Equilibrium Density Gradient Centrifugation for Introductory Biochemistry Carl Wm. Vermeulen Department of Biology, The College of William and Mary, Williamsburg, VA 23185 POLYETHYLENE BOTTLES
Isopycnography by equilibrium density gradient centrifugation has been a powerful albeit elementary tool for both preparative and analytical schemes in biochemistry for more than two decades but generally has been beyond the experience of students because of costly cesium salts, expensive ultracentrifuges, and durations incompatible with normal laboratory schedules. T h e accompanying protocol circumvents these problems mainly by being concerned with larger particles than with the macromolecules usually studied in the research laboratory. Yet despite its simplicity, worthwhile experiments can be run that ask significant biochemical questions. I n essence the protocol described here consists of determining the specific gravity of a common bacterium. This is done by using common desktop clinical centrifuges fitted with swinging buckets containing preformed density gradients of aqueous sodium bromide solutions to form isopycnotic bands that can be located visually. Experimental An overnight culture of prototypic E. coli in tryptone broth (8 g tryptone 7 g glucose 7 g NaCl 1000 ml water) is prepared in advance. Aliquots of this culture are then taken by the students and diluted with water and saturated aqueous NaBr, 131, respectively, to yield an overall specific gravity of about 1.1as saturated NaBr is near 1.5 glcc at room temperature. The making of replicates is re& ommended in case of tube breakage or other mishap. With this suspension next added to the distal side of a lineargradient maker such as shown in Figure 1, and an equal volume of saturated NaBr solution added to the proximal side, the effluent forms a linear gradient in the centrifuge tube. Glass tubes must be used so that bands can be seen after the centrifugation.The gradients are then run for 75 min at 2500 r.p.m., which is the top speed for IEC model CL desktop centrifuges. Higher speed centrifuges fitted even with slant head rotorsalso work well. We frequently use 12mlheavy-walled Corex tubes spun at 11,000r.p.m.for 8 min in either Sorvall SS-l or SS-34 rotors. After centrifuging,the readily visible bands may have their specific gravities ascertained by either of two alternative methods (Fig. 2). In either ease the students should be warned not tohurry because this is a sensitive procedure. The first method is to withdraw a drop of the band with a Pasteur pipet and read it on a refractometer. Indices of refraction are compared with a standard CuNe of known NaBr solutions. In the event that a refractometer is not available, determinations may be made by using 2-3 mm diameter droplets of various solutions of immiscible organic liquids as markers. These droplets must he carefully inserted beneath themeniscus of the gradient and allowed to settle for 5-10 sec to their buoyant levels. The densest marker should be inserted first, md so on, so that the droplets do not interfere with one another and fuse. Interpolation between these droplets quickly gives very good results. The table shows a representative series of chloroform-benzene solutions for use as markers. Despite the miserly economy of this droplet procedure, variation between student determinations approaches zero for any given hatch of cells even to three significant figures.
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Discussion Since the earliest days of molecular biology, the ultracentrifuge has been a basic tool, which has been used in two different ways that must be clearly differentiated. On the one hand, i t has been used to obtain the velocity zonal sedimentation bands by which proteins or nucleic acids have been _separated according to their molecular weights. This method
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Figure 1. A linear gradient making device can be constructed readily hom two small polyethylene bonles, pinch clamps, and short lengths of plastic tubing. Once the device has been secured in a frame with clamps pinching off both tubes, saturated NaBr solution is added to the proximal chamber.An equal auanlitv of a more dilute solution of NaBr contsinino the E. col is added to the caoillarv. distal chamber. A SDecifIc oravitv " . of 1.1 is remrniended. With the~~~. t ~ o i n gleadingto the dpper. mner lip of the centr luge 1.m. the clampsare removed and a 10- hab graden slowk 1-3 over a period at 5 rnrn in me fube. M xing in me proximal side 13 iacilMted by buobl'ngand stmng wma propel er magnetic bar ~
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Internal Denslty Standards lor Use In lsopycnography Cn orolorrn Benzene Spec~f~c Solul~an NO. (ml) (ml) Gravity 1 2 3 4
20 18 16 14
0 2 4 6
1.46 1.40 1.34 1.28
of study has generally used preformed sucrose gradients. This is a dynamic system and is nor used to reach a state ofequilihrium. The second method of use is the eauilihrium bandine of isopycnography, of which the describeci experiment is a; example. Most frequently gradients of the very dense cesium salts are used in such determinations as DNA base composition. the fraction of the genome codin2 for various functions and D N A ~ N A via (he differential handing O ~ D N ARNA, , hvhrid. or the isolation of olasmid and reromhinant DNA &om the bulk cellular D~A.'Unlikethe described experiment, the madients of cesium salts are not nreformed but are allowed to divelop during the run as the consequence of an equilibfium between the two counteractina forces of centrifueal field and diffusion over a period of daysrunning at very hiih r.p.m. In the resulting gradient the experimental biochemicals isoVolume 59 Number 12 December 1982
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Figure 2. The determiretionof specific graveies of resultant bands hesh hom the cemrifuge (a) can be made either refractometricaliy by using very small samples withdrawn from Ihe bands, or by the insertion of droplets of immiscible organic liquids of known specific gravities into the gradient. In (b). droplets of four different chloroform-benzene solutions mentionedin Me table float at the ieveis indicated at the side of the tube. Two bands are shown for Illustrative purposes only. The upper band has a specific gravity centering upon 1.36glcc. This band is diffuse because of great heterogeneity in cellular composition Ihroughout the popdation. The lower band, which is wmposedof cells having a high degree of homogeneity, has a specific gravity of 1.42 glcc.
pycnotically position themselves. Preformed isopycnotic eradients have nevertheless also been used to eood advantaee yn the case of handing viruses and viral components in hoih
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Journal of Chemical Education
sodium and potassium bromide gradients. But here too, len&hv ultracentrifueation has been necessarv due to the still small Hize of even whole virions. Thus, with an increase in size of three orders of mamitude over that of viruses, we have been able to use E. coli as an experimental obiect, and allow the heainnina student to aain a personal expe;ience with i s o p y c n o ~ r a p h ~an n inexp&sive and rapid manner. Beyond the mere acquaintance with the extremely simple experiment outlined above, the student may wish t o delve further into isopycnic analysis of intact cells such as several of my students have done. Because E. coli fortunately are totallv ~ermeableto sodium halides.' anv s~ecificeravitv chan&as measured in NaBr are a reflection i f chan;ng in"tracellular "dry wei~ht"com~ositions.This has been found to exist over the gro&h cycle ifthe bacterium and has led, for example, to some interesting observations concerning the controls over the synthesis of ribosomes, the seat of protein ~ynthesis.~.~ Isopycnography is well within the grasp of the average students' abilities, and, with imagination on the part of the instructor, many doors may he opened even onto questions a t the cutting edge of biochemistry in an introductory course.
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Ledebo, I., and Ljunger, C., Physlol. Plant., 28, 535 (1973). 2Patterson. B., Czerkawski, J.. Howard, S., and Vermeulen, C., Blochem. Biophys. Res. Comm., 95,958 (1980). (hrann, C.. Pandak. H., and Vermeulen, C., Bnchem. Biophys. Res. Comm., 97,520 (1980).
