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PubMed2002

Solution structures of complement components by X-ray and neutron scattering and analytical ultracentrifugation.

The short consensus/complement repeat (SCR) domain (also known as the complement control protein domain) is the most abundant domain type in the complement system. Crystal and NMR structures for proteins that contain single and multiple SCR domains have now been published. These contain inter-SCR linkers of between three and eight residues, and the structures show much variability in inter-SCR orientations. X-ray and neutron scattering, combined with analytical ultracentrifugation and constrained modelling based on known subunit structures will yield a medium-resolution structure for the protein of interest. The fewer parameters that are associated with the structure of interest, the more defined the structure of interest becomes. These solution studies have been applied to several SCR-containing proteins in the complement system, most notably Factor H with 20 SCR domains, a complement receptor type 2 fragment with two SCR domains, and rat complement receptor-related protein (Crry) which contains five SCR domains. The results show great conformational variability in the inter-SCR orientation, and these will be reviewed. Even though the rotational orientation cannot be modelled, it is nonetheless possible to measure the degree of extension of the multi-SCR proteins and, from this, to obtain functionally useful results.

Animals↗
PubMed1996

Activated factor VIII in therapeutic preparations: analysis by ultracentrifugation.

Activation of factor VIII is signaled by an increase in the 1-stage factor VIII activity and release from von Willebrand factor. Ultracentrifugation in 10-40% sucrose gradients was used to identify such activation in therapeutic concentrates. Plasma-derived factor VIII lots were examined and factor VIII sedimenting independently of von Willebrand factor was identified in some of the preparations. In addition there was slower sedimentation of factor VIII by the 1-stage assay than by the chromogenic assay. These results are consistent with a factor VIII cleaved at residue 1689, a site important for von Willebrand binding. This activated form leads to some of the assay discrepancies between the 1-stage assay and the chromogenic or the 2-stage assay. There is more rapid sedimentation of the factor VIII measured by the chromogenic assay than the von Willebrand factor in some manufacturers' samples indicating that the method of fractionation may select a low molecular weight von Willebrand factor which does not bind factor VIII. Routine comparison between the 1-stage and chromogenic assays during fractionation may be able to identify such activated preparations. Other assay discrepancies may be due to structural differences between the standards and the tested product.

Factor VIIIa↗
PubMed1976

[Electronmicroscopic characterisation of human plasma lipoproteins after isolation by zonal ultracentrifugation (author's transl)].

Human plasma proteins were isolated by zonal ultracentrifugation and the fractions obtained examined under the electron microscope. HDL2 had a high degree of uniformity in normal persons, characterised by numerous two-dimensional, hexagonal arrangements. Optical diffraction analysis of HDL2 gave a centre distance of 11.3 nm, individual HDL2 particles being spherical with a diameter of 9.8 nm. HDL3 represented a somewhat less uniform lipoprotein population with a particle diameter between 6.5 and 9.5 nm. LDL had a diameter of 20-25 nm. In patients with hyperlipoproteinaemia type III a third peak was isolated, in addition to the two normal lipoprotein peaks VLDL and LDL. It lies between them and has been called Lp III. VLDL from type III plasma consisted of spherical particles, diameter 31-81 nm, morphologically indistinguishable from normal VLDL. Lp III consisted of a relatively homogeneous spherical lipoprotein population with a diameter of 25-30 nm. LDL consisted of spherical particles whose size was highly homogeneous (18-23 nm diameter), indistinguishable from normal LDL.

Adult↗
PubMed2004

Studying multiprotein complexes by multisignal sedimentation velocity analytical ultracentrifugation.

Protein interactions can promote the reversible assembly of multiprotein complexes, which have been identified as critical elements in many regulatory processes in cells. The biophysical characterization of assembly products, their number and stoichiometry, and the dynamics of their interactions in solution can be very difficult. A classical first-principle approach for the study of purified proteins and their interactions is sedimentation velocity analytical ultracentrifugation. This approach allows one to distinguish different protein complexes based on their migration in the centrifugal field without isolating reversibly formed complexes from the individual components. An important existing limitation for systems with multiple components and assembly products is the identification of the species associated with the observed sedimentation rates. We developed a computational approach for integrating multiple optical signals into the sedimentation coefficient distribution analysis of components, which combines the size-dependent hydrodynamic separation with discrimination of the extinction properties of the sedimenting species. This approach allows one to deduce the stoichiometry and to assign the identity of the assembly products without prior assumptions of the number of species and the nature of their interaction. Although chromophoric labels may be used to enhance the spectral resolution, we demonstrate the ability to work label-free for three-component protein mixtures. We observed that the spectral discrimination can synergistically enhance the hydrodynamic resolution. This method can take advantage of differences in the absorbance spectra of interacting solution components, for example, for the study of protein-protein, protein-nucleic acid or protein-small molecule interactions, and can determine the size, hydrodynamic shape, and stoichiometry of multiple complexes in solution.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Characterization of the self association of Avian sarcoma virus integrase by analytical ultracentrifugation.

Retroviral integration protein (IN) has been shown to be both necessary and sufficient for the integration of reverse-transcribed retroviral DNA into the host cell DNA. It has been demonstrated that self-assembly of IN is essential for proper function. Analytical ultracentrifugation was used to determine the stoichiometry and free energy of self-association of a full-length IN in various solvents at 23.3 degrees C. Below 8% glycerol, an association stoichiometry of monomer-dimer-tetramer is observed. At salt concentrations above 500 mM, dimer is the dominant species over a wide range of protein concentrations. However, as physiological salt concentrations are approached, tetramer formation is favored. The addition of glycerol to 500 mM NaCl, 20 mM Tris (pH 8.4), 2 mM beta-mercaptoethanol significantly enhances dimer formation with little effect on tetramer formation. Furthermore, as electrostatic shielding is increased by increasing the ionic strength or decreasing the cation size, dimer formation is strengthened while tetramer formation is weakened. Taken together, the data support a model in which dimer formation includes favorable buried surface interactions which are opposed by charge-charge repulsion, while favorable electrostatic interactions contribute significantly to tetramer formation.

Avian Sarcoma Viruses↗
PubMed1962

The encephalomyelitic activity of myelin isolated by ultracentrifugation.

A relatively simple preparation of guinea pig brain myelin, free of gross contamination by other cellular elements has been described. Electron microscopic evidence of the predominance of membranous (lamellar) forms was used as the criterion of purity of this fraction. The slight mitochondrial contamination of the myelin fraction was confirmed by its low succinic dehydrogenase activity. Quantitative bio-assay of the encephalitogenic activity of myelin showed it to have a higher specific activity than whole guinea pig brain. The low encephalomyelitic activity of the other subcellular constituents (nuclei and mitochondria) which were removed from myelin by ultracentrifugation in 30 per cent sucrose could be explained by a small amount of myelin contamination. A basic protein of high specific encephalitogenic activity has been isolated from myelin by methods previously applied to whole brain. Although the protein is similar to nuclear histones, the following facts point to certain significant differences. Nuclei prepared by a different procedure from the one developed for the isolation of myelin were found to be non-encephalitogenic. Although basic protein could be extracted readily from these nuclei by dilute HCl, the same extraction procedure yielded little extractable protein from whole myelin. Myelin which had been defatted by cold chloroform-methanol yielded a basic protein which was highly encephalitogenic. The evidence presented thus supports the view that there exists in myelin a new basic protein responsible for the induction of experimental allergic encephalomyelitis, which is distinctly different from nuclear histones. The possible relationship of this protein to myelin structure and function has been discussed.

Animals↗
PubMed1995

Comparison of an immunoprecipitation method for direct measurement of LDL-cholesterol with beta-quantification (ultracentrifugation).

A direct LDL cholesterol assay was evaluated using immunoprecipitation (Sigma Diagnostics, St. Louis, MO) with beta-quantification obtained by ultracentrifugation. Excellent intra- and interassay coefficients of variation were obtained (< 4.5%). There was a good correlation (r = 0.88, P < .0001) between the two methods for low-density lipoprotein cholesterol (LDL-C) in 249 samples with triglyceride levels ranging from 13 mg/dL to 2,236 mg/dL and LDL cholesterol levels ranging from 28 mg/dL to 290 mg/dL. Similar correlations were seen for patients with triglyceride levels < 400 mg/dL (r = 0.89, n = 174) and > or = 400 mg/dL (r = 0.89, n = 75). However, using the Friedewald equation, there was a good correlation only in samples with triglyceride levels < 400 mg/dL. No significant differences were found between LDL-C quantitated by the direct LDL assay and beta quantification for patients with dysbetalipoproteinemia (Type III disorder). However, calculated LDL values using the Friedewald equation were found to be significantly higher when compared to beta-quantification in patients with the Type III disorder. There was a slight but significant decrease in LDL-C determined by direct LDL cholesterol assay for non-fasting versus fasting serum (4.7%) despite a strong correlation between these samples (r = 0.98, P < .0001). In addition, freezing samples for 30 days resulted in a significant decrease in levels (15.1%). Thus, this direct LDL cholesterol assay is recommended in place of beta-quantification in hypertriglyceridemic samples (TG > or = 400 mg/dL) and to monitor LDL cholesterol levels in patients with Type III dyslipidemia, because it is less time consuming, more cost-effective and can be adapted to the clinical laboratory.

Cholesterol, LDL↗
PubMed1988

FUGE: an analytical ultracentrifuge simulation for teaching purposes.

An IBM-compatible microcomputer program for teaching purposes is described which stimulates the operation of a sedimentation velocity determination of a protein in an analytical ultracentrifuge using schlieren optics. The program operates in speeded-up time and simulates the major procedures which would need to be carried out to operate such an instrument. The position of the sedimenting boundary can be observed at any time during the run, and up to six 'photographs' can be recorded for subsequent analysis. Calculation of sedimentation coefficient, diffusion coefficient and mol. wt can be made from a dot-matrix printout. Ten representative proteins are stored within the program, but provision exists for user-supplied data.

Computer Simulation↗
PubMed1980

Specific modification of hepatic triglyceride lipase activity by ultracentrifugally separated serum lipoprotein fractions.

Hepatic triglyceride lipase (H-TGL) obtained from postheparin plasma is first stimulated and than progressively inhibited by addition of an increasing amount of serum. To solve the mechanism of this modification, serum fractions isolated by ultracentrifugation were added to the assay mixture and their effects on H-TGL activity were determined. We demonstrated that the addition of the d=1.21 bottom fraction together with HDL almost fully reproduces the effect of the whole serum.

Heparin↗
PubMed1991

Comparison of ultracentrifugation and polyethylene glycol precipitation for concentration of hepatitis B virus (HBV) DNA for molecular hybridisation tests and the relationship of HBV-DNA to HBe antigen and anti-HBe status.

A 32P-labelled DNA probe was used to examine 50 hepatitis B surface antigen (HBsAg)-positive sera for the presence of hepatitis B virus (HBV) DNA. HBV-DNA was detected in all 21 HBeAg-positive samples, in one out of 21 anti-HBe-positive samples and in three out of eight HBeAg- and anti-HBe-negative samples. The results of this DNA hybridisation test correlated well with HBeAg status and could be used to determine infectivity in HBeAg- and anti-HBe-negative samples. Ultracentrifugation was marginally superior to polyethylene glycol precipitation for concentrating HBV-DNA from serum.

Chemical Precipitation↗
PubMed2002

Modern analytical ultracentrifugation in protein science: a tutorial review.

Analytical ultracentrifugation (AU) is reemerging as a versatile tool for the study of proteins. Monitoring the sedimentation of macromolecules in the centrifugal field allows their hydrodynamic and thermodynamic characterization in solution, without interaction with any matrix or surface. The combination of new instrumentation and powerful computational software for data analysis has led to major advances in the characterization of proteins and protein complexes. The pace of new advancements makes it difficult for protein scientists to gain sufficient expertise to apply modern AU to their research problems. To address this problem, this review builds from the basic concepts to advanced approaches for the characterization of protein systems, and key computational and internet resources are provided. We will first explore the characterization of proteins by sedimentation velocity (SV). Determination of sedimentation coefficients allows for the modeling of the hydrodynamic shape of proteins and protein complexes. The computational treatment of SV data to resolve sedimenting components has been achieved. Hence, SV can be very useful in the identification of the oligomeric state and the stoichiometry of heterogeneous interactions. The second major part of the review covers sedimentation equilibrium (SE) of proteins, including membrane proteins and glycoproteins. This is the method of choice for molar mass determinations and the study of self-association and heterogeneous interactions, such as protein-protein, protein-nucleic acid, and protein-small molecule binding.

Animals↗
PubMed2006

Molecular architecture of E. coli purine nucleoside phosphorylase studied by analytical ultracentrifugation and CD spectroscopy.

Purine nucleoside phosphorylase (PNP) is a key enzyme of the nucleoside salvage pathway and is characterized by complex kinetics. It was suggested that this is due to coexistence of various oligomeric forms that differ in specific activity. In this work, the molecular architecture of Escherichia coli PNP in solution was studied by analytical ultracentrifugation and CD spectroscopy. Sedimentation equilibrium analysis revealed a homohexameric molecule with molecular mass 150+/-10 kDa, regardless of the conditions investigated-protein concentration, 0.18-1.7 mg/mL; presence of up to 10 mM phosphate and up to 100 mM KCl; temperature, 4-20 degrees C. The parameters obtained from the self-associating model also describe the hexameric form. Sedimentation velocity experiments conducted for broad protein concentration range (1 microg/mL-1.3 mg/mL) with boundary (classical) and band (active enzyme) approaches gave s(0)20,w=7.7+/-0.3 and 8.3+/-0.4 S, respectively. The molecular mass of the sedimenting particle (146+/-30 kDa), calculated using the Svedberg equation, corresponds to the mass of the hexamer. Relative values of the CD signal at 220 nm and the catalytic activity of PNP as a function of GdnHCl concentration were found to be correlated. The transition from the native state to the random coil is a single-step process. The sedimentation coefficient determined at 1 M GdnHCl (at which the enzyme is still fully active) is 7.7 S, showing that also under these conditions the hexamer is the only catalytically active form. Hence, in solution similar to the crystal, E. coli PNP is a hexameric molecule and previous suggestions for coexistence of two oligomeric forms are incorrect.

Circular Dichroism↗
PubMed1976

The ultrastructure of the human epidermis following ultracentrifugation.

Ultracentrifugation of the human epidermis was undertaken to uncover the forces responsible for the establishment and maintenance of the supranuclear melanin cap in basal cells. The wide spacing and elasticity of the tonofibrils caused a selective trapping of melanosomes in this region. At the same time the experiments produced pictures resembling dedifferentiation of the plasma membrane as in the fetal state, and differentiation of the tonofibrillar mass resembling the granular and horny layers. Once the tonofibrils had retracted and massed together, the order of intracellular sedimentation was: melanosome, chromatin, mitochondrion, tonofibril and ribosome.

Cell Membrane↗
PubMed1984

Preparative isolation of immune complexes from serum by sucrose gradient ultracentrifugation.

Immune complexes were isolated by ultracentrifugation in sucrose gradients (20-65% (w/w]. the centrifugation procedure was demonstrated to be isopycnic. The banding density of the complexes was influenced by the chemical nature and molecular size of the antigen and by the antigen to antibody ratio. The method was applied for preparative isolation of immune complexes from patients with systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, and uncomplicated psoriasis.

Antigen-Antibody Complex↗
PubMed1985

Isopycnic ultracentrifugation of immune complexes.

Isopycnic ultracentrifugation is frequently applied for preparative isolation of macromolecules. Using bovine serum albumin (BSA)-anti-BSA antibody complexes as a model system, isopycnic banding of complexes was observed in CsCl, Nycodenz, and sucrose gradients. In CsCl gradients, free antigen or antibody could not be separated from the immune complexes. Variations in antigen to antibody ratio from equivalence and in the amount of complement present during complex formation resulted in zone broadening and banding at slightly lower densities in Nycodenz. This was not observed in sucrose. Serum immune complexes were isolated from patients with rheumatoid arthritis or ankylosing spondylitis. Banding of in vivo-formed immune complexes was observed more frequently in sucrose than in Nycodenz.

Animals↗
PubMed1983

A comparison of the analytical ultracentrifuge and high-performance liquid chromatography for the determination of molecular weight distributions of plasma protein fraction (human).

The use of high-performance liquid chromatography (HPLC) with size exclusion column packings is investigated as an alternate assay method to the analytical ultracentrifuge, when assaying for the high molecular weight components of human plasma protein fraction. Results indicate that HPLC is a more accurate and precise method for assaying high molecular weight components in plasma protein fraction, even when present in small amounts.

Blood Proteins↗