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At least 109 records · Page 6Linked to original sources
PubMed1996

Beta-lactoglobulin B: a proposed standard for the study of reversible self-association reactions in the analytical ultracentrifuge?

Bovine beta-lactoglobulin B is proposed for use as a standard in the measurement of reversible self-association reactions in the analytical ultracentrifuge. The protein is well understood on a molecular level, is readily obtainable, and is stable under harsh conditions. Bovine beta-lactoglobulin B undergoes a simple monomer-dimer equilibrium which can be predictably controlled and consistently reproduced. In this investigation bovine beta-lactoglobulin B has been studied via sedimentation equilibrium experiments in the XL-A analytical ultracentrifuge at 5-30 degrees C in buffers of ionic strength 0.1-0.2 M and pH 2.0-3.0. Samples subjected to a number of different treatments and storage methods all yielded similar results. Molar equilibrium constants for the association reaction were determined by nonlinear regression fitting of a monomer-dimer model of association either to concentration versus radius data, using the programs NONLIN and ORIGIN, or to Omega versus concentration data using the program DUGOM. At 20 degrees C and pH 2.6, over the ionic strength range 0. 1-0.2 M, the equilibrium constant for the association reaction ranges between 1 x 10(4) and 5 x 10(4) M-1. The parameters of nonideal self-association behavior were found to be independent of the particular analysis strategy. Fitting to the concentration distribution, the apparent weight-average molecular weight, or the Omega function all returned identical parameters.

Animals↗
PubMed2002

Oligomeric state of membrane transport proteins analyzed with blue native electrophoresis and analytical ultracentrifugation.

Blue native electrophoresis is used widely for the analysis of non-dissociated protein complexes with respect to composition, oligomeric state and molecular mass. However, the effects of detergent or dye binding on the mass and stability of the integral membrane proteins have not been studied. By comparison with analytical ultracentrifugation, we have evaluated whether the oligomeric state of membrane transport proteins is reflected reliably with blue native electrophoresis. For the analysis we have used two well-characterized transporters, that is, the major facilitator superfamily protein LacS and the phosphotransferase system EII(Mtl). For another member of the major facilitator superfamily, the xyloside transporter XylP from Lactobacillus pentosus, the complete analysis of the quaternary structure determined by analytical ultracentrifugation and freeze-fracture electron microscopy is presented. Our experiments show that during blue native electrophoresis the detergent bound to the proteins is replaced by the amphipathic Coomassie brilliant blue (CBB) dye. The mass of the bound CBB dye was quantified. Provided this additional mass of bound CBB dye is accounted for and care is taken in the choice and concentration of the detergent used, the mass of LacS, XylP and EII(Mtl) and four other membrane (transport) proteins could be deduced within 10 % error. Our data underscore the fact that the oligomeric state of many membrane transport proteins is dimeric.

Bacterial Proteins↗
PubMed1977

Accessibility of proteins in 50S ribosomal subunits of Escherichia coli to antibodies: an ultracentrifugation study.

The accessibility of each of the proteins on the 50S ribosomal subunit of Escherichia coli was investigated by establishing whether immunoglobulins (IgG), specific for each of the 34 proteins from the 50S subunit, were able to bind to the 50S subunit. The main criterion for accessibility was the formation of specific antibody-50S subunit complexes that could be detected by means of analytical ultracentrifugation. The proteins fell into two main groups. Immunoglobulins against proteins L1, L2, L3, L4, L5, L6, L7/L12, L8, L9, L10, L11, L14, L15, L16, L17, L18, L19, L20, L21, L22, L23, L25, L26, L27 and L30 gave large amounts of complex (20-100%) and, therefore, these proteins were considered to be accessible on the surface of the 50S ribosomal subunit. The antibodies against the remaining proteins L13, L24, L28, L29 and L31 to L34 produced small amounts of complexes (10-20%). Since their effects were unequivocably stronger than those obtained with IgG's from sera of non-immunized animals, the results indicate that these proteins are probably also accessible. Nonetheless, from the ultracentrifugation studies alone definite conclusions about the exposure of the latter group of proteins could not be drawn.

Antibody Specificity↗
PubMed1999

Hydrodynamic properties of mucins secreted by primary cultures of guinea-pig tracheal epithelial cells: determination of diffusion coefficients by analytical ultracentrifugation and kinetic analysis of mucus gel hydration and dissolution.

We have used two different approaches to determine hydrodynamic parameters for mucins secreted by guinea-pig tracheal epithelial cells in primary culture. Cells were cultured under conditions that promote mucous cell differentiation. Secreted mucins were isolated as the excluded fraction from a Sepharose CL-4B gel filtration column run under strongly dissociating conditions. Biochemical analysis confirmed the identity of the high molecular weight material as mucins. Analytical ultracentrifugation was used to study the physical properties of the purified mucins. The weight average molecular mass (Mw) for three different preparations ranged from 3.3 x 10(6) to 4.7 x 10(6) g/mol (corresponding to an average structure of 1-2 subunits), and the sedimentation coefficient from 25.5 to 35 S. Diffusion coefficients ranging from 4.5 x 10(-8) to 6.4 x 10(-8) cm2/s were calculated using the Svedberg equation. A polydispersity index (Mz/Mw) of approximately 1.4 was obtained. Diffusivity values were also determined by image analysis of mucin granule exocytosis captured by videomicroscopy. The time course of hydration and dissolution of mucin was measured and a relationship is presented which models both phases, each with first order kinetics, in terms of a maximum radius and rate constants for hydration and dissolution. A median diffusivity value of 8.05 x 10(-8) cm2/s (inter-quartile range = 1.11 x 10(-7) to 6.08 x 10(-8) cm2/sec) was determined for the hydration phase. For the dissolution phase, a median diffusivity value of 6.98 x 10(-9) cm2/s (inter-quartile range = 1.47 x 10(-8) to 3.25 x 10(-9) cm2/sec) was determined. These values were compared with the macromolecular diffusion coefficients (D20,w) obtained by analytical ultracentrifugation. When differences in temperature and viscosity were taken into account, the resulting D37,g was within the range of diffusivity values for dissolution. Our findings show that the physicochemical properties of mucins secreted by cultured guinea-pig tracheal epithelial cells are similar to those of mucins of the single or double subunit type purified from respiratory mucus or sputum. These data also suggest that measurement of the diffusivity of dissolution may be a useful means to estimate the diffusion coefficient of mucins in mucus gel at the time of exocytosis from a secretory cell.

Animals↗
PubMed1978

A rapid method for the isolation of intracytoplasmic membranes from Rhodopseudomonas sphaeroides using an air-driven ultracentrifuge.

A method has been developed for the isolation intracytoplasmic (ICM) vesicles (chromatophores) from Rhodopseudomonas sphaeroides using an air-driven ultracentrifuge. Application of conventional techniques used for preparative scale equipment to the air-driven ultracentrifuge allows the rapid isolation of ICM vesicles from reduced quantities of starting material. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis profiles of ICM vesicles isolated in this fashion are essentially indistinguishable from those isolated by conventional means.

Cell Fractionation↗
PubMed1984

Rapid precision interferometry for the analytical ultracentrifuge. II. A laser controller based on a rate-multiplying circuit.

A laser controller that uses a fixed frequency clock and a digital rate-multiplying circuit to synchronize the triggering of a pulsed laser to the spinning of an analytical ultracentrifuge rotor has been designed. The circuit is simple, inexpensive, and virtually free of any adjustments. It tracks rotors undergoing full acceleration or deceleration. At constant rotor speed it provides triggering that is accurate and reproducible to better than 0.5 microseconds. The settings of this controller are independent of rotor speed over the full range of the ultracentrifuge.

Interferometry↗
PubMed1984

Rapid precision interferometry for the analytical ultracentrifuge. III. Determination of period of rotation, frequency of rotation, and elapsed time.

The approaches presented in this series of papers make possible rapid gathering, reduction, and analysis of data from the Rayleigh interference optical system of an analytical ultracentrifuge. Instrumentation described in this paper provides some of the timing and measurement circuits necessary for a microprocessor or minicomputer to determine the rotor frequency, rotor period, and elapsed time of an experiment. It includes simple but effective circuits to generate precise rotor timing pulses that are useful for synchronization of pulsed light sources. Circuits to control photographic operations in the ultracentrifuge are described briefly. All of these circuits are interfaced to a simple microcomputer address/data bus. An adapter between this bus and a Q-bus (for a DEC LSI-11/2 or LSI 11/23 microcomputer) is also described. The circuits presented have been used in this laboratory over a 3-year period. They have proven reliable and form an integral part of the real-time data acquisition systems that have been constructed.

Interferometry↗
PubMed1981

HDL3 and HDL2 determination by a combined ultracentrifugation and precipitation procedure.

The aim of the present study was to evaluate a method to separate lipoproteins by ultracentrifugation simultaneously at density 1.006 and 1.125. This procedure combined with heparin-MnCl2 precipitation would facilitate the simultaneous determination of lipid levels of high density lipoproteins (HDL) and its main subfractions (HDL2 and HDL3, including very high density lipoproteins (VHDL)) and of very low (VLDL) and low density lipoprotein (LDL) fractions. Centrifugation at 105 500 X g (mean) for 24 h in a Beckman L5-50 ultracentrifuge with a Ti50 rotor seemed to give an adequate separation. The correlation coefficients for duplicate samples were 0.95 and 0.96 for HDL3-cholesterol and HDL3-phospholipids, respectively. The error of the method for HDL, HDL2 and HDL3 lipids was around half that of the intra-individual variation and comparable to the results for determination of conventional lipoprotein fractions. Therefore the suggested method seems applicable for evaluation of HDL2 and HDL3 levels in selected clinical material.

Chlorides↗
PubMed1981

A micro-method for the rapid ultracentrifugal separation of human plasma high density lipoprotein subfractions, HDL2 and HDL3.

Currently available ultracentrifugal procedures for separating the two major density subfractions of HDL (HDL2 and HDL3) are time-consuming and expensive, and require relatively large volumes of plasma. In order to overcome these problems, a rapid and simple procedure, requiring only 0.5 ml plasma, has been developed. After precipitation of apoprotein B-containing lipoproteins with heparin and MnCl2, 120 microliters of the supernatant is adjusted to a density of 1.125 g/ml with aqueous NaCl-NaBr, and centrifuged for 3.5 h at 160000 x g in an air-driven bench-top ultracentrifuge (Beckman Airfuge). The HDL2 is then removed in a tube-slicer, constructed to permit six Airfuge tubes to be sliced simultaneously, and the cholesterol content assayed by a cholesterol oxidase procedure. The method provides an alternative to the recently described heparin/MnCl2/dextran sulphate double-precipitation procedure, with the advantage of permitting quantitative recovery of intact HDL2.

Humans↗
PubMed1986

Solution structure of halophilic malate dehydrogenase from small-angle neutron and X-ray scattering and ultracentrifugation.

Data from small-angle X-ray and neutron scattering and ultracentrifugation experiments on solutions of malate dehydrogenase from Halobacterium maris mortui are analysed together to yield a model for the enzyme particle formed by the protein and its interactions with water and salt in the solvent. The halophilic enzyme is stable only in high concentrations of salt and the model has structural features that are absent from non-halophilic malate dehydrogenase. The complementarity of the information derived from the three experimental methods is discussed extensively and quantitatively. It derives from the fact that mass density (ultracentrifugation), electron density (X-rays) and neutron scattering density are independent of each other. Each method gives a different "view" of the same particle, and an analysis of the combined data provided thermodynamic and structural parameters with, apart from the chemical composition of the solutions, only one other assumption: a constant partial specific volume for water equal to 1.00 cm3 g-1. Both the insights gained by this novel approach and its limitations are carefully pointed out. In solvents between 1 M and 5 M-NaCl, the enzyme forms a particle of invariant volume, consisting of a protein dimer (87,000 g mol-1) with which are associated 0.87 g of water and 0.35 g of salt per gram of protein. The partial specific volume of the protein calculated from the combined experimental data is 0.753(+/- 0.030) cm3 g-1, in good agreement with the value calculated from the amino acid composition. The particle has a radius of gyration of 32 A and an equivalent Stokes radius of 43 A. By combining the data from the X-ray and neutron scattering studies, the radii of gyration of the protein moiety alone and of the associated water and salt distribution were calculated. They are 28 A and about 40 A, respectively. The large-angle scattering curves show that the shapes of the particle and of the protein moiety alone are similar. At very low resolution they can be approximated by an ellipsoid of axial ratio 1:1:0.6 (or 1:1:1.5). At higher resolution, it becomes apparent that the particle has a significantly larger interface with solvent than an homogeneous ellipsoid or globular protein. The model has a globular protein core similar to non-halophilic malate dehydrogenase, with about 20% of the protein extending loosely out of the core, forming the large interface with solvent. The main interactions with water and salt take place on this outer part.

Halobacterium↗
PubMed1983

A high molecular weight chemoattractant generated from C5 by ultracentrifugation of mouse serum without activation of complement.

After ultracentrifugation of normal mouse serum, we found chemoattractant activity in the high molecular weight protein region at the bottom of the tube, which was comparable in amount and potency to the attractant in endotoxin-activated serum. This was not a pre-formed attractant, but was generated from serum reactants at least one of which was inactivated by heating at 56 degrees C. Analysis of sera from 10 different mouse strains for hemolytic C5 activity and for capacity to generate chemoattractant on ultracentrifugation showed that the 4 strains without C5 were the only strains that failed to generate the attractant. Thus, the attractant precursor is C5. Since the activity was generated in the presence of 0.01 M EDTA, classical or alternative complement activation was not required. The chemoattractant product had a mol. wt of approximately 170,000; it was therefore not free C5a. These results, and data recently published on digestion of purified human C5 by trypsin, suggest that limited proteolysis of C5 can produce a chemoattractant molecule without release of free C5a.

Animals↗
PubMed1990

Comparison of polyethylene glycol precipitation and ultracentrifugation for recovery of cytomegalovirus from urine prior to detection of DNA by dot-blot hybridisation.

Polyethylene glycol (PEG) precipitation provided a useful alternative to ultracentrifugation for recovering cytomegalovirus (CMV) from clinical specimens prior to DNA hybridisation studies. The conditions for use of PEG are described. In a study of 61 urine samples from patients suspected of CMV infection 18 yielded a positive DNA hybridisation result. Fifteen were positive after both concentration procedures, two after PEG precipitation only and one after ultracentrifugation only. The DNA hybridisation results are discussed in the light of the results of virus isolation by cell culture.

Chemical Precipitation↗
PubMed1985

Influence of fibrinogen on fibrin polymerization. Ultracentrifugation studies.

During the transformation of fibrinogen to fibrin, excess fibrinogen suppresses further polymerization of fibrin, thereby enabling the nascent fibrin to be transported in a soluble form in blood. The question of possible complex formation between fibrin and fibrinogen was addressed by analyzing fibrin/fibrinogen (1:20, mol/mol) mixtures in the presence of calcium ions in stable linear sucrose density gradients by ultracentrifugation at 37 degrees C. During the period of ultracentrifugation in independent experiments, 40% of desAA-fibrin and 30% of desAABB-fibrin, respectively, precipitated without the participation of fibrinogen. The desAABB-fibrin, recovered in the gradient fractions, appeared as high-molecular-weight polymers (22 S), whereas the recovered desAA-fibrin exhibited only a slight increase in molecular weight (9 S) compared to fibrinogen (8 S). In contrast to this finding, both types of fibrin were totally recovered in gradient fractions provided that fibrinogen was present in the gradient at a uniform concentration of 2 mg/ml. In addition, the presence of fibrinogen but not human serum albumin reduced the size of desAABB-fibrin polymers (17 S). However, stable fibrin-fibrinogen complexes could not be demonstrated, since cosedimentation of differently labelled desAABB-fibrin and fibrinogen was not detectable. These studies suggest a specific but weak interaction of the solubilizing fibrinogen with the soluble fibrin polymers as demonstrated by a rapid exchange of both macromolecules.

Centrifugation, Density Gradient↗
PubMed1976

UV laser scanning and fluorescence monitoring of analytical ultracentrifugation with an on-line computer system.

A new optical system for the analytical ultracentrifuge is described, which permits sedimentation to be monitored by fluorescence. The optical system is based on a laser light source, which is focused to a narrow (50 micron) beam. The radical scanning of the beam provides information on the distribution of fluorescing material with distance in the centrifuge cell. Data collection and processing are performed in conjunction with an on-line computer system which sorts incoming fluorescence pulses according to rotor hole and cell sector, averages families of pulses to improve signal to noise ratios and fits the data (in the experiments reported here) to equations to determine sedimentation coefficients. Initial experiments with the system have been performed with bovine serum albumin and indicate that sedimentation can be readily monitored by fluorescence with solutions at concentrations as low as 20 micrograms per ml, with excitation at 257 nm. At these concentrations, the optical density is only in the 0.01 range, too low for experiments with absorption-scanner optical system. Even lower concentrations can be used when fluorescent labels are used with excitation in the visible region of the spectrum. The preliminary studies indicate that fluorescence monitoring of sedimentation will substantially enhance the range of experimental possibilities in ultracentrifugation by improving both the sensitivity of measurements and the discrimination between sedimenting species on the basis of their fluorescence characteristics.

Computers↗
PubMed1976

Molecular weights and molecular-weight distributions from ultracentrifugation of nonideal solutions.

Ultracentrifugation, membrane osmometry and capillary viscometry experiments have been performed on two dextran samples, which have molecular-weight distributions (MWDs) similar to those of dextrans used as blood plasma extenders. The manufacturer reported values of Mn and MW, determined by end group analysis and by light scattering, respectively. Our values of Mn, determined by osmometry, and MW, calculated from ultracentrifugal and viscometry experiments, agreed quite well with the manufacturer's results. Good agreement was obtained with values of MW and BLS (the light scattering second virial coefficient) obtained from sedimentation equilibrium experiments at different speeds using sector or nonsector-shaped centerpieces. Several ways of obtaining MW, MZ and BLS from sedimentation equilibrium experiments are presented. We have also shown how to obtain the speed-dependent term of the sedimentation equilibrium second virial coefficient. Both BLS and the speed-dependent nonideal terms could be used to correct the sedimentation equilibrium data, so that ideal values of d in c/d(r2) or dc/d(r2) could be estimated and used to obtain the MWDs of the dextran samples. Both Donnelly's and Scholte's methods were used with the sedimentation equilibrium data. With both methods, unimodal MWDs were encountered, which gave good agreement with the manufacturer's MWDs, obtained by a combination of analytical gel chromatography and light scattering. Uncorrected sedimentation equilibrium data gave MWDs quite different from the manufacturer's results. The MWD calculated from the differential distribution of sedimentation coefficients also gave a unimodal MWD, but this MWD did not give a good agreement with the sedimentation equilibrium results or with the manufacturer's results.

Dextrans↗
PubMed1995

Separation of lipoproteins, albumin and gamma-globulin by single-step ultracentrifugation of human serum. Application. I: Binding of hematoporphyrin to human serum and to albumin.

Previous studies of the serum binding of the photosensitizer hematoporphyrin (Hp) have given widely different results. The serum binding of Hp is therefore further illuminated by experiment and discussion. Ultracentrifugal separation of serum is improved and applied to study the binding of Hp to human serum and HSA. The observed distribution of Hp among the serum proteins is compared with the distribution expected from available association constants for Hp binding with individual proteins. The lipoprotein classes and the two major high density proteins (HDP), albumin and gamma-globulin, were separated in a NaCl-KBr gradient by single spin ultracentrifugation (SW 40; 30,000 rpm). HSA- and HDP-bound Hp were similarly distributed in the centrifuged gradient. Centrifugation of Hp-doped HSA separated the unbound Hp (75%) and the HSA-bound Hp (25%). The present association constant for the Hp-HSA complex (10(3)/M) was much lower than earlier published ones (10(6)/M) found by other techniques. The association of Hp with HDP in serum was much stronger than the association of Hp with the isolated HSA (electrophoretic grade). The estimated ratio of HSA-bound to LDL-bound HP in serum was at least 25 times larger than the experimental value. The percentage of LDL-bound Hp decreased with increasing Hp concentration. The serum binding of Hp is the same as that found previously using another rotor and another salt gradient (70.1 Ti, 70,000 rpm, NaCl-CsCl). LDL has high-affinity-low-capacity binding sites for Hp. HSA is the major HDP protein that binds Hp in human serum. The strength of the HSA-Hp complex may depend on the batch of HSA used and upon the absence/presence of other proteins. Proteins may interact in serum in manners that affect the binding of certain drugs. Neither the type of gradient salt nor the field of gravity affected the serum binding of Hp.

Blood Proteins↗
PubMed2004

Limitations of the ultracentrifugal approach for measuring the effective net charge of a macroion.

Limitations have been detected in a recently published method for macroion valence determination by an ultracentrifugal procedure for quantifying the Donnan distribution of small ions in macroion solutions dialyzed against buffer supplemented with chromate as an indicator ion. The limitation reflects an implicit assumption that sedimentation velocity affords an unequivocal means of separating effects of chromate binding from those reflecting the Donnan redistribution of small ions. Although the assumed absence of significant Donnan redistribution of small ions across the sedimenting macroion boundary seemingly holds for some systems, this approximation is demonstrably invalid for others. Despite preliminary signs of promise, the ultracentrifugal procedure does not afford a simple, readily applied solution to the problem of unequivocal macroion valence determination.

Anions↗