lsopycnic Ultracentrifugation A Simple and Inexpensive Demonstration of Its Principles Pedro E. Berlot and Guillerrno A. Locascio Laboratow of Bioloaical Instrumentation, Facultad d e Cienclas Exactas Y Naturales. Universidad de Buenos Aires. Ciudad ~niversitiria,~uenosAires (1428), Argentina As i t is well known, isopycnic ultracentrifugation is an analytical as well as a separative technique based on the equilibrium attained when the driving force acting on a particle or macromolecule under a radial gravitatory field created by centrifugation reaches zero value in a given point of a densitv madient. where the densitv of the surroundine medium e;&tly balances the densityof the parti~1e.l.~ most important and classical application of this technique in molecular biology has been the elucidation of the mechanism of replication of DNA bv Meselson and StahL3 a real practical demonstration of this technique involves the use of expensive equipment (preparative ultracentrifuges with swinging bucket Cotors) rkn-ing a t least for several hours and a rather laborious preparation of special materials, which generally precludes such a demonstiation to he made. T h e present demonstration has been devised for our students with inexpensive, readily available materials and can be nerformed in 1h aonroximatelv. It allows one verv clearlv to iisualize density gradient separations and to unierstani the differences between the principles of eauilibrium and sedimentation velocity techniques.
Resin Number
Lower Density ~imn
upper Densny
Mean
Limit
Densiw
Gmuo
>1.3599
>1.3599
F
ow ever,
Materials Ion exchange resins: (1) Amberlite IRA 401 (C11 121 Amberlite IR-48 (OH-) i3j Dowex ~ ~ 1(dl-) x 8' (4) Cbelex 50-100 (Cl-) (5) Dowex 1x8 400 Mesh (CI-) (6) Dawex 21K 20-50 Mesh (CIY) (7) Amberlite IRA 400 (Cl-) (8) Amberlite IRA 938 (CI-) (10) Dowex 50x8 (Nail (11) Dowex 50WX4 (H+) 112) Dowex 1x4 400 Mesh (CIH (iaj Dowex 50W50X8 400 ~ e s (H+) h (14) Amberlite MB3 (Ht OH-) (15) Amberlite IR 20 (Ht)
15
'Resin numbsn as llsled under "Materials".
the sample (whether homogeneous or not). Analogously, the upper limit was determined by sucessive additions of 1mL of concentrated (density = 1.3599) solution to the lighter final solution described above until all the components of the sample float. The results, as well as the mean density value in each case, are shown in the table. Within the above estimated limits for each type of ion-exchange resin we needed to choose intermediate values required to form a density gradient, adequate to separate each component-if more than one. From tahles5relatine densities (.d.)of sucrose solutions,concentrationa (c) in g sugariL. solution, and percent weight (w) in g sucrose Ob g ~olution,polynomial approximations were developed by leastsquares methods:
-
Cane sugar (reagent grade not required) Teat tubes, 13-mmad.. 100-mmlength Clinical bench-toocentrifuge, oarillstins:tube holdern Mohr density baiance' Methods Ion-exchaneeresins were hvdrated for 24 h at room temoerature. Due to the f a 2 that the above-mentionedcommercial ion-khanre resins are often a mixture of products of different densities, some of them occasionally behaving like polydisperse systems, s previous estimation of the upper and lower densities encompassing these products was carried out as follows. A sample of 0.5-1 g of each &nmercial hydrated resin was placed ona pieceof thick lilter paper (01 blotting peperj to remove excess water and added to about 25 m L ofa 72%rucruse solution in water (979dl.s o h . densitv 2014 = 1.3599). Most of the resins float on this coicentrated, highIdensity solution. Then 1 mL water is added, homogenized and allowed to reach equilibrium. The procedure is repeated until all the resin sediments. The density of this final solution is determined with a Mohr balance, and this value was taken as the lower density limit of ~
~~~~
~~~~
~~~
+
d X 1713.0295 + d2 X 421.243666 Correlation coefficient: 0.99999 Estimated standard error: 0.75
e = -2128.31053
X
131.372738 Correlation coefficient:0.99999 Estimated standard error: 0.094
Data of w are included for indicative purposes only but were not used in our calculations. It is apparent from the mean density values that they can he grouped around six points A, B, C, D, E, F.A seventh,lighter density
'
Schachman, H. K. UlbecsntrifugationhBiochemisbx Academic: 1959. Tanford, C. Physical Chemistry of Macmmolecule~: Wiley: 1966. Meselson, M.; Stahl, F. W. Proc. Natl. Acad. Scl. 1958, 47, 671. Requlred only if resins other than those llsted here are employed. Weast, R. C., Ed. Handbook of Chemistry and Physics; CRC: 1987-1988; Sect D. p 262. Volume 67
Number 7
July 1990
627
10 DEF . .-- FN - - C(D1) = -2128.31053
+ (~1-2)*421.24366
+ D1*1713.02905
20 INPUT "DENSITY OF MOTHER SOLUTION: ";DE 30 C1= FN C(DE) 40 PRINT "CONCENTRATION IN G I L OF SOLUTION. "; C1 50 INPUT "FINAL VOLUME OF THE LIGHTEST SOLUTIONS: ".V OF SOLUTION: -;D 60 INPUT 70 C2=FN C(D) 80 ML=C2/Cl*V 90 PRINT "DILUTE TO ";V;"MILLILITERS ";ML;" MILLILITERS OF MOTHER SOLUTION " 100 GOT0 60
"DENS'ITY
The seven solutions are then prepared, and a drop of clear concentrated (155%)household detergent or shampoo is added to each 100 mL to nrevent resins from floating because of possible artifacts due to suriace tension. To each test tuhe 1mL of each sucrose solution is slowly added with a syringe or pipet, touching the inner wall with the tip, in decreasing order of densities and taking care not to disturb the successive layers. After the addition of the last and lightest layer (E), a small amount (e.g., 2W-300 mg) of hydrated resin drained as meviously described is added on top of the discontinuous density b d i e n t so formed. The set of tubes is then centrifuged far 10 min at 4CQO rpm (aooroaimatelv 3000 e for a standard clinical centrifuge). The rei z & ~ a rshown e in theffigure.
Isopycnic Separetions. Above: before centrifugation. Below: alter cenlrilugation. From len to right: upper rows. resins 1-8, lower rows, resins 9-15.
value (E), was included to have a top solution in which no sample floats. The values of those seven densities were chosen as 1.300, 1.280,1.260,1.170,1.125,1.110,and 1.100. A simple program, written in BASIC for an Apple II Euraplus microcomputer, was used to calculate the concentration of a given hiph-density suerose solution (A) and the volume of that mother solution required to make a fixed volume of each solution of lower density (B to E).
628
Journal of Chemical Education
Comments I n order t o visualize further the principles involved in isopycnic centrifugation, a very demonstrative additional experiments are strongly recommended. A resin containing several well-defined comDonenta, ex., resin # 3 or #5, is best selected for this purpose. Two tubes will show the same band distribution after centrifugation regardless of the initial position of the sample. It is suggested that in one of them the resin be placed "ah initio", before starting the addition of the densest solution and in the other one previously described, on top of discontinuous gradient. If desired, still another tube with a quasi-continuous gradient - . can be ~ r e.n a r e d24 h before. and the almost identical positions of the bands in this tube as compared with those of the discontinuous gradient can be observed after centrifugation of the same multihand sample, previously placed carefully on top of each tube.
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