1984
NORMAN G. AXDERSON
Vol. 66
THE ZONAL ULTRACENTRIFUGE. A NEW INSTRUMENT FOR FRACTIONATIKG MIXTURES OF PARTTCLES BY KORMAX G. ANDERSON Biology Division, Oak Ridge Bational Laboratory, Oak Ridge, Tennessee Receaoed Marcli I, 1962
The zonal ultracentrifuge is a new biophysical tool which allows particles to be separated on the basis of sedimentation rate or particle density. The rotor is a hollow cylinder with a total capacity of 1625 ml. divided by vertical septa into sectorsha ed compartments. Two fluid-handling lines through the upper shaft connect the edge of the rotor and the central core wit! a flat rotating seal which presses against an external static seal. All operations, including introduction of the density gradient and the sample layer, and recovery of the gradient with its separated zones of particles, are carried out with the rotor spinning a t speeds up to 5000 r.p.m. in a refrigerated evacuated chamber. The gradient is stabilized at all times by the centrifugal field. Particle separation is accomplished a t speeds up to 30,000 r.p.m. (51,161 X 9 a t the maximum radius of 5.08 cm.). Subcellular components, including nuclei, cell membrane fragments, mitochondria, microsomes, ribosomes, and macroglobulin have been isolated using aqueous sucrose gradients. Tobacco mosaic virus and brome mosaic virus have been recovered in a high state of purity from crude extracts in one run. Types I, 11, and I11 polio virus have also been isolated in quantity from tissue culture fluid. A number of alternate rotor configurations now under consideration are presented together with data on the limits of rotor diameter attainable with aluminum and steel rotors.
A preparative ultracentrifuge capable of achieving zonal separations with a resolution approaching that of the analytical ultracentrifuge has been required for the isolation of subcellular particles and viruses, and for the separation of proteins and other macromolecules which differ in sedimentation rate or density. The development of such a zonal ultracentrifuge capable of separating particles down to the S20,w20 size range is described in this paper. It appears that zonal separations in centrifuges can only be made in the presence of density gradients. The term zonal ultracentrifuge has therefore been adopted here in preference to the term density gradient ultracentrifuge since the former term implies the presence of a density gradient. A preliminary report of this work has appeared.2 Density-gradient centrifugation using swingingbucket rotors has been extensively reviewed.3--6 The principles of zonal stability are largely derived from work on density gradient electrophoresis.6 Ideally, sedimentation should occur in sectorshaped tubes or compartments to avoid undesirable wall effects. Early low-speed studies in glass centrifuge tubes of modified sector shape7 demonstrated that excellent separations of liver cell components could be obtained; however, runs as long as 18 hr. were required, and only 2 ml. of sample could be separated per tube. To eliminate the long interval required for filling with the density gradient, a method was devised for loading the tubes while the centrifuge was running, using centrifugal force to prevent mixing.8 Recovery of the separated zones was still made with the tubes a t rest. Since most of the difficulties encountered centered around the use of tubes, the possibility of eliminat(1) Operated by Union Carbide Corporation for the United States Atomic Energy Commission. (2) N. G. .4nderson and C L Burger, Science, in press (1962). (3) (a) N. G. Anderson in “Physical Techniques in Biological Research,” Vol. 111, Gerald Oster a n d Arthur W. Pollister, Editors, Academic Press, Ino., New York, N. Y., 1956,p. 299; (b) Ch. de Duve, J. Berthet, and H. Beaiifay, PTOQT. Bzophys. and Bzophus. Chem., 9, 326 (1950). (4) J. F. Thomson, Anal. Chem., Si, 836 (1959). (5) M. K.Brakke. Adoances i n Virus Research, 7 , 193 (1960). (6) H.Svensson in “A Laboratory Manual of Analytical Methods in Protein Chemistry Including Polypeptides,” Vol. I, P. Alexander and R. J. Block, Editors, Pergamon Press Ltd., London, 1960,p. 195. (7) N. G. Anderson, Ezptl. Cell Res., 9,446 (1965). (8) J. F. Albright and N. 0. Anderson, zbzd., 18, 271 (1958).
ing them entirely was next considered. It appeared that a hollow rotor, with the internal volume divided by vertical septa into truly sector-shaped compartments, could be used if (1) the gradient and sample could be introduced, and (2) the separated zones were recovered while the rotor was spinning. A low-speed rotorg demonstrated the feasibility of this concept, and a medium-speed 625-ml. rotor operating in air showed that gradients could be introduced and recovered with very little change a t speeds up to 18,000 r,p.m. I t is essential in rotors of this type to ensure that tangential flow in the rotor is eliminated. Fluid flowing t o the rotor edge must be suitably channeled so that it has been accelerated to the tangential velocity obtaining a t the edge of the fluid chambers. Similarly, as the gradient is displaced in toward the rotor core during unloading, it is greatly decelerated. In the absence of vertical septa, the energy involved will be dissipated as heat in the process of swirling the rotor fluid. Therefore, compartmentalization of the rotor is essential. Design Considerations.-The configuration chosen (Fig. 1) for initial high speed studies mas dictated largely by the necessity of using existing components as far as possible. A Spinco Model L ultracentrifuge was modified by extending the vacuum chamber 9.9 cm. and adding a rotor temperature sensing and control system. A liquid nitrogen trap was inserted in the vacuum line, and the drain which normally returns oil from the rotor chamber to the vacuum pump was clamped off. The plate containing the overspeed safety pin was also modified to prevent liquids in the chamber from draining down to the drive shaft. Since the drive and support system was below the rotor, it was necessary to make fluid line connections a t the top, and to provide an upper bearing to center the rotor and provide a vacuum seal. The rotor chamber is 25.72 cm. long, with an inside diameter of 10.16 cm. and a wall thickness of 1.27 cm. The interval volume of 1625 ml. is divided by vertical septa into 36 identical sector-shaped compartments. The upper shaft is hollom? with an additional small center tube so that fluid may be pumped into and out of the rotor a t the same time. (9) N. G. Anderson, Bull. Am. Phys. Soc., I, Series 11,267 (1956).
Oct., 1962
1985
THEZONALVLTRACENTRIFUOE zu-l ROTOR
l
/-“PA-
/” P
60
25
30
I
3
,
35 40 45 ROTOR RADIUS (rnm) 5
7 9 VOL (100rnl).
11
50 1 3 1 5
Fig. 2.--Comparison of density gradient produced by gradient engine arid gradient recovered after 4 hr. a t 20,758 r.p.m.
0 9-
0.870
0.7-
60
$06-
-#s
50 -’
Fig. 1.-Cutaway drawing of zonal ultracentrifuge rotor. Note divisions of internal volume into sector-shaped compartments lby vertical septa, central collecting core, and coaxial lines leading to upper seal: 1, tubular rotor wall of 7075-T6 aluminum; 2, lower rotor cap; 3, upper rotor cap; 4,rotor cor(.; 5, septa; 6, fluid line crossover; 7 , upper rotor shaft; 8, center upper fluid line; 9, upper bearing; 10, coolant lines; 11, oil line; 12, rotor chamber top; 13, upper bearing housing; 14, rotating seal cup; 15, rotating seal; 16, upper static seal; 17, fluid line leading to rotor center; 18, fluid line leading to rotor edge; 19, Real pressurizing cylinder; 20, fluid line to rotor center running through pressurizing cylinder; 21, fluid line to rotor edge running through pressurizing cylinder; 22, fluid line opening a t rotor edge; 23, fluid line opening in central core.
The center line connects to the rotor wall through four horizjontal channels in the rotor cap. The outer line in the upper shaft connects to the central rotor core. The upper shaft ends in a flat Pickels seal made of Teflon filled with a metallic oxide. It presses against a flat static seal of aluminum coated with tungsten carbide. The upper seal and the upper bearings are both cooled with circulating ice water. Operation.-Irt operation the empty prechilled rotor is accelerated to 5000 r.p.m. and filled completely a t this speed. This is done by pumping a 1200-ml. density gradient to the rotor edge, light end of the gradient first. The gradients most used ranged from 17 to 55% sucrose (w./w.) for tissue fractionation and 12 to 30% sucrose for viral and ribosomal separations. After the gradient was in the rotor a “cushion” of concentrated sucrose (55
40
30
E
cn 3
20 IO
18
22
26 30 34 ?8 4 2 ROTOR RADIUS (rnrn).
46
Fig. 3.-Widening of Sam le zone in zonal ultracentrifuge. Sample introduced as g r a i e n t containing 10 ml. of 8% bovine serum albumin and 10 ml. of 17% sucrose (w./w.). Bar indicates width zone would have in the absence of any diffusion or mixing. Sample introduced a t 5000 r.p.m., accelerated to 25,000 r.p.m. for 5 min., and unloaded a t 5000 r.p.m., 5’.
or 66%) was pumped into the rotor edge until the light end of the gradient began to flow out the core exit line, indicating that the rotor was completely full. The sample (contained in a short density gradient) was then pumped into the rotor through the core, forming a sample zone centripetal to the density gradient. This is followed by an “overlay” solution having a density less than that of the sample layer, thus displacing the sample zone away from the core, but leaving the rotor full. The centrifuge is then accelerated to operating speed (up to 30,000 r.p.m.), After the desired
NORMAN G. AXDERSON
1986
Vol. 66
used, or when the septa were omitted. The came of this instability is not fully understood, but it is RAT RED CELLS largely overcome by using a very stiff upper shaft with a lightly mounted upper bearing. 60 06 Performance Studies. Gradient Stability.-The gradient actually introducad into the rotor was analyzed refractometrically before being pumped in, 40 and as recovered from the rotor. The results are shown in Fig. 2. When the short radius of the rotor ii6l is considered, the small changes are probably no more than might be expected. To see whether a ul concentrated sample layer maintains its position in 20 the centrifuge, a sample gradient made from 10 ml. b of 8% bovine serum albumin in distilled water and 10 ml. of 17% sucrose was layered over a 1200-ml. gradient varying linearly with rotor radius from IO 17 to 55% sucrose (w./w.). After the sample was introduced, the rotor was accelerated to 25,000 r.p.m. for 5 min., decelerated to 5,000 r.p.m., and unloaded. The ultraviolet absorbance of the effluent stream (Fig. 3) was recorded and the results were recalculated in terms of the rotor radius. The sample zone at a radius of 25.3 mm. would be 0.65 I 100 300 500 700 900 1100 1220 mm. thick if no mixing or diffusion occurred. The VOLUME (rnl). peak recovered had a width of 1.6 mm. at half Fig. 4.--Chilled rat red cells centrifuged to equilibrium height, indicating that only a small amount of bandposition 44.7% sucrose (w./w.) in zonal ultracentrifuge. spreading had occurred, due in part to the fact that Centrifuged 60 min. a t 20,480 r.p.m. Effluent analyzed a t the core used assumed vertical zones, 280 mp using 0.2 cm. cell; 5”. Separations may be made in density gradients on the basis of sedimentation rate or, if the equilibrium or isopycnic position is reached, on the basis of density alone. To see whether sharp separations 08 could be obtained with both methods, the following 265 rnp, 0 2 crn PATH experiments were performed. Chilled rat red cells 06-1 I become paracrystalline and cease to behave osmotically.10 The equilibrium peak position of these cells is at 44.7% sucrose and shows a very sharp peak (Fig, 4). Whether the tailing of this peak is caused by mixing by laminar flow in the lines or because of inhomogeneities in the density of these cells remains to be determined. It is evident, however, that sharp banding based on approach to equilibrium may be obtained. For rate studies, polio virus particles were used. These are homogeneous particles with a sedimentation coefficient of approximately 160. A partially purified suspension containing, in addition, soluble io 30 ’ 40 45 50 proteins and nucleic acid was separated as shown in I ROTOR POSITION-RADIUS (mrn). Fig. 5. The solution represented by the small peak 500 io00 !io0 was concentrated and photographed in the analytiVOLUME (rnl). cal ultracentrifuge (Fig. 6) and in the electron Fig. 5.-Separation of Type I1 polio virua from soluble microscope. While the preparation appeared to be proteins and nucleic acid. Centrifuged 180 min. at 27,800 homogeneous by these two met hods of observation, r.p.m.; 5’. it should be emphasized that the criteria used apply separation has been completed, the centrifuge is only to the physical properties of the particles and decelerated to 5,000 r.p.m., and the gradient dis- would not distinguish any non-viral particles presplaced out through the central core by pumping a ent having these properties. Thus far, polio virus very dense solution to the rotor edge. As the Types I, 11, and I11 have been isolated in this gradient flows out of the rotor it is analyzed con- manner as well as tobacco mosaic virus and brome tinuously for ultraviolet absorbance and, if desired, mosaic virus, It is apparent that the density grafor protein concentration or enzymatic activity us- dient ultracentrifuge is useful for the isolation of ing automated analytical systems to be described viruses. More complex mixtures, including homogenates elsewhere. In initial studies carried out a t Spinco Division of of mammalian tissues, have been successfully reBeckman Instruments, Inc., considerable instability (IO) E. Ponder, “Hemolysis and Related Phenomena,” Grune and was observed when a flexible upper shaft was Stratton, New York, N. Y.,1948. 400
W
g
:
I
’
solved into their respective components using a gradient in which the larger particles are separat,ed on the basis of their densit.y and thc smaller particles on the basis of their sedimentation rate. Thc six fractions seen (Fig. 7) in liver preparations include nuclei uncontaminated with whole cells, a fraction composed predominantly of "cell wall" mat,erial, mitochondria, endoplasmic reticulum with attached granules, ribosomes, and soluble materials. 3'incc these are seen in a single analysis, the method constitutes a new way of visualizing the cell parts. Characteristic differences, including a diminution in the amount of mitochondrial matcrial in tumor tissue, have been seen. At the speeds prcscntly availablc, laigc protcins, such as the macroglobulin of rat serum, which has a sedimentation coefficient of 18 (ref. 11) may be partially separated after prolonged 15-hr. runs. The usefu! range of the present machine, therefore, is down to approximately &20. We consider it important to develop much higher centrifugal forces in zonal rotors for the following reasons. First,, in many colloidal systems, especially synthetic polymer preparations, it is important to be able to fractionate mixtures on the basis of particle size and to obtain sufficient material from several fractions for chemical analysis. Sccondly, in the fractionation of protein mixtures, most isolation methods are strongly charge-, or chargoto-mass-ratio depcndent. Thus the sequence of serum proteins observed near neutrality in the Tiselius apparatus, the sequence eluted from diethylaminoethylcellulose, l2 and the order in which constituent proteins are precipitated with ammonium sulfate show certain similarities. Not infrequently an isolation procedure yields a mixture of proteins resolvable only in the analytical ultracentrifuge. If a preparative zonal ultracentrifuge of sufficient speed wcre available, separation on the basis of sedimentation coefficient could be one of the early steps. We have inquired, therefore, into those factors which might limit thc speed and resolution obtainable. Rotor Configurations.--A number of rotor configurations have been considered (Fig. 8). (A) Rotor with vertical axis, supported and drivrii from below, with coaxial fluid line connect.ionsa t the bop of rotor (at end of upper shaft). (R) Rot,or similar to A, but inverted. (C) Vertically mounted rotor, with shafts and bearings at each end, with one fluid line connection at each end. (D) Rotor suspended from above, with removable coaxial filling prohe which is inserted for filling at low speed. The prohe is removed and the rotor is capped for high speed operation, and then reinserted at, low speed for unloading. (E) Internal skimmcr rotor. (F) Any of the above systcms having two Ilearings may be mounted, in inst,ancca dcscrihed Idow, to rotate along a horizontal axis. Configuration A has been used here to allow use of existing equipment. It is believed, however, that configuration B may prove t.0 he more stable at high speed. I t is hoped that a facility for testing L
(11) N. 0.Anderson. R. E. C a n n i n ~ .M. 1.. Anderaun. sntl R. 11. Slrelihanmr. R z d l . Cell Rea.. 16. 292 (1959). (12) R . A. S d w r eiiti I$. A. i'rlerson. Fndcrolion i ' w ~ . ,l?. i l l 6 (IHS8).
Fig. 6.--Polio virus from small p w k in run shown in Fig.
X ol,served ia the anxlytiasl ultrarcntriiugr. KO contamination was detectable; sIu = 14% eo10-
Q-xLuar
UIERIILB
"a"Yr (-8
I
Fig. 7.--Scpamtion of rat. liver brei in zonal ultracentrifngc. Centriiugnl 240 min. :tt 20,758 r.p.rn. z t 5 O .
a number of the ahove rotor systcms can be constructed in the near future. A t very high speeds it may be advisable to remove all fluid line connections as suggcstcd in D ahovc and let the rotor hang free, as is done with the Spinco analytical ultracentrifuge. The internal skimming system will probably find its greatest use at speeds below 20,000 r.p.m., where evacuation of the rotor chamber is not essential. The skimmer consists of two stationary disks with an exit port in the center of one. As the fluid reaches the disks it is decelerated, producing a local decrease in the rotational vcIocit,y of t,he liquid and hence in the centrifugal force and pressure in the fluid between the disks. Fluid pressure a t the same radius outside the skimmer forces the fluid in the skimmer out the exit port. This principle has been previously used in the gas ultracentrifuge. One of the greatest difficulties in designing these so-called "hoop-stress" rotors arises from the differential expansion of the rotor cylinder and the cap. In Rotor I1 used in the present studies the
NORMAN G. AXDERSOX
1988
Vol. 66
length is limited only by total rotor weight, rotor stability at high speed, and by the loading and unloading speed, which in turn affects the design of the rotor core. Future core designs must take into account the fact that no equal-density zone in the ultracentrifuge is ever truly vertical. Rather, each zone will form part of a paraboloid of revolution having the same axis as the rotor according to the equation
L
=
u2r2/2X 980
(1)
where L = length of parabola in cm., from vertex to the top of the rotor chamber r = rotor radius in cm. w = angular velocity in radians per second
With the core of rotor 11, the maximal diameter is 3.785 cm. A rotor having a core of this upper diameter, according to eq. 1, cannot be longer than 500 cm. if it is to be loaded and unloaded at 5000 r.p.m. The difference in radial position between the top and the bottom of the rotor a t the maximal core diameter is approximately 1mm. For maximal resolution the core should have a large number of collecting bands and should follow eq. 1 in its overall shape.
TYPE A
R.4DIUS
ROTORS FOR Speed (r.p.m.)
-
-
TYPE D TYPE C Fig. S.-Rotor configurations for zonal ultracentrifuges; description included in text: 1,static seal; 2, rotating seal; 3, bearing; 4, drive system; 5, air turbine; 6, removable probe for filling and em tying rotor; 7 , stationary skimmer system for making fluid [ne connections t o rotor.
cap is held on by buttress threads on the inside of the cylinder. The cylinder expands during acceleration, and at maximal speed makes contact with an outer rim on the cap. The cap, therefore, is accurately positioned only a t rest and a t top speed. While there are several ways to predeform the cylinder, or to compensate for expansion by deformation a t top speed, these are not satisfactory solutions. Maximum Speeds.-To obtain information on the speeds obtainable, equations which give the optimum wall thickness and maximal inside diameter a t a given rotational speed have been solved on a computer, Some of the results are listed in Table I. Limitations on Rotor Length.-In cylindrical rotors spinning about a vertical axis, the rotor
TABLE I THICKNESS O F CYLINDRICAL BOWL THE ZONALULTRACENTRIFUGE^
AND WALL
-Steel (Hytuf a1lov)Inside Wall radius thickness
(om.)
(om.)
-Aluminum Inside radius (om.)
(7075-T6)Wall thickness (om.)
12.3 4.42 3.51 15.21 15,000 6.12 2.21 1.75 7.62 30,000 1.17 4.09 1.48 5.08 45,000 1.17 0.879 3.25 3.81 60,000 0.935 .704 2.59 75,000 3.05 2.16 .780 .587 2.54 90,000 1.63 .587 ,439 1.91 120,000 1.07 ,384 .295 1.27 180,000 a Kindly computed by Mr. A. A. Brooks, Flow Research Department, Technical Division, Oak Ridge Gaseous Diffusion Plant. Average fluid density assumed to be 1.7. Calculations also assume that the septa are centrally supported.
The optimal gradient to be used in a given instance is, in part, a function of the distribution of particle sizes in the sample. The following considerations favor a convex gradient: (1) the greatest particle capacity (-1/2 (dC/dr) x zone thickness) is needed near the sample zone since the largest concentration of particles will occur in this area; ( 2 ) the concentration of particles in a given zone diminishes as it moves centrifugally, according to the familiar equation13
and (3) the capacity of the gradient increases rapidly as the difference between particle and solution densities approaches zero. In practice, linear (with radius) gradients have proved very useful because analysis of sedimenta(13) T. Svedberg and K. 0. Pederson, "The Ultracentrifuge," Oxford, Claresdon Press, 1940.
Oct., 1962
THEZOXALULTRACENTRIFUGE
tion data is simpler, and because they give good resolutioii of mixtures containing particles having a very large size range. For example, extremely dense solutions are required to keep cell nuclei from reaching tbe rotor wall, while very dilute solutions are needed if ribosomes are to be separated in a reasonable time. A linear gradient with a dense “cushion” allows both to be isolated from the same mixture. The results obtained to date indicate that the zonal ultracentrifuge is a biophysical tool capable of achieving sharp separations of particles on the basis of sedimentation rate or density. NOTE ADDED IN PROOF.-During 102 runs with this instrument, marked instability above 24,000 r.p.m. has been observed in several instances, resulting in excessive vibration and bearing heating. Self-balancing rotors with no upper bearing are under construction at the present time.
Acknowledgments.-During the development of this centrifuge, discussions with Drs. J. W. Williams, J W. Beams, M. L. Randolph, E. G.
1989
Pickels, and D. G. Sharp have been most helpful. Construction of the rotor, drive system, and upper bearing assembly was carried out under subcontract by the Spinco Division of Beckman Instruments Inc., with additional modifications being made at Oak Ridge. I am indebted to Dr. Jonas Salk for stimulating discussions and for quantities of polio virus, to Dr. Myron Brakke for TMV- and BMVinfected plant tissues and advice on the isolation of viruses from them, and to Dr. C. A. Knight for a large sample of purified TMV. Rotor I1 and associated equipment were designed by Richard Stallman. DISCUSSION W. J. CARTER(MSA Research Corporation).-Have purified any of the bacteriophages-T-3,
you in particular?
S. G. ANDERSON.-\ve have concentrated T-3 by using the zonal rotor as a continuous flow centrifuge, and then purified by zonal sedimentation in a density gradient.
