Anomalies in sedimentation. IV. Decrease in sedimentation coefficients of chains at high fields

A theory is presented for the decrease of sedimentation coefficient at high centrifugal fields recently reported for samples of DNA by Rubenstein and Leighton and others. The theory uses the model of a chain of beads and springs to represent the molecule. Kirkwood's approximation is used for the sedimentation coefficient. The decrease in sedimentation coefficient with field comes about as a result of the uneven frictional forces in the chain, which on the average are less on segments near the center of the chain than on those near the ends. As a result the ends of the chain tend to drag behind the center, and the average intersegment distances are increased; consequently the hydrodynamic shielding of one segment by another is reduced, and the average friction is increased. The effect is thus characteristic of single molecules; intermolecular interaction is not involved. The sedimentation coefficient, S, varies as a power series in a parameter y that measures the distortion produced by the uneven friction: S = S⁰(1?D₂y² + D₄y⁴ ? ·). where S⁰ is the limiting value of S at zero centrifugal field and D₂ and D₄ are constants; y is proportional to the cen speed squared tunes the molecular weight squared divided by S⁰. It has been observed that the effects of centrifuge speed on S are negligible below certain critical values of the speed and molecular weight, but increase dramatically immediately above these values; this follows naturally from the high powers of the speed and molecular weight that appear in the above equation.     

Advances in sedimentation velocity analysis.

On February 20, 1996, a workshop titled "Advances in Sedimentation Velocity Analysis" was held at the Biophysical Society meeting in Baltimore, Maryland, in honor of Professor David Yphantis's 65th birthday. Although he is known more for his work with sedimentation equilibrium, David's work on instrumentation and data analysis is the foundation for many of the recent advances in both equilibrium and velocity sedimentation. Over the years he has trained numerous graduate students, most of whom have gone on to emphasize the use of analytical ultracentrifugation to answer biochemical questions involving macromolecular assembly. His laboratory was one of very few that continued to use and develop analytical ultracentrifugation during its nadir in the 1970s and early 1980s. The rebirth and resurgence of analytical ultracentrifugation owe a great deal to his persistence and enthusiasm. These efforts have borne fruit. In the last five years, through his work at the National Analytical Ultracentrifugation Facility, he has helped train nearly 100 individuals in the delicate art of nonlinear least-squares analysis of equilibrium sedimentation data. Furthermore, the number of researchers using the ultracentrifuge and the number of papers published has skyrocketed in the last few years. This workshop, then, was a way to thank David for his years of devotion to analytical ultracentrifugation.     

Quantitation of the rate-zonal sedimentation spectrum.

Rate-zonal sedimentation of cells from the female genital tract has been employed as a means of separating many of the different cellular components from each other. Ultra-slow streak photography of forward scattered light from the sedimentation spectrum has been used as a monitor of the success of the individual sedimentation maneuver. The presence of convention currents vitiates the separation potential of the technique, and this is readily detected by the photographic monitor. In addition, these photographs clearly indicate the positions of bands of cellular entities of different sedimentation velocities. Further study of the quantitative aspects of the light scattered from each part of the spectrum reveals that it possesses a quantitative representation of the concentration of cells at each level of the spectrum. The photographs and densitometric scans support these expectations. To avoid the limitations of film as a data-logging medium and to obtain real-time system utilization, a direct photoelectric scanner has been assembled. Preliminary tests indicate ready feasibility.     

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⁺.     

Anomalies in sedimentation. II. Testing the entanglement hypothesis

Zones of T2 DNA were sedimented through uniform solutions of T7 DNA to determine if the smaller DNA molecules would become entangled in the larger. No entanglement could be demonstrated even at high DNA concentrations. It is suggested that molecular entanglement is not responsible for the sudden loss of DNA from solution which occurs in high centrifugal fields. This communication also includes observations on the effects of rotor speed on the sedimentation behavior of DNA in high centrifugal fields, distortion of zone shape at high concentrations, and hydrodynamic interactions between DNA and MS2 bacteriophage particles.     

Seston sedimentation

SUMMARY. Three experiments are described in which the rates of loss from suspension of Lycopodium spores introduced at the water surface in a large butyl rubber enclosure ('Lund Tube') were monitored. Rates of arrival at the mud surface and in sediment traps placed in the water column were also measured. Results obtained by each method are compared with the predetermined intrinsic sinking behaviour of the individual particles. The experiments, which were carried out under differing conditions of water-column stability (isothermal mixing, thermal microstratification, and autumnal destratification), demonstrate the importance of turbulent mixing in determining the effective settling rate of seston. A simple predictive model is developed to relate sinkinglosses to the vertical extent of broadly defined water layers. Recoveries in sediment traps can give an accurate synopsis of net sedimentation when they are operated in non-turbulent water layers, but trap performance in turbulent flow is significantly modified by hydrodynamic perturbation generated by the traps themselves.     

Normal erythrocyte sedimentation rate in the elderly.

Two hundred subjects aged 60-89 were selected for a study aimed at defining a reference range for the erythrocyte sedimentation rate in the elderly. The study extended a previous survey in subjects aged 20-65. The results confirmed that the sedimentation rate increases with age and that women have higher values than men but suggested that over half of elderly patients with disease would have rates within the previously defined "normal" range. It is therefore suggested that an erythrocyte sedimentation rate exceeding 19 mm in the first hour in elderly men and 22 mm in the first hour in elderly women warrants investigation.     

Determination of sedimentation coefficients for small peptides.

Direct fitting of sedimentation velocity data with numerical solutions of the Lamm equations has been exploited to obtain sedimentation coefficients for single solutes under conditions where solvent and solution plateaus are either not available or are transient. The calculated evolution was initialized with the first experimental scan and nonlinear regression was employed to obtain best-fit values for the sedimentation and diffusion coefficients. General properties of the Lamm equations as data analysis tools were examined. This method was applied to study a set of small peptides containing amphipathic heptad repeats with the general structure Ac-YS-(AKEAAKE)nGAR-NH2, n = 2, 3, or 4. Sedimentation velocity analysis indicated single sedimenting species with sedimentation coefficients (s(20,w) values) of 0.37, 0.45, and 0.52 S, respectively, in good agreement with sedimentation coefficients predicted by hydrodynamic theory. The described approach can be applied to synthetic boundary and conventional loading experiments, and can be extended to analyze sedimentation data for both large and small macromolecules in order to define shape, heterogeneity, and state of association.     

Dynamic model for sedimentation of supercoiled molecules.

An experimental curve for the sedimentation of SV40 DNA has been fitted by using a three parameter theory. The chain stiffness parameter is evaluated at each point, and the general behavior as a function of the number of loops (superhelix density) has been described. The chain stiffness, like the sedimentation ratio, also depends on the superhelical density in a complicated way.     

Cortical cell elution by sedimentation field-flow fractionation.

As a cell sorter, Sedimentation field-flow fractionation (SdFFF) can be defined as an effective tool for cell separation and purification, respecting integrity and viability as well as providing enhanced recovery and purified sterile fraction collection. The complex cell suspension containing both neurons and glial cells of all types, obtained from cerebral cortices of 17-day-old rat fetuses, is routinely used as a model of primary neuronal culture. Using SdFFF, this complex cell mixture was eluted in sterile fractions which were collected and cultured. SdFFF cell elution was conducted under strictly defined conditions: rapid cell elution, high recovery (negligible cell trapping), short- and long-term cell viability, sterile collection. After immunological cellular type characterization (neurons and glial cells) of cultured cells, our results demonstrated the effectiveness of SdFFF to provide, in less than 6 min, viable and enriched neurons which can be cultured for further investigations.     

Numerical solutions of the Lamm equation. II. Equilibrium sedimentation

The Lamm equation has been solved numerically for conditions corresponding to equilibrium runs for a nonlinear concentration dependence of the form s/s₀ = (1 + kc)^?1. It is shown that the approach to equilibrium is very close to being exponential (in time) as in the case k = 0. We also compare results for the nonlinear case given above with results obtained for linear c-dependence of the form s/s₀ = 1 – kc. For relatively high speeds the time required to attain equilibrium may be greatly underestimated by use of the latter approximation. Finally, we present analytic approximations for the concentration distribution at equilibrium and as a function of time.