Buoyant density separation of human blood cells in renografin gradients

Renografin, because of its high density and low viscosity, has been shown to be suitable as a supporting medium for the construction of continuous density gradients. The conditions necessary for the isopycnic banding of cells were determined, initially, using human erythrocytes. In the order of increasing density, human blood cells were separated into relatively pure bands of nongranular leucocytes, erythrocytes, and granulocytes. Their relative positions in the gradient were affected, however, by the high osmolarity of Renografin. Renografin is not cytotoxic and does not aggregate cells. It has the additional advantages of being stable, readily obtained in sterile ampoules, and inexpensive.     

Band centrifugation of macromolecules in self-generating density gradients. II. Sedimentation and diffusion of macromolecules in bands

Three approaches to the simultaneous sedimentation and diffusion of hands or zones of noninteracting homogeneous macromolecules are examined: (1) The authors' method of moments: (2) the transport me of Sehumaker and Rosenbloom; and (3) the stochastic solution of the Lamm equation due to Gehatia and Katehalski. All three methods indicate that the motion of the maximum of the hand may be used to evaluate the sedimentation coefficient. The moment, method provides relations which appear to be useful for measuring diffusion coefficients. Relations are given for the analysis of resolved components. The problem of measuring sedimentation coefficients of macromolecules with concentration-dependent sedimentation coefficients is examined. Methods are described for evaluating the sedimentation coefficient in these systems and for obtaining the sedimentation coefficient at infinite dilution. Methods are described for determining the weight-average sedimentation coefficient in Multi-component systems, and the differential and integral distribution of sedimentation coefficients of macromolecules with low-diffusion coefficients.     

Concentration dependence in three-component systems in sedimentation equilibrium in buoyant density gradients

The data on the band widths and band shapes of several DNA's at various concentrations in sedimentation equilibrium in a CsCl density gradient have recently become available. In the present report, these literature data are treated in the following manner: (1) based on a theory of isotope-substitution, calculations are made of the molecular weights at infinite dilution, and (2) to explain the concentration dependence of band widths and band shapes, a theory of charge and hydration is put forth, and it is shown that by retaining the terms involving the charge of the macromolecules, it is possible to account for most of the concentration dependence.     

A model for sedimentation in inhomogeneous media. I. Dynamic density gradients from sedimenting co-solutes

Macromolecular sedimentation in inhomogeneous media is of great practical importance. Dynamic density gradients have a long tradition in analytical ultracentrifugation, and are frequently used in preparative ultracentrifugation. In this paper, a new theoretical model for sedimentation in inhomogeneous media is presented, based on finite element solutions of the Lamm equation with spatial and temporal variation of the local solvent density and viscosity. It is applied to macromolecular sedimentation in the presence of a dynamic density gradient formed by the sedimentation of a co-solute at high concentration. It is implemented in the software SEDFIT for the analysis of experimental macromolecular concentration distributions. The model agrees well with the measured sedimentation profiles of a protein in a dynamic cesium chloride gradient, and may provide a measure for the effects of hydration or preferential solvation parameters. General features of protein sedimentation in dynamic density gradients are described.     

Mathematics of band centrifugation: Concentration-independent sedimentation and diffusion in shallow density gradients

Integral expressions for concentration as a function of time and distance are derived from the continuity equation for centrifugation in a sector-shaped cell for a macro-molecular solute initially contained in a finite upper layer and a solute of low molecular weight in the supporting liquid. Computer patterns based on the sedimentation and diffusion coefficients of sucrose and of spherical and randomly coiled model solutes illustrate: (1) the time course of redistribution of both banded and supporting solutes from initial uniform concentrations; (2) the influence of the initial concentration, width, and solute concentration of the upper band; and (3) the effect of restricted diffusion at the meniscus on subsequent band shape. A Gaussian, approximation to band shape is derived and graphically tested. Rapid methods, not requiring computers, are out lined for the estimation of sedimentation and diffusion coefficients, where their concentration dependence is negligible, by band centrifugtion. The theoretical resolution of mixtures attainable by this technique is compared with moving-boundary centrifugation, with the use of both integral (interferotmetric or absorption) and derivative (schlieren) optics.     

Buoyant density of DNA-Hoechst 33258 (bisbenzimide) complexes in CsCl gradients: Hoechst 33258 binds to single AT base pairs.

Buoyant density of DNA in CsCl gradients with Hoechst 33258 (bisbenzimide) was investigated as a function of guanine plus cytosine content of the DNA (%GC; in mole percent). A formula for calculating %GC from the refractive index (nD) of the isopycnic CsCl/Hoechst 33258 solution over the range of 0-75 %GC was established: %GC = 351762.28 X nD - 123778.66 X nD2 - 249789.47 (the coefficients must not be rounded off). The shape of this curve indicates that under these conditions, in contrast to dilute buffers, Hoechst 33258 binds to single AT base pairs on DNA. Resolution of DNA bands in CsCl/Hoechst 33258 gradients is 1.6 to 2.1 times better than comparative CsCl gradients without the dye. Potential application to %GC determination is discussed.     

Buoyant density and solvation of magnesium DNA

The buoyant density of T-4 DNA was determined by equilibrium sedimentation in a density gradient, of mixed solutions of cesium and magnesium chlorides and bromides. The preferential hydration was calculated from these data, allowing appropriately for the exchange equilibrium of DNA with Cs⁺ and Mg⁺⁺ ions. The charge and intrinsic solvation of the counterions were found to have no appreciable effect on the hydration of the DNA, the extent of solvation depending only on the thermodynamic, activity of the water. Various reasonable hypotheses are discussed to account for these results.     

Sedimentation equilibrium of proteins in density gradients.

The technique of sedimentation equilibrium in density gradients in the analytical ultracentrifuge has been applied to the study of proteins. A variety of effects and procedures including the use of density marker beads, the effects of pressure on buoyant density and pH, and the calculation of compositional density gradient proportionality constants and density--refractive index relations have been developed. The buoyant densities of twenty-four proteins have been measured and hydration values computed. The buoyant titrations of six proteins have been measured. These data have been interpreted in terms of the buoyant titrations which have been obtained for six ionizable homopolypeptides, five copolypeptides, two non-ionizable homopolypeptides and three chemically modified proteins. Spectropolarimetry and potentiometric titrations were employed to further interpret these data. Approximate values for dissociation constants, numbers of ionizable residues, and the nature of ions bound or dissociated upon ionization have been obtained. The relation between potentiometric and buoyant titrations and the use of density gradient centrifugation as a probe for protein structure have been explored.     

Buoyant density analysis of myeloid colony-forming cells in germfree and conventional mice.

Granulocyte-macrophage colony-forming cells (CFUc), in the bone marrow of germfree and conventioal CBA mice, were compared quantitatively and qualitatively. Cells were separated on the basis of their buoyant density by equilibrium centrifugation in continuous albumin density gradients. CFUc in the density subpopulations were detected by culture in agar containing three different types of colony stimulating factor (CSF). The sources of the CSF were post-endotoxin mouse serum (CSFES), mouse lung conditioned medium (CSFMLCM) and human urine (CSFHU). Mice were removed from the germfree environment and the buoyant density status of their CFUc was examined 1, 4 and 8 weeks later. No difference was found between germfree and conventional mice in the number of nucleated cells per femur or in their modal density. Neither was the number of CFUc per femur different. The cell cycle status of CFUc, as determined by the thymidine suicide technique was not significantly different. Functional heterogeneity was found among the density subpopulations for both groups of mice. This depended on the type of CSF. The density distribution of CFUc was significantly different in germfree mice. There were proportionately more low density CFUc. The mean modal density of CFUc under CSFES stimulation was less by 0.0045 g/cm3 in germfree mice. The removal of mice from the germfree environment resulted in a shift of the distribution to higher densities. The trend was towards the conventional situation. The significance of the buoyant density status of CFUc is discussed.     

Boundary centrifugation in isovolumetric and isokinetic cesium sulfate density gradients: application to cartilage proteoglycans and other macromolecules

A boundary sedimentation methodology is described that avoids plateau dilution and simplifies the calculation of centrifugal parameters. The technique is designed for the preparative ultracentrifuge and uses a newly developed sectorial cell. It is based on previous developments of the transport method and depends on isokinetic or isovolumetric Cs2SO4 density and viscosity gradients. These gradients are prepared with a single-chamber mixing device, and the only two parameters required for their calculations are presented in a tabulated form for general use with most available rotors and cell sizes. Conditions are specified (1) to assure that the density and shape of the sedimenting molecules remain invariant through the selected electrolytic gradient, (2) to monitor the gradient profiles, and (3) to verify attainment of isokinetic or isovolumetric sedimentations. A set of equations is presented to calculate the average and transport sedimentation coefficients and the differential sedimentation coefficient distribution for both the isokinetic and isovolumetric centrifugal regimes. The method was applied to slowly diffusing polydisperse proteoglycan monomers, to a paucidisperse DNA from bacteriophage PM2, and to a diffusible monodisperse system (purified bovine serum albumin). In all cases, the expected results were obtained.     

Generation of density gradients. Approximations to equivolumetric gradients for zonal rotors

The use of a “density gradient generating function” allows the concentration profile of a density gradient to be written explicitly in terms of the required distribution of sedimentation coefficients in place of the previous implicit formulations. This function, which is easily implemented in a computer program, permits calculation of density gradients for a number of applications. This approach is applied to computation of a variety of equivolumetric gradients of sucrose for zonal rotors and yields a formula for the calibration of such gradients. An accurate approximation has been found which allows the generation of virtually all equivolumetric gradients of sucrose for a given rotor using a single program for the gradient generator employed; the adjustment for different particle densities and for different concentrations at the top of the gradient is made by varying only the initial and final concentrations of sucrose used.     

Buoyant density studies of several mecillinam-resistant and division mutants of Escherichia coli.

The buoyant density of wild-type Escherichia coli cells has previously been reported not to vary with growth rate and cell size or age. In the present report we confirm these findings, using Percoll gradients, and analyze the recently described lov mutant, which was selected for its resistance to mecillinam and has been suggested to be affected in the coordination between mass growth and envelope synthesis. The average buoyant density of lov mutant cells was significantly lower than that of wild-type cells. Similarly, the buoyant density of wild-type cells decreased in the presence of mecillinam. The density of the lov mutant, like that of the wild type, was invariant over a 2.8-fold range in growth rate. In this range, however, the average cell volume was also constant. Analysis of buoyant density as a function of cell volume in individual cultures revealed that smaller (newborn) lov mutant cells had higher density than larger (old) cells; however, the density of the small cells never approached that of the wild-type cells, whose density was independent of cell size (age). A pattern similar to that of lov mutant cells was observed in cells carrying the mecillinam-resistant mutations pbpA(Ts) and rodA(Ts) and the division mutation ftsI(Ts) at nonpermissive temperatures as well as in wild-type cells treated with mecillinam, but not in mecillinam-resistant crp or cya mutants.     

Sucrose density gradient sedimentation of E. coli ribosomes.

The behavior of E. coli ribosomes during sedimentation on sucrose gradients is predicted under a variety of conditions by computer simulations. Since numerous recent kinetic studies indicate equilibration in times short compared to the time of sedimentation, these simulations assume that the system attains local reaction equilibrium at every point in the gradient at all times. For any type of homogeneous equilibrating ribosome population, governed by a single formation constant at one atmosphere pressure for 70S couples, no more than two clearly defined zones will be resolved, although the presence of large dissociating effects due to pressure gradients in high speed experiments will spread the subunit zone. Normally the pattern will consist of a 30S zone and a so-called “70S” zone, which is in reality a mixture of 70S couples and 30S and 50S subunits in local equilibrium. The greater the dissociation into subunits, the more the “70S” zone will be slowed below the nominal rate of 70 Svedberg units. If ribosomes have been collected from the “70S” zone in several successive cycles of purification, the repeated deletion of resolved 30S subunits can result in a preparation with so large a molar excess of 50S subunits that the ensuing sucrose density gradient sedimentation pattern may exhibit a “70S” zone followed by zone of 50S subunits, insteadof a zone of 30S subunits. Our most important conclusion is that whenever a well-resolved 50S zone is present in a sucrose density gradient sedimentation experiment on E. coli ribosomes, in addition to a 30S and a “70S” zone, under conditions where ribosomes and subunits should be in reversible equilibrium, the preparation must be microheterogeneous, containing a mixture of “tight” and “loose” couples. Moreover in such cases the content of large subunits in the 50S zone must be derived entirely from “loose” couples whereas the 30S zone must contain small subunits derived from both “tight” and “loose” couples. Sedimentation patterns predicted for various mixtures of “tight” and “loose” couples display all the major characteristics of published experimental patterns for E. coli ribosomes, including the partial or complete resolution into three zones, depending on rotor velocity and level of Mg²⁺.