Pseudoisokinetic analytical ultracentrifugation: monitoring of bands at a fixed region in the analytical ultracentrifuge.
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Chemical Phenomena↗
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Evaluation of the dual-precipitation method by comparison with the ultracentrifugation method for measurement of lipoproteins in serum.
We evaluated the dual-precipitation method for quantitative measurement of lipoproteins as described by Wilson and Spiger [J. Lab. Clin. Med. 82, 473 (1973)] for normo- and hyperlipemic sera, by comparison with the results obtained with ultracentrifugation. If serum with an above-normal triglyceride concentration is analyzed, the very-low-density lipoprotein cholesterol value obtained with the precipitation method is usually too low. For measurement of high-density lipoprotein cholesterol the ultracentrifugation and precipitation procedures give comparable results, but the latter method is preferred because sinking pre-beta-lipoproteins present in the high-density lipoprotein fraction isolated by means of the ultracentrifuge may result in falsely high values for cholesterol in that fraction. Therefore, at least for the determination of very-low-density lipoprotein cholesterol in hyperlipemic serum, the use of an ultracentrifuge remains necessary. Because few laboratories have an ultracentrifuge at their disposal, it seemed important to look at the stability of sera in view of the forwarding of samples. Also, a way of increasing the efficiency of the ultracentrifuge was studied. Sera can be stored for a week at 4 degrees C or for 54 h at room temperature without noticeable effect on lipoprotein values. Moreover, reliable values can be obtained with an ultracentrifugation time of 8 h (0.8 X 10(8) g-min).
Chemical Precipitation↗
Ultracentrifugal inoculation of herpes simplex virus.
By ultracentrifugation of 30 ml of highly dilute suspensions of herpes simplex virus (HSV) directly onto monolayer cultures grown in centrifuge tubes, infectivity was significantly greater than without centrifugation. Ultracentrifugation at 20,000 to 25,000 rpm (28,000 to 45,000 X g) for 1.5 to 2.3 h was utilized with good preservation of cultures. With low-speed centrifugation at 3,000 rpm (1,100 X g), infectivity was almost 10-fold greater than without centrifugation. With ultracentrifugal inoculation, infectivity was about 100-fold greater than without centrifugation. Ultracentrifugal inoculation permitted the detection of HSV at concentrations as low as 0.05 plaque-forming units per ml. Similarly, ultracentrifugal inoculation of cultures was almost 100-fold more sensitive a method of detecting infectious HSV than was pelleting HSV from dilute suspensions followed by resuspension and inoculation of cultures. Ultracentrifugal inoculation of cultures may permit the isolation of HSV in situations where virus cannot be detected by ordinary means and may prove applicable to the study of other viruses.
Cells, Cultured↗
Structural and compositional changes attending the ultracentrifugation of very low density lipoproteins.
The effects of repetitive ultracentrifugation on the physical and chemical properties of very low density lipoproteins (VLDL) were investigated. VLDL recentrifuged one to seven times were characterized by chemical analyses, analytical ultracentrifugation and electron microscopy. The VLDL content of triglyceride was increased and the proportion of phospholipid decreased by ultracentrifugation. Recentrifugation of VLDL decreased the number of Sf-o 20-100 particles and generated particles of Sf-o greater than 400. The bulk of the material removed from VLDL by ultracentrifugation was lipoprotein having pre-beta mobility on paper electrophoresis, flotation rates of Sf-o 10-100 and a particle size of 300-400 A-O. Two ultracentrifugations separated an average of 14% of the starting VLDL protein. Characterization of the apoproteins in this material by polyacrylamide gel electrophoresis, gel chromatography, immunoprecipitation and amino acid analysis demonstrated a relatively high proportion of beta-apoprotein and relatively little C-apoproteins.
Adult↗
Routine titration of foot and mouth disease virus suspensions by analytical ultracentrifugation. 1: Sedimentation method.
Infectivity and Complement Fixation (CF) tests are commonly used for the routine titration of Foot and Mouth Disease (FMD) virus suspensions. Only recently were techniques published for the routine determination of the virus concentration by the physical properties of the virions (Fayet et al., 1971; Barteling et al., 1974). These techniques are based on the separation of the virions from the culture fluid by sedimentation through a sucrose gradient, in a preparative ultracentrifuge. The ultraviolet absorption pattern of the tube content is recorded by a flow colorimeter. The virus concentration is estimated using either standard curves or direct caculation by the specific extinction coefficient (Bachrach et al., 1964). In our own attempts to develop a preparative ultracentrifugation technique for the routine titration of FMD virus suspensions, we had to deal with some problems such as remixing of the virus band at the end of the run. We therefore turned over to analytical ultracentrifugation methods. The manipulations are less complicated and the virus band is traced and measured while the rotor is spinning. Four samples are analyzed simultaneously and the scans are repeated to follow the move of the virus band. The sedimentation rate of the virus band, calculated from the repeated scans, helps to detect artifacts. The present paper describes the technique we developed for the routine titration of FMD virus suspensions, by the band sedimentation method, using an ultraviolet scanning analytical ultracentrifuge.
Aphthovirus↗
Plasma lipoprotein separations by zonal ultracentrifugation.
Procedures for the separation of plasma lipoprotein classes and subclasses by zonal ultracentrifugation are described. The main density classes, very low density lipoproteins (VLDL), low density lipoproteins (LDL) and high density lipoproteins (HDL), in plasma can be separated in a single run for 20 hours. For the isolation of VLDL-LDL a centrifugation time of only 90 minutes is needed. Separations can be performed on plasma volumes varying from 10 to 400 ml in the Ti-14 rotor used; VLDL can in this way be isolated from 400 ml plasma in 30 minutes. The advantages and disadvantages of zonal ultracentrifugation in comparison with the commonly employed differential ultracentrifugation for separation of lipoproteins are discussed.
Centrifugation, Zonal↗
Turbidimetric ultracentrifugation. Application to the study of human serum very low density lipoprotein distributions.
In this communication it is shown that the sedimentation coefficient distribution may be accurately measured for very large particles using turbidimetric techniques and the ultraviolet-scanning analytical ultracentrifuge. A principal advantage is that turbidity is a function of the product of concentration and molecular weight; thus, large particles may be observed even when present in very small amounts. We propose to call this method of analysis "turbidimetric ultracentrifugation." We have used turbidimetric ultracentrifugation ot determine the sedimentation coefficient distribution for a sample of human serum very low density lipoproteins. This distribution is compared to that found with conventional schlieren techniques with good agreement.
Humans↗
Routine titration of foot-and-mouth disease virus suspensions by analytical ultracentrifugation 2nd communication: sedimentation equilibrium method.
A routine method for the determination of the virus concentration in FMD virus cultures and vaccines was developed. This method was based on sedimentation equilibrium in the analytical ultraviolet scanning ultracentrifuge. The virus suspension was first clarified. The virions were then sedimented in a preparative ultracentrifuge. The resuspended virions were diluted in a Cesium chloride solution and brought to equilibrium in the density gradient generated in the analytical ultracentrifuge. The optical density of the virus band was measured by the UV scanning system. A calculation procedure was developed to compute the density at the limits and at the maximum of the virus band. The virus concentration expressed as weight, was calculated for the original virus suspension.
Aphthovirus↗
Copper (II) induced polymerization of human albumin, and its depolymerization by diglycyl-L-histidine: a pH static and ultracentrifugation study.
Copper (II) ions successively induce dimers and tetramers of human serum albumin (L) when the Cu (II) concentration is extended beyond that of 200 muM. This is shown by emf titrations and by ultracentrifugation experiments. The emf titrations, which involve a new pH static method, were performed at 25 degrees, in a 0.5 M NaCIO4 medium at pH 6.59, using glass and copper amalgam electrodes. The total concentration of Cu(II) varied from 0.14 to 2.2 mM and the albumin concentration from 0.05 to 0.7 mM. In order to evaluate the formula of the main complexes, without using any a priori assumptions regarding their compositions, a detailed graphic procedure was used. The results, in the form of equilibrium constants for the main species, were refined by the use of a general least squares computer program. The experimental data are found to be consistent with the formation of the monomeric CuL, Cu5L, and Cu6L species and the dimeric Cu3L2, Cu4L, Cu6L, and Cu8L2 species. In addition, there is some indication for a minor species, most probably the Cu12L4 tetramer. The pH static results qualitatively agree with the findings obtained by ultracentrifugation. As indicated by distinct bands and their S-values, ultracentrifugation experiments show not only monomeric and dimeric species of albumin, but also tetrameric species. The polymerization of the albumin is reversible, since diglycyl-L-histidine, a peptide designed to mimic the Cu (II) transport site of albumin, depolymerizes the Cu (II)-albumin polymers.
Binding Sites↗
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↗
Molecular sizes of egg yolk very low density lipoproteins fractionated by ultracentrifugation.
Egg yolk very low density lipoproteins isolated from normal eggs were separated into six fractions by preparative ultracentrifugation. The sizes of the molecules in three of the fractions were estimated by gel chromatography and by electron microscopy of the lipoproteins using a negative staining technique. The three fractions had average diameters of 39 nm., 32 n., and 25 nm. and average molecular weights of 17 X 10(6), 10 X 10(6), and 5 X 10(6). There was considerable overlap of size of molecules in the three fractions. Size differences were not observed in the ultracentrifuge separated fractions of very low density lipoproteins isolated from eggs laid by hens receiving crude cottonseed oil in their diets. However, the fatty acids from these fractions differed in stearic and oleic acid contents while those from the normal egg lipoproteins did not. The floating very low density lipoproteins contained more oleic acid and less stearic acid then the sedimenting ones did.
Animals↗
[Identification of the mink alpha2-lipoprotein Lpm-allotypes by the method of preparatory ultracentrifugation].
The density class accessory of 8 mink alpha2-lipoprotein allotypes are determined by means of preparative ultracentrifugation. It is found that Lpm1, Lpm2, Lpm2, Lpm3, Lpm4, Lpm5, Lpm7 and Lpm8 are determinants of lipoproteins with density exceeding 1.210, i.e. they are VHDL. The allotypic marker 6, which has been earlier assigned by other criteria to Lpm group, belongs to mink lipoprotein, which distributes during ultracentrifugation at the region of low and, partially, high density.
Alleles↗
An ultracentrifugal study of the self-association of canine apolipoprotein A-I in solution.
The sedimentation behavior of canine apolipoprotein (apo) A-I in 0.02 M EDTA, pH 8.6, was studied as a function of protein concentration by the techniques of sedimentation velocity and sedimentation equilibrium in the analytical ultracentrifuge. At concentrations of less than 1 g/liter, apo-A-I exhibited a monomodal sedimentation pattern, with apparent sedimentation coefficients which varied from 2.3 to 3.5 S with increasing protein concentrations. Above 1.5 g/liter, apo-A-I had two well resolved peaks with s20,w values of 4.15 S and 5.75 S. The proportion of the 5.75 S component increased with increasing apo-A-I concentrations, with a concomitant decrease of the 4.15 S component. By sedimentation equilibrium ultracentrifugation with both the conventional and meniscus-depletion methods, the apparent weight-average molecular weight of apo-A-I was found to be concentration-dependent. At a protein concentration of 5.25 g/liter, an apparent weight average molecular weight of 138,000 was determined, indicating that molecular species larger than a tetramer (monomer molecular weight = 28,000) were present in solution. When analyzed in terms of a reversible self-associating system, the experimental data could best be described according to a monomer-dimer-tetramer-octamer model, as previously reported from human apo-A-I (Vitello, L. B., and Scanu, A. M. (1975) J. Biol. Chem. 251, 1131-1136). The equilibrium constants were: K2 = 4.5 liters/g, K4 = 470 liters3/g3, and K8 = 41,600 liters7/g7, respectively.
Animals↗
Identification of strains of herpes simplex virus by comparison of the density of their DNA using the preparative ultracentrifuge.
The buoyant densities of the DNA of herpes simplex virus type 1, type 2 and Pseudorabies virus, as determined in the analytical ultracentrifuge, are 1.725, 1.727 and 1.731 correlating with G+C contents of 67, 69 and 73 per cent respectively. The density differences for the DNA's of type 1 and type 2 herpes simplex viruses have been confirmed in experiments with isotopically labelled DNA from four type 1 and six type 2 strains by preparative CsCl gradient ultracentrifugation. The DNA of all the type 2 strains was denser than that of any of the type 1 strains examined. Despite these differences in DNA base composition of type 1 and type 2 strains, nearest neighbour analysis of their DNA's disclosed no obvious differences in doublet pattern or general design.
Base Sequence↗
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↗
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↗
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↗
A comparison of simplified methods for lipoprotein quantification using the analytic ultracentrifuge as a standard.
Two simplified methods for quantitative lipoprotein analysis have been calibrated and compared with each other using analytic ultracentrifugation as a standard reference procedure. The first method was the Friedewald procedure and the second was an automated agarose gel electrophoresis system. Both procedures offer comparable quantitative lipoprotein analysis with potential for large scale screening purposes at low cost ($4.00-$5.00 per analysis). There were advantages and limitations to both procedures. The Friedwald procedure can be used on frozen sera but requires 3 ml sera. In contrast, the electrophoresis system must be used with fresh serum but requires only 50 mul serum and the electrophoretic slides may be quantitatively analyzed several years retrospectively.
Adult↗