PREPARATIVE DENSITY GRADIENT CENTRIFUGATIONS
PREPARATIVE DENSITY GRADIENTCENTRIFUGATIONSBy A. FritschDepartement de Biologie MoleculaireInstitut Pasteur, Parisfor all countries except USA and Canada Copyright by Beckman InstrumentsInternational S.A., Geneva,Beckman
CONTENTSFOREWORDCHAPTER I: INTRODUCTIONCHAPTER II: ZONE CENTRIFUGATIONII.1 The principle of the methodII.2 Practical aspects of zone centrifugationa) The choice of the rotorb) The choice of the gradient materialc) Making the gradient- Swinging bucket rotors- Zonal rotorsd) Layering the macromolecular sample- Swinging bucket rotors- Zonal rotorse) The centrifuge runf) Fractionating the gradientII.3 The measurement of sedimentation coefficientsa) The sedimentation coefficientb) Isokinetic and equivolumetric gradientsc) The measurement of sedimentation coefficientsII.4 Sedimentation coefficient and molecular weight5799102342CHAPTER III: ISOPYCNIC CENTRIFUGATIONIII.1 The principle of the methodIII.2 The density gradientIII.3 Measurements and significance of the buoyant densityIII.4 The shape of macromolecular bandsIII.5 The duration of the centrifuge runa) Sedimentation equilibrium of the gradient materialb) Sedimentation equilibrium of the macromolecules- Equilibrium gradients- Preformed gradientsIII. 6 Resolving power, and rotor speeda) Equilibrium gradientsb) Preformed gradientsIII.7 Density gradient materials and their applicationsa) Nucleic acidsb) Proteinsc) Subcellular fractionsd) Virusese) CellsIII. 8 Practical aspects of isopycnic centrifugation494950525658APPENDIXA.1 Properties of rotors used for zone centrifugationA.2 Density of aqueous solutions of a few saltsA.3 Sedimentation equilibrium coefficients of the aqueous solutionsof a few saltsA.4 Relationship between the density of salt solutions and theirrefractive index79BIBLIOGRAPHY8562657528a) Proteinsb) DNA'sc) RNA'sII.5 Sedimentation and conformational changes32II.6 The resolving power34a) General notionsb) Elements for a quantitative approach- Isokinetic gradients- The gradient induced zone sharpeningII.7 The macromolecular load of the gradientsa) The hydrostatic stability criterionb) The differential diffusion effectc) The particular case of DNA
CHAPTER IForewordIntroductionSince the first edition of this monography, density gradient centrifugationmethods have undergone numerous theoretical and technical improvements. In particular, the advent of high performance rotors, especially ofzonal rotors, has led to a more thorough study of resolving power andmacromolecular load of the gradients. Accordingly, the field of applications ofthese methods has been considerably extended. In addition to their being afundamental tool in molecular biochemistry, they are more and more used forthe fractionation and characterization of subcellular particles and whole cells.Their industrial applications have been developped as well.Unavoidably, this second edition became almost a new book. But as forthe first edition, its scope is limited to the use of density gradient methodswith preparative centrifuges. They have not only their own methodology buttheir field of applications is the widest. Our aim was essentially to write ahandbook which should help in setting up experiments and in interpretingtheir results.If some properties, like the macromolecular load of the gradients, are incompletely treated, this is essentially because the experimental facts stillsuffer from a lack of good theoretical support. But as soon as this gap will befilled, one can predict that new experimental conditions will give rise to newapplications. Other shortages of this text are exclusively due to the limits ofour personal experience; we hope that we were able to compensate thempartially by our choice of bibliographic references.We are specially indebted to P. Tiollais for his criticism and invaluable suggestions during the preparation of the manuscript. We also acknowledgethe help of P. Courvalin, P. Rouget, P. Tiollais and A. Ullmann who kindlysupplied part of the experimental support. We wish especially to thank J. Freud forher expert secretarial contribution, and D. Thus/us for correcting the Englishtranslation.Paris, February 1975.During density gradient* centrifugation, one should distinguish zonecentrifugation (also called rate zonal centrifugation) from isopycniccentrifugation (or isopycnic zonal centrifugation). Despite the fact that oneuses density gradients in both cases, that the macromolecules to be studiedare always concentrated in narrow bands, and that one resorts to the samerotors, their principles are completely different.Zone centrifugation separates macromolecules according to theirsedimentation velocity, or, more precisely, according to their sedimentationcoefficients. The density of the sedimentation medium is always smaller thanthe density of the macromolecules. Accordingly, beyond a certain time ofcentrifugation, they will pile up at the bottom of the centrifuge tube, or at theedge of the rotor. The role of the gradient is secondary, albeit necessary: it isto avoid the convection currents which tend to destroy the macro-molecularzones.In the case of isopycnic centrifugation on the other hand, the density gradientconstitutes the very principle of the method. The density range which iscovered by the gradient, necessarily includes the density of themacromolecules. After a proper time of centrifugation the latter areconcentrated at a position in the gradient where their density is equal to thedensity of the sedimentation medium. Isopycnic centrifugation, thus,separates macromolecules according to their density.Zone centrifugation and isopycnic centrifugation are semi-analytical methods. Indeed, despite the impossibility to follow the sedimentation processwhile it is going on, one can measure sedimentation coefficients and densities, and sometimes molecular weights and conformational changes. Themost remarkable aspect of both methods is that these parameters can bemeasured for non-purified macromolecules at extremely small amounts. Theminimal amount depends on the sensitivity of the method used for measuringconcentrations (enzymatic activity, radioactivity, etc.)] whereas the puritydepends only on the specificity of this method for eadh kind ofmacromolecule.* In rigorous terms, the expression "density gradient" designates the slope ofthe curve which describes the density of the sedimentation medium as afunction of the distance to the rotor axis. However, it is common practice inbiochemistry to call density gradient every sedimentation medium in which adensity change occurs. We shall use the expression in both senses, itsprecise meaning being always defined by the context.
Zone centrifugation and isopycnicc nltifugation are also purification /methods at a more or less large scajefFor this application, the design (Ander-L-s"on and Burger, 1962) of the zonal rotors has led to large progress. In thelaboratory, they allow the purification of several grams of ribosomal sub-units(Eikenberry et al., 1970), whereas a battery of 48 zonal centrifuges is used forthe commercial purification of influenza vaccines (Sorrentino et al.,1973). Theimportance of zonal rotors is largely illustrated by an entire volume of Nat.Cancer Inst. Monograph (vol. 21, 1966), and by a recent meeting (Spectra2000, vol. 4, 1973; Editions Cité Nouvelle, Paris) which entirely dealt withthem.Zone and isopycnic centrifugation methods can be combined, either as ananalytical tool, for example to characterize the replicating complex of a viralgenome (Magnusson et al., 1973), or as a preparative method for the purification of large amounts of subcellula fractions. For the latter, one takes intoaccount that in the two dimensional space defined by the sedimentationcoefficient and the density, mitochondria, nuclei, viruses, etc. occupy a verydefinite position (Anderson, 1966). Both methods are sometimes combinedduring the same centrifuge run (Wilcox et at., 1969: Anderson, 1973).Our aim is to show how to use these centrifugation methods for both theiranalytical and preparative applications. Since it is impossible to mention allthe applications, we will restrict ourselves to those which appear - at least tous - to be methodologically the most significant. Number of applications areanalyzed in monographies devoted to zonal rotors (Anderson, 1967; Clineand Ryel, 1971; Price, 1972; Chervenka and Elrod, 1972) and in "Fractions"(Beckman Instruments, edt). In looking through any issue of the periodicalsdevoted to biochemistry, or molecular biology, one becomes rapidly aware ofthe variety, and of the importance of these methods.CHAPTER IIZone Centrifugation11.1 THE PRINCIPLE OF THE METHODIn order to separate macromolecules, or subcellular fractions according totheir sedimentation coefficient differences, i.e. most often according to theirmass differences (section II.3.a), two methods can be used. The first one,called moving boundary centrifugation, or differential centrifugation, consists incentrifuging a homogeneous solution of macromolecules. At the time wherethe most rapidly sedimenting molecules are pelleted at the bottom of thetubes, part of the more slowly sedimenting ones will still be in solution. If theratio of the respective sedimentation velocities is equal to, say 5, the pelletwill be contaminated by 20% of the slower macromolecules, whereas onlyless than 80% of them can be recovered in purified form.The second method is zone centrifugation, still called rate zonal centrifugation. After the pioneering work of Brakke (1951; 1953), Britten and Roberts(1960), and Martin and Ames (1961) gave zone centrifugation its presentshape. Its principle is the following:A very narrow layer, or zone, of a macromolecular solution is layered on topof an appropriate medium. During centrifugation, macromolecules with thesame sedimentation velocity move through this medium as a single zone. Itwill appear as many zones as the initial layered contained different types ofmacromolecules. Each zone sediments at the speed characteristic of themacromolecules which it contains. The centrifuge run is stopped before thefastest zone has reached the bottom of the tube (or rotor). The content of thetube is then fractionated into layers perpendicular to the direction of thecentrifugal force field, and the macromolecular content of each fraction ismeasured.In order to keep the zones stable, i.e. as narrow as possible, their sedimentationshould obey a certain number of criteria.First, the density of the initial layer should always be smaller than thedensity of the sedimentation medium just below the layer. Otherwise, thecontent of the layer would immediately spread into the sedimentation medium.Second, as soon as the macromolecular sample solution has been layeredon top of the supporting medium, a negative density gradient is generated onthe leading edge of the zone. In order to maintain the stability of the zone.
this negative, macromolecular gradient has to be compensated by a positivedensity gradient introduced into the supporting medium (Figure 1). This positive gradient is obtained through the addition to the supporting medium ofa solute (for example, sucrose) whose concentration increases progressively in the direction of the centrifugal field.The positive density gradient will also largely reduce the convective currents due to the "wall effect" (section ll.4.a) of the swinging bucket tubes,or to slight temperature differences in the supporting medium.With regard to moving boundary centrifugation, zone centrifugation hasthe following advantages: a) a high resolving power, since macromoleculeswhose sedimentation coefficients differ by as little as 15% can be separated;b) all the macromolecules remain in solution; c) it is easy to measure relatively precise sedimentation coefficients and, from these, to obtain sometimes good estimates of molecular weights or conformational changes ofthe macromolecules.II.2 PRACTICAL ASPECTS OF ZONE CENTRIFUGATIONa) The choice of the rotorZone centrifugation, necessarily .requires the use of swinging bucketrotors, or zonal rotors (Figure 2). These are the only rotors in which thedensity gradient is always parallel to the force to which it is submitted, and inwhich, for this reason precisely, the zones don't suffer any major distortion.After several unsuccessful trials, it is now well established (Castañeda et al.,1971) that fixed angle rotors are not suited for zone centrifugation.The choice of a particular rotor depends essentially on the amount ofmacromolecules to be centrifuged, on the resolving power, and secondarilyon the centrifugation time. Most often, the best compromise has to be foundbetween these three parameters; they will be discussed in later sections.Among the zonal rotors, other factors have to be considered. The reorienting zonal rotor has the two advantages that contamination with pathogenic substances is largely limited, and that shearing of DNA in the rotatingseal is avoided. With the edge unloading rotors, instead, one saves thesubstances needed to obtain high density solutions. Independently of theirbetter resolving power and lower centrifugation time, titanium rotors differfrom aluminium rotors in their good resistance towards the corrosive actionof highly concentrated salt solutions, or solutions with extreme pH's.b) The choice of the gradient materialIn zone centrifugation, density gradient materials are always used at concentrations giving solutions whose density is smaller than the density of themacromolecules, or particles to be centrifuged. They should also satisfythe following criteria: good solubility in water, electrical neutrality, andtransparency to UV-light. Whereas their viscosity should be relatively smallfor the centrifugation of macromolecules, this restriction is less stringentfor viruses or large cellular organels.10Figure 1. Why a density is a prerequisite to zone centrifugationThe figure represents the density variation of a centrifuge tube content as a function of thedistance to the rotor axis. If the density of the sedimentation medium were constant, its localperturbation by a macromolecular zone [(a) and (b)] would always give rise to a negativedensity gradient on the leading edge of the zone; from the hydrostatic point of view, thiswould be an unstable situation, and the zone would spread out, until the negative gradientwould have completely disappeared. If, instead, a positive density is incorporated in the sedimentation medium, and if the amount of macromolecules is small enough, this positive gradient will compensate the negative gradient due to the zone [(a')]. But beyond a certain amountof macromolecules (see section III.6), the resultant density gradient on the front of the zonebecomes again negative [(b')], and the zone will spread.Figure 2. Swinging bucket rotor, and zonal rotorThe swinging bucket rotors (a), are essentially characterized by a set of buckets which hangin the vertical while the rotor is at rest, and which come to the horizontal position as soon asthe rotors spin at a few hundreds of rpm. Hence, the tubes placed inside each bucket arealways submitted to a force (earth's gravitation, or centrifugal force) which is parallel to their axis.Zonal rotors (b), instead, are cylinders which spin around their revolution axis; they can beconsidered as a swinging bucket rotor, where one of the buckets (in the horizontal position)has been opened to 360 . Zonal rotors contain a central core with four vanes whose functionsare: force the rotor content to spin with the rotor, and allow communication with the centerand the edge of the rotor. These operations, also require a rotating sealassembly (not represented).11
Since the first experiments of Britten and Roberts (1960), the most usedsubstance is sucrose. For certain experiments, it is necessary to usVglycerol,which is a distillation product, and thus devoid of impurities, in particularnucleases (Williams et al.,.1960; Orth and Cornwell, 1961). In order to\ btaina better resolving power than in sucrose, Kaempfer and Meselson N971)have used cesium chloride gradients at low temperature. For the centrifugation of RNA, the use of sulfolane, trimethyl phosphate, or urea has beenproposed (Parish and Hastings, 1S66; Hastings et al., 1968) the two formerproducts have the advantage not toVteract with the liquid scintillation countxing process. Centrifugation of DNA' above pH 12 leads to strand breaks,and in order to avoid them, Gaudin and Yielding (1972) centrifuge singlestranded DNA in 90% to 100%, or 25% to\50% formamide gradients. Snyderet al. (1972) have studied the dissociation of alkaline phosphatase in Trisgradients. Sodium bromide gradients have been used for the fractionationof lipoproteins (Wilcox et al., 1969). Centrifugation of low molecular weight4macromolecules (less than 10 daltons), leads to run lengths and rotor speedswhich are such that the initial shape of the density gradient is modified (ittends towards the equilibrium gradient, see chapter III). Since zone centrifugation very often implies an accurate knowledge of the gradient shape(sections II.3 and II.4), McEwen (1967, a) suggests the use of equilibriumsodium chloride gradients; it is obvious that the macromolecules shouldthen remain soluble, and stable at high ionic strength.For the Centrifugation of subcellular particles sensitive to high osmoticpressure, sucrose can be replaced by sorbitol (Neal et al., 1970,1971), or byFicoll (Boone et al., 1968; Pretlow, 1971). None of these two substances penetrates into the cells. Ficoll solutions, at a weight to weight concentration of25%, have an osmotic pressure similar to the physiological pressure. Mixturesof sucrose, sorbitol and Ficoll have also been used (Vasconcelos et al., 1971).In all cases, the density gradient materialis dissolved in a buffer solutionsuited for each particular experiment. In order to increase the density of thesolutions, they are sometimes prepared with heavy water (Beaufay et al.,1959; Kaempfer and MeselsorX 1971).\In orderta solidify the conteYvt of the centrifuge tubes at the end of thecentrifuge runTsome photopolymerizable acrylamide can be added to\thegradient (Cole, 1971).\\c) Making the gradientIn this section, we shall only describe the methods used to obtain differentgradient shapes. Their properties, instead, will be given in later sectionsof this chapter.- Swinging bucket rotorsWith this type of rotor, the gradients are established before centrifugation. They are obtained upon mixing in the proper way two solutions of thedensity gradient material at the appropriate concentrations. Before filling,the centrifuge tubes should sometimes be treated with silicone; this will12avoid sticking of the macromolecules on the tubes (Burgi and Hershey, 1968).It is recommended that the density gradient be established at the temperature of the centrifuge run.Figure 3 shows a very simple apparatus (Britten and Roberts, 1960; Martinand Ames, 1961), which is commercially available under different versions.It gives gradients in which the concentration of the gradient material varieslinearly with distance, or certain convex gradients. More generally, if the2two reservoirs, Ri and R2 have respective sections of Ai and A2 cm , theshape of the gradient will be given by:II-1z1and z2 are the initial concentrations of the two gradient material solutionsin each reservoir, and v1 and v2 are their initial volumes; z1and z2 are alsothe two extreme concentrations of the gradient, z is the concentration of themixture when the centrifuge tube has received a gradient volume equal tov. (v1 v2) is equal to the final gradient volume. Equation II-1 shows that theconcentration varies linearly for A1 A2. This case is the most widely used,especially in order to obtain the isokinetic (section II.3b) 5% to 20% sucrose,or 10% to 30% glycerol gradients, and for certain Ficoll gradients (Pretlow,1971). For A1 A2, one obtains convex gradients with which the macromolecula
CHAPTER III: ISOPYCNIC CENTRIFUGATION 49 III.1 The principle of the method 49 III.2 The density gradient 50 III.3 Measurements and significance of the buoyant density 52 III.4 The shape of macromolecular bands 56 III.5 The duration of the centrifuge run 58 a) Sedimentation equilibrium of the gradient material .
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Giving preparative thin layer chromatography some tender loving care. John J. Hayward*, Lavleen Mader & John F. Trant* 1Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario, Canada jhayward@uwindsor.ca; j.trant@uwindsor.ca Abstract: Preparative thin layer chromatography (prepTLC) is a commonly used method of .
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