate formation even with single component hydrocarbon fuels. Formation of large particles has been observed in the neighborhood of burning droplet arrays, particularly when the droplets are closely spaced. To provide insight into the potential for particulate formation during the combustion of fuel droplet sprays in gas turbine combustors, a mathematical framework is developed for examining the formation of soot nuclei in droplet combustion. A simplified model of the chemistry of fuel pyrolysis and nuclei formation is used and a series of calculations is made to explore the sensitivity of soot nuclei formation to conditions typical of gas turbine combustion systems.

scholarly journals Domain structure of human complement C4b extends with increasing NaCl concentration: implications for its regulatory mechanism

2016 ◽  
Vol 473 (23) ◽  
pp. 4473-4491 ◽  
Author(s):  
Ka Wai Fung ◽  
David W. Wright ◽  
Jayesh Gor ◽  
Marcus J. Swann ◽  
Stephen J. Perkins
Keyword(s):  
Crystal Structure ◽  
Domain Structure ◽  
Conformational Changes ◽  
Analytical Ultracentrifugation ◽  
Sedimentation Coefficient ◽  
Salt Bridge ◽  
X Ray ◽  
X Ray Scattering ◽  
Nacl Concentration ◽  
Dual Polarisation Interferometry

During the activation of complement C4 to C4b, the exposure of its thioester domain (TED) is crucial for the attachment of C4b to activator surfaces. In the C4b crystal structure, TED forms an Arg104–Glu1032 salt bridge to tether its neighbouring macroglobulin (MG1) domain. Here, we examined the C4b domain structure to test whether this salt bridge affects its conformation. Dual polarisation interferometry of C4b immobilised at a sensor surface showed that the maximum thickness of C4b increased by 0.46 nm with an increase in NaCl concentration from 50 to 175 mM NaCl. Analytical ultracentrifugation showed that the sedimentation coefficient s20,w of monomeric C4b of 8.41 S in 50 mM NaCl buffer decreased to 7.98 S in 137 mM NaCl buffer, indicating that C4b became more extended. Small angle X-ray scattering reported similar RG values of 4.89–4.90 nm for C4b in 137–250 mM NaCl. Atomistic scattering modelling of the C4b conformation showed that TED and the MG1 domain were separated by 4.7 nm in 137–250 mM NaCl and this is greater than that of 4.0 nm in the C4b crystal structure. Our data reveal that in low NaCl concentrations, both at surfaces and in solution, C4b forms compact TED–MG1 structures. In solution, physiologically relevant NaCl concentrations lead to the separation of the TED and MG1 domain, making C4b less capable of binding to its complement regulators. These conformational changes are similar to those seen previously for complement C3b, confirming the importance of this salt bridge for regulating both C4b and C3b.

Bovine Hageman Factor: Physicochemical Properties and Mechanism of Activation

1975 ◽  
Author(s):  
A. Molla ◽  
H. Claeys ◽  
D. Collen
Keyword(s):  
Conformational Changes ◽  
Analytical Ultracentrifugation ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sedimentation Coefficient ◽  
Factor Xii ◽  
Hageman Factor ◽  
Terminal Residue ◽  
Inactive Form ◽  
Fresh Plasma

Bovine Hageman factor (factor XII) was obtained in inactive form from fresh plasma with a recovery of 54.7±9.0 per cent and a yield of 16.3±2.9 mg per liter. The specific activity after activation with kaolin, determined with Hageman factor deficient human plasma, was 35.6±2.6 units (the activity of 1 ml fresh BaSO4-adsorbed bovine plasma) per mg protein.Analytical ultracentrifugation revealed a homogeneous protein with a sedimentation coefficient of 4.36. The extinction coefficient (A280 nm %) was 14.4, and the NH2-terminal residue Ala (0.6 mole/mole determined as Dns-Ala). SDS-polyacrylamide gel electrophoresis (PAGE) in the presence of dithiothreitol revealed a doublet with an average estimated mol wt of 76,000 and a difference of less than 5,000 between the two forms. Chemical analysis revealed (in moles per mole) Met: 2; His: 59; Lys: 28; Arg: 32 and sialic acid: 13 (thio-barbituric acid assay), hexose: 26 (orcinol method). PAGE at pH 2.5 revealed two and PAGE at pH 8.3 six main bands.Over 90 per cent of the purified iodine-labeled protein was adsorbed on celite. About 95 per cent of the enzyme activity was destroyed by incubation with 10−2 M di-iso-propylfluorophosphate (DFP) for 24 hr at room temperature. Between 65 and 85 per cent of the protein was eluted from the celite with 5% SDS. Both the NH2-terminal residue and the mol wt of the eluted protein were unchanged suggesting that activation of the proenzyme is the result of conformational changes with exposure of an active center serine residue. Measurements of radioactive DFP incorporation and COOH-terminal analysis, to prove this hypothesis are in progress.

Evidence for the release at low salt concentration of a lipid–protein–carbohydrate complex from isolated envelopes and whole cells of a marine pseudomonad

1971 ◽  
Vol 17 (5) ◽  
pp. 713-723
Author(s):  
F. L. A. Buckmire ◽  
Robert A. MacLeod
Keyword(s):  
Salt Concentration ◽  
Analytical Ultracentrifugation ◽  
Divalent Cations ◽  
Sedimentation Coefficient ◽  
Whole Cells ◽  
Carbohydrate Complex ◽  
Low Salt ◽  
Cell Envelopes ◽  
Isolated Cell

The effect of divalent cations on the turbidity and pH of suspensions of isolated cell envelopes of a marine pseudomonad has been examined. As the concentration of Mg2+, Ca2+, or Mn2+ was increased the turbidity and pH of the suspensions increased. A concentration of 0.010 M of divalent cations produced the same turbidity in a suspension of cell envelopes as 1.0 M NaCl. The envelopes released soluble non-dialyzable material (Ndf) when suspended in 0.01 M NaCl but not when suspended in 0.001 M solutions of the divalent cations. Whole cells of the marine pseudomonad released an Ndf when suspended in 0.5 M sucrose. The Ndf from both cells and envelopes contained lipid, protein, and carbohydrate. Analytical ultracentrifugation of the Ndf from both cells and envelopes revealed the presence of only one major component with a sedimentation coefficient of 9.7 ± 0.07. Electrophoresis of Ndf gave rise to a component which stained for carbohydrate and another which stained for protein. Both moved to the anode.

Masking of the Fc region in human IgG4 by constrained X-ray scattering modelling: implications for antibody function and therapy

2010 ◽  
Vol 432 (1) ◽  
pp. 101-114 ◽  
Author(s):  
Yuki Abe ◽  
Jayesh Gor ◽  
Daniel G. Bracewell ◽  
Stephen J. Perkins ◽  
Paul A. Dalby
Keyword(s):  
Concentration Dependence ◽  
Solution Structure ◽  
Analytical Ultracentrifugation ◽  
Sedimentation Coefficient ◽  
Radius Of Gyration ◽  
X Ray ◽  
X Ray Scattering ◽  
Starting Point ◽  
Self Association ◽  
Ray Scattering

Of the four human IgG antibody subclasses IgG1–IgG4, IgG4 is of interest in that it does not activate complement and exhibits atypical self-association, including the formation of bispecific antibodies. The solution structures of antibodies are critical to understand function and therapeutic applications. Thus IgG4 was studied by synchrotron X-ray scattering. The Guinier X-ray radius of gyration RG increased from 5.0 nm to 5.1 nm with an increase of concentration. The distance distribution function P(r) revealed a single peak at 0.3 mg/ml, which resolved into two peaks that shifted to smaller r values at 1.3 mg/ml, even though the maximum dimension of IgG4 was unchanged at 17 nm. This indicated a small concentration dependence of the IgG4 solution structure. By analytical ultracentrifugation, no concentration dependence in the sedimentation coefficient of 6.4 S was observed. Constrained scattering modelling resulted in solution structural determinations that showed that IgG4 has an asymmetric solution structure in which one Fab–Fc pair is closer together than the other pair, and the accessibility of one side of the Fc region is masked by the Fab regions. The averaged distances between the two Fab–Fc pairs change by 1–2 nm with the change in IgG4 concentration. The averaged conformation of the Fab regions appear able to hinder complement C1q binding to the Fc region and the self-association of IgG4 through the Fc region. The present results clarify IgG4 function and provide a starting point to investigate antibody stability.

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