Oct., 1962
SUBFRBCTIOKATION O F
LIPOPRWEIXS
2007
SUBFRACTIOXATION OF THE sf 2 0 - 1 0 5 LIPOPROTEINS IN A SWINGIKG BUCKET ROTOR1 BY F. T. LINDGREN, A. V. NICHOLS,F. T. UPHAM,A N D It. D. WILLS Donnar Laboratory, Lawrence Radiation Laboratory, Univeysitv of California, Berlceley, California Received March 10, 1068
Human serum lipoproteins of the Sr 20-105 class have been subfractionated by accumulative flotation rate techniques employing a NaC1 density gradient and a specially designed swinging bucket rotor. A feature of this rotor is that it allows the use of full length 1.27 X 8.89 CML. preparative tubes, providing an increased radial flotation path. Studies of 7s os. p allow estimations of hydrated density for lipoproteins within the Sf 20-400 class. Over the Sf.20-105 lipoprotein spectra, lipid chemical data on isolated lipoprotein subfractions indicate a progressive increase in glyceride content with increasing Sr rate. Although imperfect fractionation was achieved, this type of subfractionation or modification of it may be useful in exploring accumulative flotation rate separation techniques of particles or macromolecules which differ in size or molecular weight, but which, because they are less dense than water (or the usual solvent), may not be separated on the basis of their hydrated density.
Introduction Subfraationation of the low-density lipoproteins less dense than serum background (1.006 g./m1.)2 has consistently presented difficulty. In contrast, the fractionation of lipoproteins of higher density may be achieved by appropriate density adjustments in the background small molecule (salt) solution. Thus, broad lipoprotein classes may be fractionated by differential density ultracentrifugat i ~ n . ~ Further, -~ it is theoretically possible to make very delicate lipoprotein fractionations on a density gradient provided, of course, the solution background in a region of the preparative tube can be properly adjusted to equal the hydrated density of the lipoprotein itself. In practice, this gradient may be a temporary 0 1 1 8 ~which ~~ necessarily will be changing slowly with time or, more ideally, a gradient which corresponds closely to the sedimentation equilibrium gradient* characteristic of the particular salt, the salt concentration, and the centrifugal conditions. However, once fractionation has been achieved on the basis of hydrated density, physical homogeneity of the macromolecular preplaration is not necessarily assured, since there may exist a considerable degree of particle size inhomogeneity within the same hydrated density class. On an ultracentrifugal basis the technique of rate separation by sedirnentati~n~~'" or flotationll potentially rallows further subfractionation of a given lipoprotein preparation which is either less dense than 1.006 g./ml., or which already has been frac(1) Work irupported in part b y Research Grants H-1882 (C7) and H-2029 (C4) from the National Heart Institute, Public Health Service, Bethesda, Md., and by the U. S. Atomic Energy Clommission, Washington, D. C. (2) Unless otherwise indicated all densities are given a t Z O O . (3) R. J. Havel, H. A. Eder, and J. H. Bragdon, J. CZzn. Inuest., 34, 1345 (1955). (4) L. A. l~illyard.C. Entenman, H. Beinberg, and I. L. Chaikoff, J . BzoZ. Chenr., 214, 79 (1955). (5) F. T. Lindgren, A. V. Nichols, and N. K. Freeman, J. Phys. Chem., 69, 930 (1955). (6) E. M. IZuss, J. Raymunt, and D. P. Barr, J. Cltn. Invest., 36, 133 (1956). (7) J. L. Oncley, K. W. Walton, a n d D. G. Cornwell, J. Am. Chem. Soc., 79, 4666 (1957). (8) F. T. Lindgren, A. V. Nichols, T. L. Hayes, N. K. Freeman, and J. W. Gofman, Ann. N . Y . Acad. Sci., l a , 826 (1959). (9) M. K. Brakke. .I. A m . Chem. Sac., 7 3 , 1847 (1951). (IO) N. G. Anderson, Ezpll. Cell Res., 9, 448 (1955). (11) F. T. Lindgren, H. A. Elliott, and J. W. Gofman, J . Phys. Chem., 66, 80 (1951).
tionated om a density gradient. These techniques for separation are also applicable for mixtures of proteins of closely similar hydrated densities which cannot be subfractionated on a density basis, but nonetheless may differ substantially in molecular weight from one another. In order to achieve optimal conditions for rate separation, it is desirable to use a swinging-bucket type rotor to avoid detrimental convective disturbances inherent in angle preparative ultracentrifugation.12 Also, if rate fractionation is employed, the resolvabili1,y is greater the longer the path length over which the rate separation occurs. Experimental Apparatus.-Although swinging-bucket type rotors have been designedla or are available commercially, such as the Spinco (SW 39 I,., SW 25.1, and K6) and Serval (HS) rotors, none provide for use of both fullsize 6-ml. and 9-ml., 12.7 mm. diameter preparative tubes. Use of these fulllength tubes, particularly the 9-ml. tubes, would resent a long radial path (8 cm.) in which to achieve near$ ideal radial sedimentation or flotation. Because of the widespread use of Spinco ultracentrifuges, it seemed desirable to design a swinging-bucket type of rotor that would operate in either the standard Model L or Model E ultracentrifuge. Since these ultracentrifuges utilize a high vacuum for operation, a necessary requirement for the swinging-bucket assembly would be for an effective vacuum seal to prevent possible escape of fluid from the centrifuge preparative tube into the vacuum chamber during operation. Another desirable feature indudes the capability for sealing the preparative tubes with st,andard preparative cap agsemblies to achieve an additional safety vacuum seal as well as to provide for ease of insertion and removal of the preparative tube from the centrifuge bucket. These features have been incorporated into a low-speed swinging-bucket rotor described below. Construction and Testing.-Because of the convenience of either two- or four-bucket operation, it seemed advantageous to design a four-place rotor employing 90" symmetry. The rotor assembly shown in Fig. 1 consists of three parts: a rotor base, a cylindrical rotor, and a locking cylinder which also serves as a suspension coupling for use on a $lode1 E ultracentrifuge. The rotor base, allowing operation in the Spinco Model L ultracentrifuge, was turned down from a retired Spinco preparative rotor. The cylindrical rotor was made from a forged billet of 7075 aluminum. After the cylindrical rotor had been machined slightly oversize, it was subjected to a T-6 heat treatment. This process consisted of maintaining the rotor at 471" for 1 hr., followed bx a 52" water quench. The rotor then was reheated to 121 for 24 hr. and air quenched. A final machinigg of the tempered rotor was made to the specified tolerance (d~0.05 mm.) for all dimensions. (12) E. G. Pickels, J . Gen. Physiol., 26, 341 (1943). (13) H. Kahler and B. J. Lloyd, Jr., J . Phys. Chem., 66, 1345 (1951).
F. T. LINDGREN, A. V. SICHOLS, F. T.UPHAM,ASD R. D. WILLS
2008
Vol. 66
Each trunnion was tested in a special support fixture and a hydraulic press subjected each trunnion to a full 9,070 kg. stress. The stress-strain relationship as determined by the downward deflection of the center of the trunnion is
On; trunnion was &rr;ed to failure. which occurred a t
CAP
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10
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loading of 15,060kg. Although s r a d i d cromsection throu h the center of the preparative tube hole was zpproximat$y 90% of the combined area of the two aupport points, failure occurred simultaneously across these two support points. Examination of the trunnion subjected t o failure suggested that one pivot failed in shear, where= the other pivot failed as a result of the severe bending moment developed in the region of failure. Thus, i t would appear from this failure ( a t approximately 10,550 kg./cm.z) that enlarging the radius in the region joining the support pivot and the body of trunnion would potentially increase trunnion strength. The expected ultimate strength of Allegheny Ludlam GO9 with our heat treatment is in the neiehborhood of 21,000 kg./cm.'. The 6- and 9-ml. swinging buckets and caps were machined a u t of ST24 duraluminum and balanced to within 10.020 g. A t,hrust shoulder of 2.38 mm. radius was machined on each of the buckets, matchina a comolementarv SUDDOItinK -.. .. r~~~~
E*"lfractions I, 11, and 111. Approximati: concrmtrations for each fraction from top to lmttom w w :qqxoximaI,i+ 4, fi, 15, nrid Ifi( 6 (COwferring to initid scrim concentration). Snmplrs were ultmcentrifuwd in II 4-plecr an;rlytic:rl rotor at a ttq,wature of 26.0". An nccclcrabion timr of ilpprasimstely 5.20 min. was rcquircd to achievr frill speed (52,640 r.p.m.). 5 ) 20 - 10' LIPOPROTEIN SUBFRACTIONATION Flololion r d e
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tion gradient is progrensively reduced and may ultimately be reversed. Thus, u t the end of the first run (2 hr.) tho Sr 15!10-10' lipoproteins hnvc all thmrrtically migrated to the ton of the prewrative tube, yet there remains B %exative" % i o roGin-density gmdicnt a4 the rrault of an .incream in {poprotein eoneentrxtion v.ith depth. Without thc salt madient to stahilizr the svstem. serious eonvcctivc clisturbanccs would rpsult. Ail.u
