INSTR UMENTATI0 N
Advisory Panel Jonathan W. Amy Glenn L. Booman Robert L. Bowman
Jack W. Frazer Howard V. Malmstadt William F. Ulrich
Ultracentrifuge Some Analytical and Preparative Uses in Biomedical Research Ronald J. Casciato Director, Research Applications, International Equipment Co., A Division of Damon, Needham Heights, Mass.
The ultracentrifuge is an increasingly important instrument in biomedical research. Separations of cellular and subcellular species are exceedingly important to a better understanding of human disease mechanisms
ODAY’S
SCIENTIFIC
RESEARCH
com-
Tmunity is experiencing a revolution within its ranks. Fewer than 20 years
ago not only were basic research and medical research widely separated regarding the level of activity and direction, but within each group, subdivisions of specific subgroups were well defined. Research was carried on almost in competition with other disciplines in the same major group. As the level of technology and research activit y increased, it became clear that this division was not always in the best interest of research, and growing interrelation between groups has evolved. Dr. N. G. Anderson, chairman of the MAN program a t the Oak Ridge National Laboratories, has aptly stated, “Each science, as it matures is concerned with defining those basic units which in turn are definable by the next subjacent discipline.” The effect is seen in many of today’s important scientific achievements, both technological and theoretical. To make the system work effectively, each discipline must work toward diminishing its boundaries to achieve a more complete understanding of today’s research problems. This interdisciplinary approach has provided many new and exciting approaches to old problems. Separation Necessary in Cell Research
It is now generally accepted that the majority of human diseases are ultimately to be understood at the molecular level, and it must be concluded that
Editor‘s N o t e : T h i s article is primarily intended as background information for a comprehensive feature article on clinical analysis systems t o appear in AXALYTICAL CHEMISTRY early in 1970.
separation and cataloging the molecular constituents of human cells are necessary groundwork for attempts to describe human pathological states in molecular terms. It is clearly this attempt t o separate, purify, isolate, and ultimately characterize the molecular constituents of a cell that occupies much of the research attention today. Separations are made by utilizing one or more of the characteristic properties of the specific entity, whether it is an organelle, such as a mitochondrion, or soluble serum protein. Some useful properties include surface potential, ionic charge, membrane permeability, enzymatic specificity, solubility and/ or insolubility, color, absorptiveness in light, morphological appearance, size, shape, density, and sedimentation coefficients. It is these latter properties that almost totally govern the effectiveness of the ultracentrifuge in biomedicine. Many cellular components, including macromolecules, exhibit characteristic size, shape, and density phenomena, I n combination, these parameters coupled with a specific technique of sedimentation result in a “sedimentation-coefficient” which is in itself, characteristic of the component. By
utilizing centrifuge technology along with sedimentation coefficients, a separation or fractionation of a mixture of cellular components may be effected. Pertinent to this discussion is the development of some principles of centrifugal force and technology of design, as well as discussion of the application to biomedical research problems. Centrifugal force, simply stated, is an enhancement of gravitational force. The concept of using gravitational force for separation of particles is easily envisioned when one considers a cylindrical glass tube of water into which is dropped a mixture of sand and gravel. As the mixture sediments, the gravel stones are only slightly retarded in the water and rapidly drop to the bottom. The sand, however, settles more slowly and after a short time a visible separation is evident with the sand layered on top of the gravel and the liquid above is again clear. If the process had been stopped after the gravel had reached bottom and before the sand had fully sedimented, a separation would have been accomplished. This separation is the total result of various forces (including gravity) on the size, shape, and density of the suspended material. This example is directly related to the separation of cellular particles or organelles and components, where the differences are magnitudes of the size, shape, and density of the parameters; in cellular particles they are thousands of times smaller than those of the example above. The ability t o separate cellular entities depends on the ability
ANALYTICAL CHEMISTRY, VOL. 41, NO. 13, NOVEMBER 1969
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to increase the effective force of separation, in this case gravity. The most desirable means from an engineering point of view, is to increase the force by rotation. Not only are space requirements lessened, but resulting instrumentation facilitates easy sample handling. By rotating a cylinder about an axis a t one end it is evident that the force generated a t the opposite end is increased. By increasing the force, the time of separation can be lessened and/ or the sizes of the particles to be separated may be much smaller. If increased g force is to be achieved, thus permitting the separation of cellular components, certain design requirements must be satisfied. By briefly considering existing commercially available equipment, it becomes evident that the ultimate ultracentrifuge is a very complex interrelated laboratory unit. -4variable-speed drive system is required. Currently available units have speed ranges to 60,000 rpm (approximately six times that of a jet aircraft turbine engine) and equal to 405,000 X g. To maintain the physiologicaI state of the samples, refrigeration systems must be considered; sample temperatures should be held constant at a range from 0-20 O C . Rotation a t controlled high rpm alone is an arduous task. Add to this the load forces of a rotor and sample material, and the task becomes impossible. I t is therefore necessary to construct the instrumentation so that the rotation systems operate in a vacuum, thus eliminating the windage forces acting upon the rotor. High vacuum is also essential for ultimate temperature control. Current vacuum levels approach 0 . 1 ~ . This provides essentially a cabinet housing a vacuum system, refrigeration, controls, and drive system, all operative to a chamber into which the rotor and samples will be placed. Rotor Types
For detailed literature and technical assistance, write:
The samples themselves are self-contained in a variety of “rotors” in either tubes or bottles in which the actual sedimentation takes place. The design of centrifuge rotors is very complex. Rotors have historically taken only a few configurations: fixed angle, horizontal, or swinging bucket, and more recently, zonal. The fixed angle rotor is one in which the cavity that holds the tube is set a t an angle to the central axis (the angle is determined by the types of pelleting material desired). It is useful for pel-
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ANALYTICAL CHEMISTRY, VOL. 41, NO. 13, NOVEMBER 1969
leting materials a t all speeds, and if the sample suspension is homogeneous, a concentration of material is obtained by decanting the supernatant. I n contrast, the horizontal or swinging bucket rotor is one in which the centrifuge tube hangs in a vertical position a t rest, and during acceleration the tube swings out to a horizontal position. In this rotor the material is rarely pelleted but separations are based on liquid density gradients. The zonal rotor is basically a largevolume cylindrical pressure vessel rotating on an axis. The design of zonal rotors permits larger volumes to be used at existing speeds, using materials such as titanium and aluminum. I n all cases it is important that the design of the rotating system be sufficient t o withstand the repeated stress of cycling a rotor t o high rpm’s and g levels. I n practice, the ultracentrifuge is utilized for pelleting or separation of subcellular components or particulate material. I n fixed-angle rotors the operation is straightforward-homogeneous suspensions of sample material, whole tissue homogenates, or cellular components are contained within a sealed tube at a fixed angle to the rotating axis. The fluid within the tube is affected by convective forces that set up a swirling motion from the top t o bottom of the tube. As the heavier material in the suspension nears the tip of the tube, increasing g force causes pelleting as the suspension fluid is centrifuged for longer periods of time; smaller fragments are subsequently pelleted as a result of high g force for an extended period of time. This process removes suspended particulate material from a homogeneous solution. It is, however, of little value in the separation of subgroups within the sample, since pelleting causes overlapping of the material. Types of Centrifugation
Sample fractionation may be accomplished by the technique of differential centrifugation. I n this method a given sample suspension is spun in a horizontal tube, and since the particles in the suspension are distributed uniformly, the material at the bottom of the tube experiences the highest g force and sediments (pellets) rapidly. This initial pellet captures with it some smaller material and soluble nonsedimenting material. Continuing centrifugation causes smaller particles t o layer over the initial material. The process is continued, but contamination of each layer with its subsequent layer is approximately proportional t o the differences in sedimentation rates. The net result is that only the smallest particles may be obtained in a pure state in a single centrifuge run. Since many cel-
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INSTRUMENTATION
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lular components have a rather wide range of sedimentation rates, it has been, in many cases, sufficient to use differential centrifugation for preparation. This can easily be seen when whole cells, organelles, and soluble proteins are centrifuged in the same run. It is increasingly difficult, if not impossible, to satisfactorily use this method for the subsequent separation of particles with a narrow sedimentation-rate range. Particle-sizing studies are also cumbersome using this method. It is difficult, from an experimental point of view, to consider successive stages of differential centrifugation, such as a resuspending and recentrifuging, until additional separations are achieved. An excellent method for sophisticated separations using liquid density gradients is, however, in widespread use. Liquid-density centrifugation mas initially employed t o provide a means for selectively separating families of particles during a single run. The liquid density gradient is simply made by placing a lighter liquid over a heavier liquid so that two layers are formed. Diffusion at the interface gradually lessens the sharpness of the boundary between layers and after some time a more less-continuous transition in density from the top to bottom can be seen. I n this gradient it is possible to effect separations based on either size and/or density of the suspended particles, The density of the gradient is centrifugally stable, and the sample is layered on top of the gradient. During centrifugation, material is separated into bands of material spread proportional to the sedimentation rates. Two types of separations are poasible. Isopycnic, or equilibrium centrifugation is based on the principle that particles sediment to their own density in a liquid density gradient whose density range encompasses extremes within which the density of the particles lie. As the centrifugal force acts upon the particles, their immediate location may be either above or below their specific gravity. They mill be forced in a n axial direction until the density of the surrounding fluid matches the specific gravity of the particles. Rate-zonal centrifugation uses the relative size of the particles as well as density t o effect separations. If a gradient is constructed over a narrow density range, a shallow gradient, and several size particles are centrifuged, the resulting separation will be based on the rate a t which each particle travels against the viscous forces of the medium. This type of separation is usually more efficient. Both of these methods offer ad102A
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ANALYTICAL CHEMISTRY, VOL
Non-aqueous Atomic Absorption Three sound reasons why non-aqueous atomic absorption techniques make sense even if you’re dealing with aqueous samples: 1. Enhanced sensitivity (up to a hundredfold). 2. Simplified sample preparation, since sample digestion is eliminated. 3. Elimination of interfering substances which necessitate elaborate reagent blanks. . . . and a lot more reasons why Eastman Organic Chemicals should be your source for non-aqueous A.A. reagent supplies.
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Tantages for increased separation capabilit,y. This “resolution” is the narrowest distance t’liat separates two different particles in a specific system. Since resolving power is oftentimes the desired result of any cellular separation, rate-zonal centrifugation has become the method of choice for research use. This is due mainly to the use of a n “isokinetic gradient” developed by Dr. Hans Xoll ( 1 ) . This gradient is shaped to compensate for the increasing velocity of sedimentation as the particle enters a n increasing g field during t,he run. The classic example of this technique is seen in the experimental separation of ribosomal aggregates, called polysomes. Polysomes are groups of ribosomal particles connected by a strand of messenger RSA. These aggregates are seen within the cytoplasm of cells and under certain conditions may be isolated intact. Since the number of attached ribosomes is a measure of the position of the messenger RiSA in the system of protein synthesis, it would be helpful to achieve separations whose length showed sequential variation, By use of 14.5 X 96-mm thin-walled tubes in a swinging bucket rotor rotating a t 41,000 rpm (283,000 g) , an analysis of consecutive polysome bands shows separations of up to 10 ribosomal subunits, demonstrating that the instrument is capable of resolving two macromolecular objects of equal densit y and similar shape whose mass differs by as little as 10%. Techniques are currently available to increase even this measure of resolution. There are, however, serious limitations to either of these methods of separation. They stem more from the physical conditions imposed on the system than from limitations in the application of the theory. For example, any zone centrifugation was originally performed in a tube inserted into the rotor. Since sedimentation occurs radially from the axis of rotation, it is clearly seen that t’he tubes provide an impedance to the centrifugal path of all particles with the sample. The area encompassed by a group of particles a t the top of a tube increases directly with a n increase in radius of the tube. It follows directly then, that’ if the tip of a centrifuge tube is twice the radius of the meniscus of the fluid a t the top of a tube, approximately 25% of the particles will impact the wall and give rise to incomplete separation. Also, construction of a static density gradient, on top of which a sample of material is to be placed, has proved to be a cumbersome and a n experiment,ally limiting condition. Berman ( 2 ) shows that t,he t,heoretical capacity-mass of the sa’mple to be separated on swinging bucket gradients-is never approached even when optimum conditions are
ANALYTICAL CHEMISTRY, VOL. 41,NO. 13, NOVEMBER 1969
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INSTRUMENTATION
AN ADVANCED OSMOMETER IS FIT TO BE TRIED
maintained. This reaction attributed to the uneasy stability of the gradient and excessive manipulation in handling and sample loading. Added to this difficulty is the additional handling of the gradients in the recovery phase of the run. Another problem is the decrease in usable sample volumes in rotors when higher rpm and g force are required. At high rpm’s the volumes usable for separations become increasingly small. This seriously limits the amount of material that can be used in any separation requiring high speed and g forces. It is not difficult to see why this technique has been used more in analytical than in preparative-type systems. Zonal Rotor System
We’ll prove it. Advanced Osmometers are the most reliable instruments you can buy to determine the osmolality of solutions w i t h greatest accuracy. Eight models are available - each designed for varying laboratory requirements Read about them. Then try one in your o w n laboratory and see for yourself. Send the coupon today for a free brochure and/or a trial.
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In 1964, however, a major breakthrough in the field of centrifugal technology was presented by Dr. K . G. Anderson at Oak Ridge. This was the development of the zonal system of rotors for use in ultracentrifugation. The invention of a zonal rotor system and subsequent commercial production by ultracentrifuge manufacturers has provided a state-of-the-art approach to the solution of the inherent problems with earlier gradient systems. As discussed previously, the zonal rotor is a cylindrical pressure vessel-the most recent design can be seen in Figure l. Nore than 7 5 rotor designs have been considered. This bowl-shaped rotor rotates about is central axis. Inside the rotor are compartments interconnected through fluid channels in the vanesepta. This design eliminates several of the aforementioned problems (such as wall effects). The septa form essenA D I V . OF DAMON
~ m m m m ~ Mail to: Advanced instruments, Inc., 44 Kenneth Street, Newton Highlands, Massachusetts, 02161
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Please send the Free Osmometer brochure. 0 Please arrange an Osmometer demonstration in my laboratory.
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IYAIVIC
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POSITION ORGANIZATION RES:::
STATE
Figure 1. A commercial rotor constructed of titanium. Edge unloading is facilitated through the channels at the rim of the septa. The tapered wall of the bowl facilitates better resolution
Circle NO. 11 on Readers’ Service Card
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ANALYTICAL CHEMISTRY, VOL. 41, NO. 13, NOVEMBER 1969
tially sector-shaped compartments which do not allow a large percentage of the sedimenting particles to impact the wall. Rotor capacity is increased a t operating speeds many times that of existing rotor volumes. Gradient materials may be introduced into the rotor while it is spinning a t low speed through fluid channels and an effluent feed seal system, causing the gradient to be less sensitive to manipulative operations since the gravitational field stabilizes the fluid. Increased gradient capacity is also facilitated by this system. Since fluids may be interchanged without stopping the rotor, the recovery phase becomes simply a pumping operation with a flow monitor and a collection device. -4n added advantage of this system of rotors is the flexibility in gradient shape us. rotor volume. Preliminary experimentls by Spragg e t al. (S) indicate that the zonal rotor may be even more quantitative as an analytical tool than other systems. I n operation (Figure 2 ) , the seal is attached to the rotor a t the beginning of the run and the rotor is accelerated to a loading speed of approximately 2000 rpm. The gradient, sample, and overlay are then pumped into the rotor through the feed and effluent seal, the seal is removed, and the rotor is capped and accelerated to its maximum operational speed. After the desired separation has been accomplished, the rotor is decelerated to its unload speed, approximately 2000 rpm. The cap is removed, and the effluent and feed seal are reattached. Heavy gradient material is then pumped to the periphery of the rotor to displace the fractionated contents to the core portion of the rotor, and through the feed and effluent seal for fraction collection and further analyses. For many specific applications, the commercial availability of zonal rotors has provided a means for the researcher to obtain large quantities of the separation need while a t the same time improving purity. Three basic types of zonal rotors are shown in Figure 3. The “A” series, low speed rotors (up to 4600 rpm) are designed for the isolation and purification of large subcellular organelles such as nuclei mitochondria, and lysosomes. The intermediate speed rotor, Z-series, allows centrifugation up to 8000 rpm and purification of subcellular components such as chloroplasts, mitochondria, and large viruses such as tobacco mosaic virus. Both the il series and Z series rotors are constructed of aluminum and plexiglass allowing visual observation and measurement of band migration during operation. The “B” series are high speed rotors -up to 50,000 rpm-that allow isolation of subcellular particles down to the
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ANALYTICAL CHEMISTRY, VOL. 41, NO. 13, NOVEMBER 1969
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ribosomal, ribosomal subunit, and macromolecule size. Particle studies with sedimentation coefficients to 10s such as large proteins, bacterial RNA, serum macroglobulin, and smaller viruses, such as polio, are ideally suited for use in these rotors. The B series rotors are constructed of titanium and afford the maximum versatility needed to perform any zonal run. The B-29 and B-30 incorporate the
latest design innovations of the Oak Ridge National Laboratories. These rotors allow the unloading sequence t o proceed through the rim line instead of the core line facilitating separations where path length is too short in standard rotors. I n practice, the “edgeloading” has the advantage of allowing removal of faster sedimenting particles while lighter and slower particles are still sedimenting. This, in effect, increases the available path for sedimentation to infinite dimensions. Rate separations based on flotation of the sample from the dense end of the gradient are now possible also. As a result of this design, exotic gradient materials,
B
Figure 3. Several different types of commercial zonal rotors
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ANALYTICAL CHEMISTRY, VOL. 41, NO. 13, NOVEMBER 1969
N( 5 , ANDERSON
Fractionation by zonal ultracentrifugation
often prohibitively expensive, do not need a piston or cushion fluid of greater density to unload the rotor, since either air pressure or water supplied t o the core is satisfactory. Zonal systems offer biomedical research a new outlook for the isolation and study of cellular and subcellular systems. The ultracentrifuge is a sophisticated research instrument whose capabilities are ever increasing t o meet the demands of research. If a complete un-
by Ralph
R.
derstanding of human disease mechanisms and their treatment is to be achieved and the level of disease mechanisms is ultimately at the macromolecular level, then separation of cellular and subcellular species is of the utmost importance. References
tional Cancer Institute Monograph 21, p 41, 1966. (3) S. P. Spragg, R. S. Harrod, and C. T. Rankin, Jr., “The Optimization of Density Gradients for Zonal Centrifugation (19691,” in press. Suggested Reading
(1) Hans Xoll, Nature, 215, 360, 1967. (2) A. S. Berman, “Theory of Centrifugation : Miscellaneous Studies,” Na-
National Cancer Inst,itute Monograph No. 21, 1966. The Development of the Zonal Centrifuge.
ings proved t o be unsatisfactory (loss of sphericity at high speed?). Rotors with definite ellipticity rather than exact cylindrical shape were capable of higher speed before fpacture occurred. More recent observations have shown a decrease in temperature of the rotor upon acceleration from rest to 60,000 rpm. This was attributed t o stretching of the rotor and a consequent adiabatic change, causing a temperature drop of about - 1 . O O C . This was contested by Hiatt ( 2 ) but completely confirmed by Biancheria and Kegeles ( 1 ) by melting point techniques and by Pickles ( 3 ) by direct measurement with a thermistor mounted in the rotor. I t has been shown that the temperature decrease is linear with the square of the rotor speed, and the cooling for acceleration t o 60,000 rpm is very close t o 0.8”C. -4ccording t o Schachman (4)much of the data in the literature must be modified because of neglect of this factor. Svedberg made extensive studies of frictional losses of all kinds. Windage losses were examined in detail. By operation in hydrogen a t reduced pressure, the loss was strictly proportional to the 3/2 power of hydrogen pressure. Today, operation in a good vacuum is preferred. Speed control and speed measurement have been a continuing problem. The earliest control method involved a differential gear arrangement in which one input shaft was driven by the main motor or turbine, the other input shaft by a synchronous motor of known speed. The output shaft of the differential gear drove a rheostat controlling the main motor. Rotational speed was measured stroboscopically. Today, the measurement and control of speed can be as precise and elegant as one can afford. For example, a single reflecting spot on the rotor can be scanned photoelectrically, yielding a n output pulse for each revolution. By
substituting an encoding disk for the single spot, several hundred pulses per revolution can be obtained. The pulses can be counted directly or after preliminary electronic pulse shaping. A scaler can indicate the total number of counts for a precisely defined time interval. I n the limit, an 8-decade scaler with crystal controlled counting of time (operation a t 100 MHz) can measure speed to 1 part in 108. This is quite feasible but not necessarily sensible economically. Speed control can be realized indirectly by other means. Thus, in the magnetically levitated rotor, designed by J. W. Beams, the rotor is brought u p to speed by an air turbine. After drive cutoff, the rotor is operating in a high vacuum and its decrease in speed can be as low as 0.3 rps per day! The researches of Jesse Beams a t the University of Virginia over a period of more than 30 years provide another classic in the field. Numerous key references are to be found in Ref. (4). The resources of physical optics have been adequate since the very beginning of ultracentrifuging techniques. Some of them, notably light absorption, have been vastly improved by modern methods of photoelectric photometry. The Jamin, Mach-Zender, and Rayleigh interferometers provide elegant means of following boundaries during ultracentrifugation. Since one can measure to within 004 of an interference fringe, with the sodium line equal t o 58936, this amounts to a distance of about 4 x 10-7 in. The original Lamm-scale method yielded excellent results but was exceedingly tedious and time consuming. The schlieren technique of T6pler and Thovert as modified by Philpot, Svensson, and Longsworth is widely used for viewing and analyzing refractive index gradients. Excellent examples of the patterns given by the various methods
H. Muller
CASCIATO’S discussion emphasizes
D the importance of large-scale zonal
ultracentrifugation permitting, as it does, the separation of cellular and subcellular species. I n his opinion, this is the ultimate role of the ultracentrifuge in biomedicine. I n a preparative sense, this might well be true, but so much information is obtained during the course of ultracentrifugation that we wonder i f biochemists would agree with the finality of this significant achievement. I n the 46 years of development since The Svedberg’s pioneer n-ork, enormous advances have been made. Fern methods or techniques in science borrow so heavily on related disciplines. The ultracentrifuge has never been a simple device. Even the mere task of spinning a relatively heavy rotor smoothly a t speeds approaching 100,000 rpm is an engineering feat of the first order. Even the earliest investigations of Svedberg could have been published as outstanding examples of engineering, applied optics, or physical chemistry, entirely aside from the main problem of ultracentrifugation. Most experts seem t o agree that the 1940 summary of the field by Svedberg and Pedersen (S)is much more than a historical review; it is a classic which embodies practices and concepts still accepted and only greatly improved upon with the passage of time. The earliest studies coincided with our graduate studies a t Columbia University and they were required rezding particularly since our research was concerned with the optical and electrical properties of colloidal dispersions of gold. -411 students were urged t o study these papers as models of skilled research, interpretation, and documentation. Detailed mechanical studies with the original oil turbine-driven centrifuge revealed interesting effects. Ball bear-
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