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At least 199 records · Page 11Linked to original sources
PubMed2005

Metal ion complexes of EDTA: a solute system for density gradient ultracentrifugation analysis of lipoproteins.

In the study reported here, we apply some of the features of coordination chemistry to solve a long-standing problem in the separation and characterization of lipoprotein particles. Lipoproteins are circulating micelle-like particles responsible for lipid transport. They exist in three major classes: very-low-density lipoprotein, low-density lipoprotein, and high-density lipoprotein in well-defined density ranges using the density gradient ultracentrifugation (DGU) method. The analytical instrumentation of DGU has improved over the years in response to clinical evidence that certain lipoprotein species are linked to a high risk for developing cardiovascular disease. A long-standing problem has been a lack of appropriate gradient-forming solutes that can generate a useful gradient from a homogeneous solution. We have found that a new class of solutes based on metal ion complexes has the potential of providing a wide selection of compounds where the features can be modulated by choice of ligand, complexing metal ion, and counterion. In this study, we have chosen the cesium salt of BiEDTA (CsBiEDTA) and have investigated the dynamics of density gradient formation in the ultracentrifuge. We show that a useful density gradient can be formed within a few hours beginning with a homogeneous solution. We also present data on the migration behavior of lipoproteins under gradient-forming conditions and show that high-resolution density profiles can be obtained with good precision. The resolution of the CsBiEDTA profile is compared with those obtained using high molecular weight organic solutes.

Bismuth↗
PubMed2005

Kinetics of shrinking of polymer gels induced by ultracentrifugal fields.

The kinetics of the shrinking of polymer gels induced by ultracentrifugal fields is investigated. A theory is proposed to describe the diffusion process of polymer networks under centrifugal fields. The initial shrinking rate is proportional to the ratio of the centrifugal force to the frictional force of networks. The shrinking attains the stationary state as a result of the balance between the centrifugal force and the swelling force of networks. The characteristic time for shrinking is of the order of a2/D where a and D are the stationary displacement and diffusion constant, respectively. We also present the experimental data for the shrinking of the poly(acrylamide) (PAAm) gels under ultracentrifugal fields. The shrinkage increases linearly with time in the initial stage whereas it reaches the steady state in the long time limit as expected by the theory. Each of longitudinal elastic modulus and friction coefficient of the PAAm gels is evaluated from the data on the basis of the theory.

Journal Article↗
PubMed1960

Studies on the fine structure of ultracentrifuged spinal ganglion cells.

THE FOLLOWING STRUCTURES WERE OBSERVED IN ELECTRON MICROGRAPHS OF THE MOUSE SPINAL GANGLION CELLS: Nissl bodies composed of both aggregated rough-type, largely oriented, membranes of the endoplasmic reticulum and discrete particles; short rodlike mitochondria with well-developed transverse, obliquely or longitudinally arranged cristae, and a relatively typical Golgi complex. The components of ultracentrifuged ganglion cells (400,000 times gravity for 20 minutes) are stratified, the layers appearing in the order of their decreasing density as follows: (1) A microsomal or ergastoplasmic layer which may be further divided into three sublayers without sharp boundaries, namely, a discrete particle layer, a layer of discrete particles and highly distorted membranes of the endoplasmic reticulum, and a layer composed of relatively intact, but stretched membranes of the endoplasmic reticulum and discrete particles. (2) Mitochondria constitute a relatively broad layer. They are sometimes stretched; however, they retain most of their fine structure. The stratified nucleus is found within the mitochondrial layer. (3) A relatively wide layer of tightly packed vesicles. (4) At the centripetal end, resting against the cell membrane, are a few lipid vacuoles. A comparison is made between the ultrastructure of the stratified layers in situ and those described by others in differentially ultracentrifuged homogenates.

Animals↗
PubMed1952

Ultracentrifuge, electrophoresis, and viscosity studies on crystalline pyrophosphatase.

Ultracentrifuge and electrophoresis studies on one preparation of crystalline pyrophosphatase showed the presence of one major component which accounted for more than 90 per cent of the protein. Combination of viscosity, diffusion, and ultracentrifuge data leads to a model of low asymmetry with an anhydrous molecular weight of about 60,000.

Electrophoresis↗
PubMed1967

Purification of the Moloney and Rauscher murine leukemia viruses by use of zonal ultracentrifuge systems.

The B-IV and B-IX zonal ultracentrifuge rotors were applied to the concentration and purification of the Moloney and Rauscher murine leukemia viruses from large volumes of infected tissue culture fluids and animal materials. Potassium tartrate, potassium citrate and sucrose gradients were used to obtain viral concentrates from the density 1.16 to 1.18 zone. Proteolytic enzyme digestion of tissue culture preparations prior to zonal ultracentrifuge processing was effective in releasing virus from cell debris and producing highly purified, though nonleukemogenic, viral concentrates. Infected Rauscher mouse plasma was processed to give highly purified infectious virus fractions. A single centrifugation of crude Rauscher mouse spleen homogenates resulted in partially purified infectious concentrates with high virus particle counts.

Animals↗
PubMed1982

Comparison of direct and indirect enzyme immunoassays with direct ultracentrifugation before electron microscopy for detection of rotaviruses.

A direct and an indirect enzyme immunoassay (EIA) were evaluated against a standard of electron microscopy after direct ultracentrifugation of the specimen for their performances in detecting rotaviruses. The indirect EIA had variable background activity which influenced test specificity. The indirect EIA control (test system without the detector antibody) plus a regression line (which reflected background noise) improved test specificity. However, the results of direct EIA (Rotazyme; Abbott Laboratories, North Chicago, Ill.) sensitivity (86%) and specificity (96%) were better than those of the indirect EIA in tests on 73 rotavirus-positive and 78 rotavirus-negative specimens. Endpoint titrations of purified SA-11 rotavirus showed greater sensitivity of the direct EIA test. Electron microscopy, performed after direct ultracentrifugation, and direct EIA were approximately 2 log10 more sensitive in the detection of purified SA-11 rotavirus than was electron microscopy with standard methods of unconcentrated specimen preparation. Direct EIA test are potentially sensitive, specific, and practical for the rapid detection of rotaviruses from human clinical specimens. Further studies are needed before EIA methods for detection of human rotaviruses can be equated with the level of reliability of results obtainable with sensitive electron microscopy techniques.

Animals↗
PubMed1979

Use of the ultracentrifuge vertical rotor in the detection of rubella-specific IgM on a sucrose density gradient.

A comparison was made of the performance of swing-out and vertical ultracentrifuge rotors in the detection of rubella-specific IgM on a sucrose density gradient. Tests were performed on 30 sera, of which 11 were found to contain rubella-specific IgM by both methods. The centrifugation time for the swing-out rotor was 16 hours at 35,000 rpm. This was reduced to 2 hours using the vertical rotor at 50,000 rpm. Routine use of the vertical rotor would allow a reduction in the time taken to perform the test, increase the number of sera tested each time, and reduce wear on the ultracentrifuge.

Antibodies, Viral↗
PubMed1978

Isolation of subfractions of human very low density lipoproteins by zonal ultracentrifugation.

Very low density lipoproteins (VLDL) have been isolated and subfractionated on the basis of their differing flotation rates. The procedure consists of a single 45-min zonal ultracentrifugation step using a linear density gradient of d = 1.00 to 1.15 g/ml. Appropriate fractions of the zonal rotor effluent containing the entire VLDL spectrum were characterized by analytical ultracentrifugation, gel filtration chromatography, and complete chemical analysis. Flotation rates of VLDL subspecies from hypertriglyceridemic and normolipemic plasmas correlated directly with their Stokes radii and triglyceride content and inversely with their proportion of cholesterol, cholesteryl esters, phospholipids, and total protein. There was also an inverse correlation of flotation rate with the fraction of tetramethylurea-insoluble protein. This procedure provides a reliable methodology for a rapid isolation of VLDL subfractions and the accurate determination of their flotation rates.

Centrifugation, Zonal↗
PubMed1989

Determination of the sedimentation coefficient and molecular weight of proteins by density gradient ultracentrifugation in fixed angle rotor.

The applicability of density gradient ultracentrifugation using fixed angle rotors for the determination of sedimentation coefficient and molecular weight of proteins was studied. Ovalbumin, bovine serum albumin, IgG-, IgA- and IgM-globulin proteins, as standards were, among others, centrifuged in a fixed angle rotor (Beckman type 50) in 5-20% (w/v) sucrose density gradient for 300 min, at a speed of 40,000 rpm, at 5 degrees C. After centrifugation, the sedimentation diagram of the above-mentioned proteins was taken by a spectrophotometer equipped by a flow-through cuvette and a compensator. The sedimentation path was measured on the diagrams. A calibration curve was drawn by plotting the sedimentation coefficients of the standard proteins against the sedimentation paths. This curve, for which the equation y = 1.74x-1.6 is valid, was used for the determination of the sedimentation coefficients of several globular proteins under the conditions described above. The values obtained this way agree within +3-10% with those determined with analytical ultracentrifuge. If the logarithms of proteins molecular weight used as standards were drawn against the logarithms of their sedimentation paths a calibration curve was obtained. This curve for which the equation ln y = 2.022 ln x + 8.71 is valid can be used for the determination of molecular weights of globular or nearly globular proteins.

Animals↗
PubMed1987

Single spin density gradient ultracentrifugation method for the detection and isolation of light and heavy low density lipoprotein subfractions.

A single spin density gradient ultracentrifugation method in a swinging bucket rotor has been applied for the detection and isolation of low density lipoprotein (LDL) subfractions. The visualization of the LDL heterogeneity was facilitated by prestaining the serum with Coomassie Brilliant Blue R prior to density gradient ultracentrifugation for 19.5 hr. A total of 13 human serum pools was analyzed. In each pool, two LDL subfractions, a lighter LDL1 subfraction, occasionally showing a subdivision into two bands, LDL1A and LDL1B, and a heavier LDL2 could be clearly distinguished by the banding pattern in the density gradient. Physicochemical characteristics of the isolated LDL subfractions were determined. The simple method for detection and isolation of these subfractions presented here may facilitate future studies on LDL heterogeneity.

Adolescent↗
PubMed1982

A one-step separation of human serum high density lipoproteins 2 and 3 by rate-zonal density gradient ultracentrifugation in a swinging bucket rotor.

A method was developed for the separation of the high density lipoprotein subclasses HDL2 and HDL3 from human serum. Six serum samples are fractionated in a single-step ultracentrifugal procedure using the Beckman (SW-40) swinging bucket rotor. The method is based on a difference in flotation rate of the high density lipoprotein subclasses. Separation of HDL2 and HDL3 is accomplished by a discontinuous NaBr density gradient applied on top of 2 ml of serum brought to a density of 1.40 g/ml. After centrifugation, high density lipoprotein subclass profiles were obtained using a specially designed gradient fractionator. Contamination of the isolated high density lipoprotein subclasses by serum albumin or by apolipoprotein B-containing lipoproteins was minimal while only a slight overlap between the HDL2 and HDL3 profiles was observed. Chemical and immunochemical analyses of the high density lipoprotein subclasses isolated by the present method were in close agreement with the results obtained by rate-zonal density gradient ultracentrifugation in zonal rotors (Patsch, et al. 1980. J. Biol. Chem. 255: 3178-3185). The major advantage of the method presented in this paper as compared with the zonal rotor method is the possibility to analyze as many as six serum samples simultaneously.

Adult↗
PubMed1991

[The determination of protein hydrophobicity. 2. Determination of the of protein binding of sodium dodecyl sulfate with the use of the ultracentrifuge].

The interaction between sodium dodecylsulphate (SDS) and the proteins bovine serum albumin (BSA) and ovalbumin, respectively, was studied using a synthetic boundary cell of an ultracentrifuge. A method for the determination of protein-bound SDS is described. The SDS binding capacity of BSA is found to be 250 +/- 50 micrograms SDS per 500 micrograms of BSA. The amount of SDS bound by ovalbumin is so low that the procedure cannot be recommended for this protein.

Ovalbumin↗
PubMedDate not supplied

Ultracentrifugal isolation of vesicular carriers of biliary cholesterol in native human and rat bile.

We have utilized ultracentrifugation of native bile-Metrizamide density gradients to isolate a vesicular transport system of biliary lipids in both man and rat. We identified vesicular structures by electron microscopy. Fresh bile specimens were obtained from bile fistula rats (unsaturated bile) and from patients 1 week after bile duct surgery (supersaturated bile). Metrizamide was dissolved in bile (33% w/v), and continuous density gradients were performed with undiluted bile (density limits = 1.020 to 1.300 gm per ml). The relative distribution of biliary cholesterol, phospholipid and bile salt was studied as a function of the density of the fractions. Approximately 50% of total rat biliary cholesterol and between 61 and 90% of human biliary cholesterol was concentrated in the lightest fractions of the gradients (density less than 1.060 gm per ml). In contrast, less than 20% of bile salts was present in fractions with densities lower than 1.060 gm per ml. The highest amounts of bile salts and phospholipids of the bile-Metrizamide density gradients were found in the density range of 1.075 to 1.100 gm per ml in both human and rat bile. More than 80% of biliary proteins was found in fractions with densities greater than 1.075 gm per ml, and only 2% was found in the cholesterol-rich fraction with density less than 1.060 gm per ml in both species. When bile salt concentration was raised in rat bile from 38 to 97 mM by adding taurocholate, the low density cholesterol-rich fraction almost disappeared. Electron microscopy of negatively stained preparations of the fractions with density less than 1.060 gm per ml showed 40 to 120 nm vesicles, which were not apparent in the other fractions. Similar vesicles were demonstrated also in fresh rat bile and within the canaliculi after acute depletion of the bile salt pool (biliary bile salt concentration of 3.45 mM; total biliary lipid concentration of 0.25 gm%). The structure of these vesicles was shown in thin sections of liver specimens. They appeared as internal cavities surrounded by a single, continuous 6-nm-thick bilayer. These studies demonstrate that a high proportion of biliary cholesterol is transported in vesicles in human supersaturated native bile and that vesicular carriers are also responsible for the transport of a significant amount of biliary cholesterol in unsaturated rat bile. The presence of vesicles in unsaturated hepatic bile strongly supports the thesis that biliary lipids may be secreted as vesicles from the hepatocyte into the canaliculi.

Animals↗
PubMed1980

Preparation of a highly purified vaccine against tick-borne encephalitis by continuous flow zonal ultracentrifugation.

By the use of continuous-flow zonal ultracentrifugation, a highly purified, inactivated tick-borne encephalitis (TBE) virus vaccine was prepared tht contained almost exclusively TBE virus particles. The purity of this preparation was 90- to 100-fold higher than that of a previously used partially purified vaccine. A mean protein content of 3-5 microgram/ml in the final vaccine represents 20 mouse protective doses50 in a mouse challenge potency test. This high degree of purity resulted in a strong reduction of side reactions observed with the less purified vaccine.

Animals↗
PubMed1975

Ultracentrifugal study of effect of sodium chloride on micelle size of fusidate sodium.

Apparent micellar molecular weights were determined with the antibiotic fusidate sodium by ultracentrifugation in varying counterion concentrations (Na+). The effects of buffer salts, pH, sodium chloride concentration, and drug concentration were studied. The results strongly support the concept of the formation of primary micelles composed of five monomer units, followed by aggregation of the pentomers into larger micelles as salt concentration increases.

Buffers↗
PubMed1977

Micellar distribution equilibria: ultracentrifugal study of apparent partition coefficients.

Ultracentrifugation was used for the partial isolation of polysorbate 80 micelles in aqueous media to determine the apparent partition coefficients of various drug species between water and the micellar pseudophase. The ratio of solute concentration in the micelles to that in water was measured for procaine, salicylic acid, sulfapyridine, sulfisoxazole, and sodium 2-naphthalensulfonate over ranges of pH, surfactant concentration, drug concentration, and micelle sedimentation. Apparent partition coefficients for the systems investigated were independent of both drug concentration and surfactant concentration, indicating that the mode(s) of surfactant-drug interaction are essentially invariant over the ranges of systematic variables studied. The method provides a relatively simple and rapid means of quantitatively evaluating drug-surfactant interactions above the CMC, when surfactant and solute can be assayed in mixtures without interference.

Chemical Phenomena↗
PubMed1987

Sizing of a vesicle drug formulation by quasi-elastic light scattering and comparison with electron microscopy and ultracentrifugation.

Quasi-elastic light scattering has been used for the sizing of a vesicle formulation. Porcine insulin was encapsulated in a vesicle preparation containing cholesterol, dipalmitoylphosphotidylcholine, digalactosyl digycerides, and dipalmitoylphosphotidylglycerol in a 40:40:15:5 ratio. The measurement requires less than 15 min to obtain mean size and distribution information and can be operated in a quality control environment. Advantages and limitations of quasi-elastic light scattering are described and this method is compared to electron microscopy and ultracentrifugation.

Chemistry, Pharmaceutical↗