Raymond E. Boudreau, Anne Heaney, and David 1. Weller Department of Microbiology and Biochemistry Universitv of Vermont Burlington, 05401
A Sedimentation Experiment Using a Preparative Ultracentrifuge
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Subcellular narticles are usuallv senarated and nuritied by centnfugal'techniques on t h e basis of their differenr sedimentation coefficients and/or densities ( I ) . The a n a lytical ultracentrifuge is used th measure physical pmperties of macromolecules i e , sedimentation coefficient, molecular weight, buoyant density). Thus, the theory of sedimentation is often presented i n biochemistry and molecular bioloev courses. Student l&oratory experiences sometimes include demonstrations of sedimentation velocity and/or equilibrium using the analytical ultracentrifuge '(2). Beckman Instruments (Wakefield, Mass.) has made available a Schlieren optics accessory for their preparative ultracentrifuge (Model L series), making it possible to do both velocity and equilibrium experiments with only slightly less accuracy (3) than the Model E. We found t h a t this accessory greatly facilitates t h e teaching of the principles of ultracentrifugation. In this paper we describe a n experiment used i n our molecular biology course t h a t illustrates the use of the preparative ultracentrifuge i n isolating and purifying bacterial ribosomes, demonstrates the use of the analytical ultracentrifuee t o determine the sedimentation coefficients of thk ribonucleoprotein particles, and demonstrates the subunit structure of the 7 0 3 ribosome and the role of Mg2+ in the association of subunits. Methods and Materials E. coli B cells were purchased as a frozen paste from Grain Processing Co. (Muscatine, Iowa). Ribosomes are prepared essentially as described by Tissieres, et al. (4). Cells are broken by grinding with bacteriological grade alumina (Alcoa A-305, Sigma Chemical Co., St. Louis, Mo.) (2.5 times the wet weight of cells) for about 15 min using a mortar and pestle. All work is done in the cold (0-5°C). The paste is taken up in a volume of solvent equal to the mass of alumina used (1 ml for eaeh gram). The extraction solvent contains 5 X M tris-HCI at pH 7.4, M Mg (CHaCO& (solvent A), plus 2 gg/ml DNAase-I. The alumina, unbroken cells, and cell debris are sedimented at 20,000 x g for 30 min. The supernatant may be further clarified by reeentrifuging. The 'supernatant is then centrifuged at 130,000 X g for 3 hr. (This is at 45,000 rev/min in the Model L2 ultracentrifuge using the type 65 rotor.) The supernatant is removed and the golden brown sediment (mostly ribosomes) is resuspended in a solvent containing 5 X M tris-HCI at pH 7.4, 8 X 10-2 M Mg (CH3C02)s1and 2 M NHICI (solvent B). The volume of solvent is halved with eaeh resuspension. We generally resuspend by gentle shaking overnight at 4'C. The suspension is clarified at 20,WO X g and again centrifuged at 130,MW) X g for 3 hr. The cycle is repeated once again: resuspension in solvent B, clarification, and recentrifugation. Following treatment with the 2 M NHICI the ribosomal sediment is colorless and transparent. The final ribosome pellet is resuspended in solvent A and the sample clarified. The coneentration of the RNP-particles is determined from Azao using k c m a - I , % = 16. The predominant form of ribosomes in 10-2 M Mg2-i is the 70-S monasome. A suspension containing only 30-S and 5 0 3 suhunits is prepared by decreasing the Mg2+ concentration in two steps by dialysis. First, the sample is dialyzed in tris buffer containing 5 X M Mg2+ for about 12 hr and second, in tris huffer containing 5 X 10.' M MgZ+-The dissociation is completely 128
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reversible; ribosomal subunits can he reassociated by dialysis in 10-ZMMgZ+ (4). Analytical ultracentrifugation is performed in a Beckman Model L ultracentrifuge equipped with the Schlieren optics attachment and a modified AnD rotor (3). During sedimentation velocity runs, the temperature of the rotor and samples is maintained as close to 20PC as possible in order to reduce the need to correct results to standard temperature. Corrections to the viscosity and density of water are unnecessary, since the solvents used do not differ significantly from water. The ribosome samples to be analyzed are diluted to 10-15 mg/ml using the appropriate solvent. In order to analyze two samples in one run, we replaced one of the plane quartz windows in each cell with one degree positive and negative wedge windows, respectively (Beckman Instruments). We also use an intermediate diameter Schlieren wire (0.007 in.) to achieve a sharper line image, although the larger diameter wire (0.01 in.) provided with the accessory gives an acceptable line for most experiments. Beckman supplies a Polaroid camera back with the accessory. Frequently, we substitute a Graflex back holding 120 film. This reduces the cost per run and improves the quality of photo reproduction. Results and Discussion Differential centrifugation yields a pure fraction of ribosomes since the soluble components sediment more slowly than t h e ribosomes and since pmcaryotes lack t h e endoplasmic reticulum, nucleus, and mitochondria typical of eucaryotic cells. Bacterial DNA, which would sediment only a little more slowly than the smaller ribosomal subunit (4), is eliminated a s a contaminant of the ribosome pellet by the addition of DNAase to the extracting solvent. T h e nuclease depolymerizes the DNA, thereby decreasing its velocity markedly. T h e 2 M salt treatment solubilizes acidic proteins t h a t adsorb to the ribosomes when the cells are broken (5). The yield of ribosomes is 1-2% of the fresh weight of cells broken. Photographs of Schlieren patterns. Upper image in each photo is of ribasoma1 subunit preparation (in 5 X 10.' M Mg2+): lower image is of manosome preparation (in M Mg2'). Rotor speed is 30.000 rev/ min. Temperature is 20PC. The photos from top to bottom were taken at 4-min intervals starting at 21 min after reaching speed. Bar angle is 60' for all photos except the last which is 50'. Scale is 1 cm.
Figure 1 shows Schlieren patterns obtained from a sedimentation velocity run. The positive wedge cell (upper image in each photograph) contained a subunit preparation and the negative wedge cell (lower image) contained a monosome preparation. The calculated sedimentation coefficients for each peak are as follows: positive wedge cell (left to right) 26-S (30-S), 38-S (50-S); negative wedge cell 35-S (30-S), 52-S (50-S), 67-S (70-S). The lower sedimentation coefficients observed for the subunits in 0.5 mM Mg(CH3C021~reflect a greater dependence of velocity on concentration at lower salt concentrations. Although this experiment may he performed as a demonstration laboratory, the ease of operation permits the students to use the equipment themselves. Thus, &dents gain some with the use of nreDarative ultracentrifugation - - ~ in the isoiation of subcellular particles and with the use of 'ltracentrifugation in the measurement of sedimentation coefficients. This experiment can he expanded
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to include the isolation and characterization of ribosomal RNA (6, 7) and protein (8-10) and can also be used to prepare for the in uitro incorporation of amino acids under the direction of synthetic messenger RNA (12). Literature Cited (11 ~ d e m . ~ . ~ . , s r i 1~1.1m11966). onro. I21 Gerbr,B.R.,andDrerkin,S.C..J.CHEM.EDUC.48,4Mi19711. 13) GlifT,th,O.M.,aodGmppr,L.,Annl. Blochem.. 31.218119681. Tissierea, Watsan. 1. D., D., and Ho~lingsamrth. B. Mal. R~OI., 1.211 119591. 151 Weller. D. L., and S j o p n , R.E.,Bioehim.Biophya. Acfo, zm, 508(1910). 161 Kurland, C.G.,JMolBiol., 2.83(1960). (71 spshr.P. F.,snd~isilier.s, A..J MOI. B~OI..I. 237 119591. (81 Spitnik-Elmn. P.. Biochem. Biwhys. Re*. Commun.. 18.557 (19651. (9) kBw. P. S.. CO% E. C.. and Fhks. J. G.. h e . No" Aeod Sci.. US., 52. 1381
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