Properties of various rotors used for zone centrifugation

The following results have been obtained from a quantitative study of zone centrifugation: (1) it is shown that the sedimentation velocity of all kinds of macromolecules is constant in 5–20% constant sucrose gradients, whatever swinging bucket or zonal rotor is being used, and at any usual temperature. (2) The proportionality constants between time of centrifugation and sedimented distance have been calculated for several rotors. They allow an estimate of relative centrifugation times. (3) An equation of the resolving power of zone centrifugation in isokinetic density gradients is used to compare the resolution of various rotors. (4) An equation of Vinograd and Bruner and Spragg and Rankin is discussed and used for the calculation of the maximum macromolecular load of the rotors. A summary of these results is presented in a table, which should help in the choice of the rotor best suited for a particular experiment.     

Rapid density gradient centrifugation using Short Column Techniques

The Short Column Technique is a useful technique for rate zonal and isopycnic density gradient runs with both the swinging bucket and fixed angle rotors, We have described and illustrated its advantages of significant time saving and the elimination of the need for tube caps. Thick-walled polycarbonate tubes are used. While some loss in resolution may result with some samples using short columns. It is our opinlon that the technique can be widely adopted to provide useful information.     

Separation of biological macromolecules in vertical rotors using sucrose gradients shaped by a microprocessor-controlled gradient former

The separation of macromolecules in vertical rotors using linear or isokinetic sucrose gradients permits shorter centrifugation times and a higher sample capacity compared to swing-out rotors. In vertical rotors appropriate gradients give a resolution almost identical to that in swinging bucket rotors. This could be demonstrated for the isolation of polysomes and oligonucleosomes from rat liver. A microprocessor-controlled gradient former is presented which produces gradients of any desired shape. This device has been applied to prepare gradients with the desired linear or isokinetic shape after reorientation in the vertical tube during centrifugation.     

Multiple alternate channel selection (MACS): new versatility for zonal centrifuge rotors

We describe a system of Multiple Alternate Channel Selection (MACS) for zonal rotors in which conical adapters, which attach to the dynamic seal, connect at different vertical heights to any pair of channels in the core. In this way one can pump fluids to and from different positions or compartments in the same rotor during a single run.The specific applications described in this paper include edge-unloading of sensitive particles through the center channel of the dynamic seal and direct insertion of samples at a predetermined radius. Other applications are outlined.     

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.     

A simplified high-resolution gradient analysis system

A simplified high-performance, semiautomatic apparatus for the analysis and fractionation of density gradient preparations is described. In this system the gradient, mounted directly under a standard recording spectrophotometer, is displaced upwards through a vertical straight-path flow cell, then downward to a fraction collector.The resolution attainable with this apparatus is greater than that required for the high fidelity analysis of small volume centrifuged density gradients. The system is easily adaptable to cesium gradients, zonal rotors, and to chromatographic columns where its high-resolution, bubble-free performance makes practical the analysis of both small and large volumes. It is significantly simpler and less costly to construct than other apparatus of comparable performance.     

Purification of Large Quantities of Influenza Virus by Density Gradient Centrifugation

New zonal centrifuges can conveniently process as much as five orders of magnitude (10(5)) greater sample volumes than conventional swinging-bucket rotors. The continuous-sample-flow-with-banding versions may be used in series with ancillary purification procedures. Here we have studied the combined process: absorption and elution of influenza virus with barium sulfate followed by concentration and isopycnic banding of the virus in a buffered sucrose gradient. Kilogram quantities of impurity have been rapidly separated from grams of purified virus, which have been conveniently concentrated several hundred-fold by the purification process. Experimental vaccines made by these procedures are being evaluated.     

Concentration and purification of two small RNA viruses: mycophage PS-1 and bacteriophage phi6

Two small RNA viruses, mycophage PS-1, and bacteriophage φ6 were concentrated and purified by the sequential use of continuous flow zonal centrifugation, high speed continuous flow sedimentation, and combined rate zonal centrifugation and isopycnic banding. The continuous flow centrifugations were done using the K-II and K-XI rotors while the BXXIX rotor was used for batch rate zonal centrifugation and isopycnic banding. The deseribed procedures are useful for the concentration and purification of virus from large volumes of culture fluid.     

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.     

Cryogenic solid state NMR studies of fibrils of the Alzheimer’s disease amyloid-β peptide: perspectives for DNP

Dynamic Nuclear Polarization solid-state NMR holds the potential to enable a dramatic increase in sensitivity by exploiting the large magnetic moment of the electron. However, applications to biological solids are hampered in uniformly isotopically enriched biomacromolecules due to line broadening which yields a limited spectral resolution at cryogenic temperatures. We show here that high magnetic fields allow to overcome the broadening of resonance lines often experienced at liquid nitrogen temperatures. For a fibril sample of the Alzheimer’s disease β-amyloid peptide, we find similar line widths at low temperature and at room temperature. The presented results open new perspectives for structural investigations in the solid-state.     

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.     

The isolation of lipoproteins from human plasma by ultracentrifugation in zonal rotors

The major classes of lipoproteins were isolated from human plasma by ultracentrifugation in continuous density gradients using the Ti-14 and Ti-15 zonal rotors. Chylomicrons + VLDL, LDL, and HDL were separated from each other and from the more dense residual proteins (albumin fraction) of plasma by rate-zonal flotation in NaBr gradients in the density range 1.0-1.4. The chylomicron-VLDL fraction was subfractionated into constituent chylomicrons and VLDL by zonal ultracentrifugation in NaBr gradients in the density range 1.0-1.1. Plasma lipoproteins were analyzed for composition of lipids and content of protein, for electrophoretic mobility on paper, and for antigenic determinants by immunoelectrophoresis and immunodiffusion. Flotation constants (S(f)) of the LDL and HDL were calculated from measurements made in the analytical ultracentrifuge. Lipoproteins isolated from plasma by zonal ultracentrifugation were identical by these criteria to lipoproteins isolated by the usual procedure of sequential ultracentrifugation in solvents of increasing density. The procedure of zonal ultracentrifugation is rapid, quantitative, and less laborious than sequential techniques. Lipoproteins isolated by zonal ultracentrifugation are relatively uncontaminated by other proteins and extensive washing is therefore unnecessary. Zonal ultracentrifugation is more than a preparative method for the plasma lipoproteins; it is also an analytical procedure in that a record is obtained of the distribution and quantity of the lipoprotein within the continuous density gradient.     

Evaluation of centrifugation parameters for density gradient experiments by means of a programmable pocket calculator

A calculation program is proposed suitable for programmable pocket calculators (e.g. HP series) to estimate s_20,w ∫ ω² dt values from density gradient centrifugation data. The program can be applied to linear or exponential density gradients prepared from sucrose or glycerol solutions spun in zonal rotors or swinging bucket rotors. A wide solute concentration range and temperature range is accounted for. Constants for empirical density calculation of glycerol and sucrose solutions concentrated in % (w/v) are estimated. Experiment verification of the program was carried out.     

DNA accessibility: a determinant of mammalian cell differentiation?

Rats bearing intracerebral 9L tumors were whole-brain irradiated with 1250 to 5000 rad, and the in situ DNA repair kinetics of the undifferentiated tumor cells and terminally differentiated cerebellar neurons were examined by alkaline sucrose gradient sedimentation in zonal rotors with gradient reorienting capability. Biphasic repair kinetics were observed for both tumor cells and cerebellar neurons. Quantitation and analysis of the slow phase of the repair process suggest that the dividing tumor cell genome is completely accessible to the enzymatic repair machinery, while it is possible that the genome of the permanently nondividing neuron may contain a region that is inaccessible to this repair machinery.     

Use of Zonal Ultracentrifuge Systems for Biophysical Studies of Rhinoviruses

This paper reports the use of zonal ultracentrifuge techniques to conduct biophysical studies of rhinoviruses grown with WI-38 cells. Good clean-out of infectivity from rhinovirus harvests was obtained with the continuous-flow B-V and B-IX rotors. Use of the B-V rotor resulted in the successful concentration of rhinovirus infectivity and antigenicity. Additional purification was achieved by the combined use of continuous-flow centrifugation and isopycnic banding procedures. Two particle sizes were found to be associated with the virus-infected cell harvests. The infectious 22-nm particle banded in density ranges of 1.38 to 1.40 g/cm(3) in CsCl and 1.26 to 1.27 g/cm(3) in potassium citrate. The 8.0 nm capsomere was composed of 2.0 nm subunits and banded with a density of protein at 1.28 g/cm(3) in CsCl. Equivalent sedimentation coefficients of 155 or 185, depending on particle density in sucrose, were calculated from rate zonal experiments by use of the B-IV zonal rotor.     

Detection of liposome membrane viscosity perturbations with ratiometric molecular rotors

Molecular rotors are a form of fluorescent intramolecular charge-transfer complexes that can undergo intramolecular twisting motion upon photoexcitation. Twisted-state formation leads to non-radiative relaxation that competes with fluorescence emission. In bulk solutions, these molecules exhibit a viscosity-dependent quantum yield. On the molecular scale, the fluorescence emission is a function of the local free volume, which in turn is related to the local micro-viscosity. Membrane viscosity, and the inverse; fluidity, are characteristic terms used to describe the ease of movement withing the membrane. Often, changes in membrane viscosity govern intracellular processes and are indicative of a disease state. Molecular rotors have been used to investigate viscosity changes in liposomes and cells, but accuracy is affected by local concentration gradients and sample optical properties. We have developed self-calibrating ratiometric molecular rotors to overcome this challenge and integrated the new molecules into a DLPC liposome model exposed to the membrane-fluidizing agent propanol. We show that the ratiometric emission intensity linearly decreases with the propanol exposure and that the ratiometric intensity is widely independent of the total liposome concentration. Conversely, dye concentration inside liposomes influences the sensitivity of the system. We suggest that the new self-calibrating dyes can be used for real-time viscosity sensing in liposome systems with the advantages of lifetime measurements, but with low-cost steady-state instrumentation.     

Separation of hepatocytes of different acinar zones by flow cytometry

Hepatocytes in the proximal (zone 1) and distal (zone 3) regions of the liver acinus are selectively stained by perfusion of the isolated rat liver with 0.2-20 microM acridine orange (AO). After 10-60 min of anterograde perfusion, AO fluorescence is visible in zone 1 cells, whereas retrograde perfusion stains cells of zone 3. In this paper, we describe a technique to isolate a mixed population of fluorescent and nonfluorescent hepatocytes (cells from all acinar zones, which do not loose the zone specific AO labeling) and to separate these cells according to their zonal origin by fluorescence activated cell sorting. The zonal populations obtained were either fluorescent or nonfluorescent (purity greater than 95%). Separated cell fractions differed in their enzyme content (5' nucleotidase, succinate-dehydrogenase, beta-glucuronidase). An unidentified AO metabolite, which is not found in bile after retrograde perfusion (not formed in zone 3 cells), is also absent after retrograde perfusion in sorted fluorescent cells (zone 3 cells), indicating zonal purity of sorted cells.     

Zonal organization of the mammalian main and accessory olfactory systems

Zonal organization is one of the characteristic features observed in both main and accessory olfactory systems. In the main olfactory system, most of the odorant receptors are classified into four groups according to their zonal expression patterns in the olfactory epithelium. Each group of odorant receptors is expressed by sensory neurons distributed within one of four circumscribed zones. Olfactory sensory neurons in a given zone of the epithelium project their axons to the glomeruli in a corresponding zone of the main olfactory bulb. Glomeruli in the same zone tend to represent similar odorant receptors having similar tuning specificity to odorants. Vomeronasal receptors (or pheromone receptors) are classified into two groups in the accessory olfactory system. Each group of receptors is expressed by vomeronasal sensory neurons in either the apical or basal zone of the vomeronasal epithelium. Sensory neurons in the apical zone project their axons to the rostral zone of the accessory olfactory bulb and form synaptic connections with mitral tufted cells belonging to the rostral zone. Signals originated from basal zone sensory neurons are sent to mitral tufted cells in the caudal zone of the accessory olfactory bulb. We discuss functional implications of the zonal organization in both main and accessory olfactory systems.     

Isolated metaphase chromosomes. II. Proteins of Chinese hamster chromosomes.

The proteins on metaphase chromosomes theoretically may be distributed ubiquitously throughout the karyotype, may be present uniquely on individual chromosomes or classes of chromosomes, or may exist in any combination of the above. Separation of chromosomes according to size using sucrose velocity gradients in high capacity zonal centrifuge rotors allows sufficient fractionation of the genome to indicate the distribution of proteins within the karyotype. Flow cytometric analysis and direct microscopic analysis were used to evaluate qualitatively the types of chromosomes present in the fractions obtained. This report is the first quantitative evidence that some of the chromosomal proteins are not distributed ubiquitously on all of the chromosomes of the karyotype.     

Separation of plant cell organelles by zonal centrifugation in reorienting density gradients

Preparative separations of plant cell organelles (glyoxysomes, proplastids, mitochondria, and endoplasmic reticulum) in an ordinary refrigerated centrifuge were obtained by sucrose density gradient centrifugation in a zonal rotor loaded and unloaded in the static manner. The quality of the separation which was monitored by marker enzymes and electron microscopy compares to analytical separations in swinging-bucket rotors. Membrane alterations observed in glyoxysomes and mitochondria are traced back to sucrose as a major component of the homogenization and density gradient medium.