Analytical techniques for cell fractions. XVI. Preparation of protein-free supernatants with a "Z"-path rotor

A new “Z”-path centrifuge rotor is described for the separation of pellets from supernatants in up to 15 samples simultaneously. The samples and precipitating solutions are stored in separate chambers at rest, and are transferred to a sedimentation chamber by centrifugal force during rotation, where mixing occurs during rapid changes in speed, followed by sedimentation of the precipitate. The supernatant decants itself at rest into a third series of chambers, where it may be transferred centrifugally into radially disposed measuring devices, transferred with suitable pipettes, or used as a sample ring for conventional analyzers.     

Perfluoroalkanes. A model for the hexagonal methylene subcell?

High resolution hkO electron diffraction intensity data from hexagonally packed helices in the crystalline perfluoroalkane, nC₁₆F₃₄, are well fit by a CF₂ rotor model similar to the CH₂ rotor model used earlier for the α-form of long chain lipids. Other crystallographic evidence for similar helical twisting of polymethylene chains is adduced in a consideration of the structure as a model for the hexagonal methylene subcell.     

Polyacrylamide gel electrophoresis on micro slabs

An electrophoretic technique using micro polyacrylamide flat gels is described and its usefulness demonstrated. The gels are vertically cast and electrophoresed in slab form (75 × 18 × 0.75 mm) in closed thin glass cells (cuvets) made from detachable microscope slides. The main features of the method are: requirement of small sample quantities (0.1–20 μg contained in <1–5 μl solution), simultaneous analysis of several samples in a single gel, relatively brief running periods, easy removal of the gel for rapid staining due to the two-piece gel mold, little pattern diffusion, convenient optical evaluation, drying, autoradiography and other contact print methods, and easy application of immunodiffusion techniques. Continuous gradient gels can be prepared. The advantage and complications of the technique are discussed and certain applications in biochemistry, clinical chemistry, and medicine are pointed out.     

Large Scale Isolation and Purification of Eyespot Granules from Euglena gracilis var. bacillaris

Large volumes of eyespot granules were isolated from homogenates of Euglena gracilis Klebs var. bacillaris Pringsheim by flotation centrifugation in a Beckman Ti-15 zonal rotor, and were further purified by centrifugation in a swinging bucket rotor. Examination with the electron microscope showed the eyespot granules to be free from other cellular material. Freezing had no apparent effect on the structure or on the absorption properties of the eyespots. Absorption spectra of pure fractions of eyespot granules free of chloroplast contamination showed the previously reported curves in the range of 360 to 520 nanometers, as well as a peak at 660 to 675 nanometers. The procedure for the large scale isolation of eyespot granules from Euglena gracilis is compared with other methods which have employed conventional centrifugation, and the significance of the use of zonal rotors for isolating large quantities of pure eyespot granules is discussed.     

Nuclear and mitochondrial deoxyribonucleic acid replication during mitosis in Saccharomyces cerevisiae.

To study nuclear and mitochondrial deoxyribonucleic acid (DNA) synthesis during the cell cycle, a 15N-labeled log-phase population of Saccharomyces cervisiae was shifted to 14N medium. After one-half generation, the cells were centrifuged on a sorbitol gradient in a zonal rotor to fractionate the population according to cell size and age into fractions representing the yeast cell cycle. DNA samples isolated from the zonal rotor cell samples were centrifuged to equilibrium in CsC1 in an analytical ultracentrifuge to separate the nuclear and mitochondrial DNA components. The amount of 14N incorporated into each 15N-labeled DNA species was measured. The extent of nuclear DNA replication per sample was obtained by measuring the amount of hybrid DNA. The percentage of hybrid nuclear DNA increased from 6 to 68% and then decreased to 44% during the cell cycle. Upon ultracentrifugation, mitochondrial DNA banded as a unimodal peak in all zonal rotor samples. Mitochondrial DNA replication could be ascertained only by the 14N level in each mitochondrial peak and not, as with nuclear DNA, by hybrid DNA level. In contrast to the nuclear incorporation pattern, the 14N percentage in mitochondrial DNA remained effectively constant during the cell cycle. Comparison of the data to theoretical distributions showed that nuclear DNA was replicated discontinuously during the cell cycle, whereas mitochondrial DNA was replicated continuously throughout the entire mitotic cycle.     

Separation of subclasses of human serum high density lipoproteins by zonal ultracentrifugation.

An improved one-step method for the preparative separation of three subfractions of high-density lipoproteins from normal human serum has been developed. It employs the method of rate zonal ultracentrifugation in a z-60 rotor using a discontinuous NaBr gradient in the density range of 1.0-1.4. The density gradients were monitored directly by a flow-through density meter allowing the direct read-out of the actual densities in the process of filling and emptying the rotor. The separation of the three density fractions from 5 to 15 ml serum was achieved during a single 12 hours run at 59.000 rpm. The three fractions showed characteristically different patterns on polyacrylamide gel electrophoresis and differences in their lipid and protein composition.     

A model for sedimentation in inhomogeneous media. II. Compressibility of aqueous and organic solvents

The effects of solvent compressibility on the sedimentation behavior of macromolecules as observed in analytical ultracentrifugation are examined. Expressions for the density and pressure distributions in the solution column are derived and combined with the finite element solution of the Lamm equation in inhomogeneous media to predict the macromolecular concentration distributions under different conditions. Independently, analytical expressions are derived for the sedimentation of non-diffusing particles in the limit of low compressibility. Both models are quantitatively consistent and predict solvent compressibility to result in a reduction of the sedimentation rate along the solution column and a continuous accumulation of solutes in the plateau region. For both organic and aqueous solvents, the calculated deviations from the sedimentation in incompressible media can be very large and substantially above the measurement error. Assuming conventional configurations used for sedimentation velocity experiments in analytical ultracentrifugation, neglect of the compressibility of water leads to systematic errors underestimating sedimentation coefficients by approximately 1% at a rotor speeds of 45000 rpm, but increasing to 2-5% with increasing rotor speeds and decreasing macromolecular size. The proposed finite element solution of the Lamm equation can be used to take solvent compressibility quantitatively into account in direct boundary models for discrete species, sedimentation coefficient distributions or molar mass distributions. Using the analytical expressions for the sedimentation of non-diffusing particles, the ls-g*(s) distribution of apparent sedimentation coefficients is extended to the analysis of sedimentation in compressible solvents. The consideration of solvent compressibility is highly relevant not only when using organic solvents, but also in aqueous solvents when precise sedimentation coefficients are needed, for example, for hydrodynamic modeling.     

Comparison of two ultracentrifugation procedures for separation of nonhuman primate lipoproteins.

A comparison of nonhuman primate plasma lipoproteins isolated by swinging bucket rotor density gradient or fixed angle rotor differential ultracentrifugation is described. Whereas these two methods produced comparable results for the composition of low density (LDL) and high density (HDL) lipoproteins, the very low density lipoprotein (VLDL) fraction isolated with the swinging-bucket rotor contained relatively more cholesterol (free and esterified) and less phospholipid and protein than that fraction obtained with the fixed-angle rotor. Estimations of lipoprotein concentration by agarose gel electrophoresis and particle size by electron microscopy coupled with molar ratios of surface to core constituents indicate that the swinging bucket procedure resulted in a more complete harvest of VLDL particles, especially those in the larger size range.     

Induction of peroxisomal beta-oxidation in rat liver by high-fat diets.

Liver peroxisomes were prepared by using a Percoll gradient in a vertical rotor. beta-Oxidation was measured in peroxisomes isolated from livers of rats fed on either high-(15% by wt.) or low- (5% by wt.) fat diets. The feeding of high-fat diets gave a 1.4-2.4-fold increase in total liver peroxisomal beta-oxidation, and a similar increase in specific activity. A 1.5-4.5-fold increase was seen in the specific activity of purified peroxisomal preparations. The reasons for these increases are discussed.     

A magnetic viscometer for shear-sensitive macromolecules

This paper is a report about a rotation-viscometer with a submerged rotor which has been developed for measuring the viscosity of biological macromolecules. The device avids the effects of surface disturbance. The rotor is centered and height-balanced electromagnetically and is controlled by a light barrier. The driving force is rotating electromagnetic field and rotor revolution periods are measured by an electronic timer controlled by a second light barrier. Shearing effects are negligible if very slow revolution are pre-selected; thus, intrinsic viscosity for DNA can be obtained by merely extrapolating the concentration dependence. In contrast to DNA, chromatin has a very low viscosity with almost no dependence on concentration. If the ionic strength of a chromatin solution is decreased, the viscosity increases due to structural unfolding.     

Anomalies in sedimentation. I. Stability theory of sedimenting entanglements in the "tight-bending" limit

A simple model is introduced to investigate the stability of a sedimenting entanglement. The sedimenting entanglement is represented by a sedimenting sieve. Solvent can pass through it, but single-chain molecules that flow into it become entangled and their flow decreases or, if permanent entanglements form, ceases entirely. With this model we are able to find the conditions under which the mass of a sedimenting entanglement remains constant, grows or decays to a stable value, grows beyond limit, or decays to the mass of a single chain. The theory is applied to the sedimentation of small concentrations of large chain molecules in solutions of small chain molecules in solutions of small chain molecules for the case in which the entanglements are long-lived. Equations are derived which, (1) give the stable entanglement mass as a function of rotor speed and concentration and, (2) for a given concentration predict the rotor speed at which the entanglement mass grows without limit. Numerical results for small concentrations of T2 DNA sedimenting in solutions of T7 DNA are presented.     

Optimizing the rotor design for controlled-shear affinity filtration using computational fluid dynamics

Controlled shear affinity filtration (CSAF) is a novel integrated processing technology that positions a rotor directly above an affinity membrane chromatography column to permit protein capture and purification directly from cell culture. The conical rotor is intended to provide a uniform and tunable shear stress at the membrane surface that inhibits membrane fouling and cell cake formation by providing a hydrodynamic force away from and a drag force parallel to the membrane surface. Computational fluid dynamics (CFD) simulations are used to show that the rotor in the original CSAF device (Vogel et al., 2002) does not provide uniform shear stress at the membrane surface. This results in the need to operate the system at unnecessarily high rotor speeds to reach a required shear stress of at least 0.17 Pa at every radial position of the membrane surface, compromising the scale-up of the technology. Results from CFD simulations are compared with particle image velocimetry (PIV) experiments and a numerical solution for low Reynolds number conditions to confirm that our CFD model accurately describes the hydrodynamics in the rotor chamber of the CSAF device over a range of rotor velocities, filtrate fluxes, and (both laminar and turbulent) retentate flows. CFD simulations were then carried out in combination with a root-finding method to optimize the shape of the CSAF rotor. The optimized rotor geometry produces a nearly constant shear stress of 0.17 Pa at a rotational velocity of 250 rpm, 60% lower than the original CSAF design. This permits the optimized CSAF device to be scaled up to a maximum rotor diameter 2.5 times larger than is permissible in the original device, thereby providing more than a sixfold increase in volumetric throughput.     

Required ultracentrifugal time for condensing solution by ultracentrifuge

Required ultracentrifugal time for condensing a solution by ultracentrifuge with an angle rotor or a swing rotor was estimated by considering the calculated distribution of concentration in an ultracentrifugal tube. From the calculated distribution of the concentration a free boundary between solvent and solution was found in the ultracentrifugal tube under the condition that t^*>0.25. A condense constant was newly defined as the ratio of the mean concentration in the ultracentrifugal tube after removing the solvent to the concentration of initial solution. The equation determining the required ultracentrifugal time for condensing a solution up to a given condense constant is derived and solved numerically. The required ultracentrifugal time for an angle rotor was shorter than that for a swing rotor.     

Purification of filamentous plant viruses by thin layer centrifugation.

The construction of a modified thin layer ultracentrifuge rotor is described. This rotor was used in the purification of five filamentous plant viruses, viz. TMV, SCMV, PVX, SGV and YMC. The purification and concentration of these viruses in their monomeric forms is hazardous when conventional "tube" rotors are used since they invariably result in dissociation and aggregation of the virus particles. Using the thin layer rotor these infective agents may be concentrated in volumes of fluid equal to approximately 1% of the starting suspension and not as pellets obtained after ultracentrifugation is conventional "tube" rotors. Electron microscopy revealed that the virus particles concentrated by thin layer centrifugation were not aggregated and that only few fragments of the virus filaments were present in the final preparations.     

5. An Insert for the Reorienting Gradient Rotor for Virus Extraction from Infected Plants

ABSTRACT

A device made of nylon and which is inserted in the bowl of the reorienting gradient rotor is described. This insert acts as a container for holding infected plant tissue for the purpose of separating virus by centrifugal force from intact and fresh plants, from frozen and thawed plants and from plant tissue disintegrated by mechanical means. The extracted fluid represents 80 to 90% of that present in the untreated plant tissue. Electron micrographs taken of concentrates of the viruses prepared by the centrifugation extraction procedure indicate that extraneous materials are reduced to a low level.     

Transport Phenomena in Zonal Centrifuge Rotors: I. Velocity and Shear-Stress Distributions of Fluid During Acceleration

An analysis is presented for the evaluation of velocity components and shear-stress distributions of fluid in zonal centrifuge rotors during acceleration. Analytical expressions for the distribution of tangential and radial velocity components and the tangential shear-stress and the radial shear-stress distributions of fluid are obtained for the transient case. Characteristics of each distribution for a typical density gradient liquid in a zonal centrifuge rotor are computed from the relations derived, and are presented as figures. An unusual phenomenon—the tangential velocity of the gradient exceeding the velocity of the rotor during a particular period of acceleration—is demonstrated.     

Isolation of intact spinach chloroplasts in the CF-6 continuous-flow zonal rotor; implications for membrane-bound organelles

Intact chloroplasts were separated from large volumes of spinach brei by continuous-flow zonal centrifugation in the CF-6 rotor in gradients of colloidal silica. Tests with the Z-15/Mark 10 rotor showed that the external membranes of chloroplasts were sensitive to high rotor speeds and especially to the bore of the fluid seal. Enlargement of the CF-6 seal channel diameter to 4 mm resulted in almost complete recovery of the intact chloroplasts.     

Optimal design of non-Newtonian, micro-scale viscous pumps for biomedical devices

The present paper addresses the numerical optimization of geometrical parameters of non-Newtonian micro-scale viscous pumps for biomedical devices. The objective is to maximize the mass flow rate per unit of shaft power consumed by the rotor when an external pressure load is applied along the channel that houses the rotor. Two geometric parameters are considered in the optimization process: (i) the height of the channel that houses the rotor (H) and (ii), the eccentricity (epsilon) of the rotor. Three different micro-scale viscous pump configurations were tested: a straight-housed pump (I-shaped housing) and two curved housed pumps (L- and U-shaped housings). The stress-strain constitutive law is modeled by a power-law relation. The results show that the geometric optimization of micro-scale viscous pumps is critical since the mass flow rate propelled by the rotor is highly dependent on epsilon and H. Numerical simulations indicate that mass flow rate is maximized when epsilon approximately 0, namely when the rotor is placed at a distance of 0.05 radii from the lower wall. The results also show that micro-scale viscous pumps with curved housing provide higher mass flow rate per unit of shaft power consumed when compared with straight-housed pumps. The results are presented in terms optimized dimensions of all three configurations (i.e., H(opt) and epsilon(opt)) and for values of the power-law index varying between 0.5 (shear thinning fluids) and 1.5 (shear-thickening fluids).     

Experiments and theoretical calculations for forming gradients for zonal rotor centrifugation

A gradient mixer operating with a compressed inert gas instead of a pump is designed particularly for work with zonal rotors. Different exponential and linear profiles are calculated as they form along the rotor radius. The centrifugation time dependent on gradient shape and temperature is estimated.