Effect of Temperature on the Buoyant Density
of Bacterial and Viral DNA in CsCl Solutions in
the Ultracentrifuge
JEROME VINOGRAD and ROBERT GREESWALD, Gates and Crellin
Laboratories of Chemistry* and Norinan W . Church Laboratory for Chemical
Biology, California Institute of Technology, Pasadena, California, arid
JOHK E. HEARST, Department of Chemistry, University of California,
Berkeley, California
Synopsis
The effect of temperature on the buoyant density of CsDNA in CsCl at equilibrium
in the ultracentrifuge has been measured between 5 and 60°C. The temperature coefficients of the buoyant density for three DNA's of varying guanine-cytosine (G-C)
contents were, within experimenhl error, +4.2 f 0.3 x
g./ml."C. This coefficient may be used to normalize results obtained between 5 and 60°C. to 25°C. The
results are explained in terms of a loss of water from the hydrated CsDNA complex.
It is estimated that about one-third per cent of the water preferentially hydrated by
the DNA is liberated per degree elevation in temperature. Significant errors in buoyant
density determinations are made if the thernial expansion coefficient of CsCl solutions
is ignored.
Introduction
The buoyant density of a macromolecule at sedimentation equilibriuin
in a density gradient is a thermodynamically defined quantity representing
the density of a complex forined from the anhydrous, electrically neutral
iiiacroniolecule and a definite amount of preferentially bound solvent. 2 , 3
The buoyant density is evaluated by measuring the density of the neutrally
buoyant binary solvent. The composition of solvated CsDNA varies
widely in different cesium
an effect attributed to the varying water
activities in the buoyant salt solutions. The buoyant density of CsDNA
has been shown to decrease with increasing pressure in CsCl so1utions.j
We now report that with an increase in temperature the density of the
CsDNA-water complex in CsCl increases and that water is lost from the
c01nplex.
Materials and Methods
Optical grade CsCl supplied by Harshaw Chemical Company was used
without further purification. FC-43 Fluorochemical from Alinnesota
* Contribtition No. 2935.
1OY
VISOGRAD, GI<EI<SWALD, A U D IIEAIIST
110
3Iiniiig aiid JIaiiufacturing Coiiipaiiy was washed with \vat cr three t iiiies
to reiiiove trace amounts of water soluble iiiipurity. The E. coli DX.4 aiid
the A t . Zysodeikticus D S A were generously supplied by Dr. 1'. 0. 1'. Ts'o
arid Dr. T . W. Thonipson. They were prepared by the niethods of JIarmur6and
respectively. The T-4 bacteriophage D S A was prepared
by extraction of the purified virus by phenol by the procedure of 3Iandell
arid Hershey.*
3Iost of the experiments were perforiiicd iii a Spiiic.0 JIodcl IS aiialytiral
ultracwitrifuge equipped with thc standard RTIC teiiiperaturc coiit rol
system aiid phase-plate sc.hliereri opt its. The first high-teniperature csperiments were performed in a ma(-hirieequipped with a high-teiiipcrat ure
heating apparatus. It was found, however, that the uppcr l i i i i i t for the
Kel-F plastics centerpieces, about 65"C., did riot exceed the oporaiiiig
capability of the standard ultrace~itrifuge.~Above 1O"C., a high-tciiiperature thermistor was used.
All the experiments were perforiiied with paired CsCl solutions run
siinultaiieously at 44,770 rpm. Both double-sector, graphite-filled Epoii
cent crpieces aiid pairs of 4" graphite-filled, Iiel-I? cent erpieces were uscd.
The double-sector c d s were unsat isfactory above 40°C. because of leakage
a(*rossthe partitioii. 111 ruiis above 33°C. liel-IT filling-plug gaskets w ~ e
used. Cells ~veretightened to l l , j in.-lh. at rootii teiiiperature. The rotor
a i d cell assenibly were preheated or cooled to a teiiiperaturc of about
5°C. above or below the desired operatirig teiiipcrature. .I thiti layer
of fluorocarbon oil was uscd at the base of each licluid c.oluiiiii to provide
an unaiiibiguous hot toiii iiicnisrus.
Densities were iiieasured 011 sainples of the original solutioiis or 011 the
homogeiiizcd c d l contents at the eiid of each run. 111thc latter rase, care
was taken to prevent the fluorocarboii oil prcseiit froiii iiitc.rfcriiig. *
100
I
pl. niicropipct used as a pycmomcter was weighed on a JIcttlcr iiiic.rohalanre at 2.j"C. Deiisities acwrate to *0.001 g./nil. ~vcrcohtailled. h
period of 24-30 hr. was allowed for the scditiiciit ing systciii to rcacah equilibrium. Hands wm usually apparent within 12 hr. at 60°C.; d1aiigc.s i n
band shape were still iioiiwable between 24 aiid :3(i hr. at 5 ° C . I'hotographs takcii with the schlicreii optical systciii were iiieasured with a
Sikori JIodel 0 projec-tioii votiiparator. Band positions werc taken i o he
the average of the posit ivc arid negative pcaks.j Readings w r c awuratc
to +0.001 i i i i i i . The huoyaiit density at hand c ~ c i i t cwas
~
cd(*ulatcdu-ith
t hr equat i o ~ i s , ~
= pe.1'
(dp/d,.)1,.?O =
+
(dp/d7.)1
(dp/d,.)+
()*0,1
-
)'p,1)
= (dp/dr)P
= p,
+ (dp/cl/.)zo
(I.",?
-
I.?,?)
(1)
=
(Pr.?O - p r , l " ) / [ ~ l s o . l
rt.l)
-
OS0.? -
).C.Jl
(4
where po" is the dciisiiy a t atiiiospheric prcssurc of a CsCl solutioti
iii
which the DXA will band at the isoconcwitratioii positioii
in the liquid caolutiiii. The dciisity of this solution is the buoyant density.
TEMPERATURE EFFECT OR T H E BUOYANT DENSITY OF DNA 111
The radial distances
and
are the equilibrium band positions, and
are the isoconcentration distances in the two solutions. In
CsCl a t a density of 1.70 g./ml., the isoconcentration distance occurs a t the
root-mean-square position in the liquid column. The densities pe,l0 and
p,,zo are the densities of the two homogeneous solutions, so selected that the
bands form both above and below the root-mean-square positions. The
densities of the paired solutions differed by about 0.06 g./ml. The DNA
concentrations were OD260 = 0.15 for the bacteriophage D N A arid ODZEO
=
0.40 for the bacterial DNA's.
In applying eqs. (1) and (2), values for the solution density at the appropriate temperature were calculated with the thermal expansion coefficient,lo
4.3 X lop4OC.-l for 55y0 CsCl solutions, pZ5 = 1.68.
Y ~ Jand
Results
Buoyant densities for three different DNA's as a function of temperature
are given in Figure 1. The data for the T-4 bacteriophage D N A are COINpiled from experiments with five different sets of starting solutions. Some
of these were run at more than one temperature.
The bacterial
DSA's were run with three separately filled sector pairs. The slope of the
best line through the T-4 points, determined by the inethod of least squares,
is 4.2 f 0.3 X lo-* g./inl."C. The slopes for the E. coli and M . Zysodeikticus D N A lines, 4.7 and 4.2 x
g./nil."C., respectively, are the same
within experimental error.
1.66
I
I
I
I
I
10
20
30
40
50
60
TEMPERATURE, OC.
Fig. 1. The dependence of buoyant density on temperature: (a) M . Zysodeiklicus
DNA; (A)E. coli DNA; ( 0 )T-4 bacteriophage DNA. The dashed line is the "apparent" buoyant density, which is the density a t 25°C. of the CsCl solution buoyant for the
DNA at temperature T.
112
VIKOGRAD, GREEUWALD, A N D IIEARST
The dashed line in the figure shows the “apparelit” buoyant density of
T-4 DNA as a function of temperature. This quantity is calculated without regard for changes in density of the CsCl solution resulting from the
changes in teinperature from 25°C. The line describes the downward displacement of the band in the liquid coluiiiri upon increasing the temperature. Its slope is 11.4 + 0.3 X lop4g. /ml.”C.
Positive values for the expaiisioii coefficieiits of the solvate water arid the
anhydrous macromolecule lead to a prediction incorrect in sign for temperature dependence of buoyant density. That the changes in buoyant density were not due to deriaturatiori was shown with the aid of spectrophotonietric heating studies. These were perforiiied with T-4 DNA i n 0.15,11 NaCI0.01M Tris buffer, pH 7.4, arid in 7,11 CsCI. A shift in the tiieltirig point
mas observed from 86°C. to ’33°C. No sigriificmit effect of the high salt
concentration was rioted except i n the region of the iiieltiiig point. The
hyperchroiiiicity was the sariie, 387, arid 40%, in the two solvents.
Discussion
If we assuiiie phase equilibriuiii between the solvate water atid the water
in solution it is possible to estiiiiate the eiithalpy change for the reaction
AH
DNA, rH20 S CSCI, H,O
Consider the following reactions:
AH
DNA, I’H20 $ H 2 0vapor
(4)
for which
and
for which
111(Pb’/Pa)
= -
(AH,,p/R) [(1/Tb)- ( l / T a ) ]
At teiiiperature
DNA, r H 2 0 is in equilibrium with CsCl, H20 solution
arid a vapor at partial pressure P,. At /6, DNA, rH20 is i i i equilibrium
with vapor a t partial pressure P b , arid CsCl, H 2 0 is in equilibriuiii with
vapor a t partial pressure Po’. Subtractiiig cq. ( 3 ) from eq. (4) yields
hI(Pb/P,’)
= AU,/U,
=
(AH1/RT2)(AT)
(6)
where a, the water activity is measured at T,. The quantity Aa, is the
difference betwem the water activity of the solution i i i equilibriuiii with
DKA, rH20 a t Tb arid the water activity at Tb of the salt solution which
remain conwas in equilibrium with DNA, r H 2 0 at T,. 111 order that
stant,
(bI’/ba,),Aa,
= -
(bl’/bT),wAT
(7)
'L'EMPEHAI'URE EFFECT ON THE BUOYAXT DENSI'I'Y OF DNA 113
Any function which is a measure of r niay replace r in eq. (7). For example po,> which is corrected for the thermal expansion of the niacromolecule and solution niay be used. This correction is made with eq. (8),
where T , = 25°C.
PO,?
= po,zs0[1
4-(YDNA - ycSci)ATI
(8)
The quantities ?DNA arid ycScl are the thermal expansion coefficients for
hydrated DNA arid CsCl solution, respectively. Finally, because the slope
of the dashed line in Figure 1, (dpoo,zs/dT),
indicates changes in salt concentration as well as temperature,
(dpo0,25/dT)(1 - a )
=
(dpoo,25/dT)azo
(9)
where CY is a quantity defined previ~usly.~Combining eqs. (7), (8), and
(9) yields
A%/AT
= -
(1 - a ) [(dpOfl,25/dT)
PoO(yDS4
-
Y C ~ C I )I/@poo/%n)r,p
(10)
The appropriate constants (from ref. 4) are substituted into eqs. (6) and
(10) to obtain (11) for AH1 in cal./mole HzO.
AH1 = 31.2 X 104[11.4 X
+
(11)
~~'(YDNA
- YC~CI)]
In a similar iiianner an expression for (dr/dT),, can be obtained. Table
I provides a summary of values calculated for these two parameters. Case
3 represents a reasonable estimate of YDNA. It has been calculated on the
basis of a value of 2.5 X
OC.-'
for the hydrate water yIIto,and 6.6 X
lop4 OC.-' for the thermal expansion coefficient of the dry DNA. The
latter figure is taken to be the same as the measured values of y for heinoglobin" and ribonucleasegwhich are the only water-soluble niacroniolecules
for which data are available.
The value of AH1 for case 3 is 27Oj, of the heat of fusion of water. This
comparison is made only to demonstrate that the order of magnitude of
AH1 is reasonable. The positive value of AH1 does show that an energetically favorable interaction of water with DNA occurs in solution. I t
must be remembered that this AH1 is only valid for r = 0.29 in a buoyant
CsCl solution, arid that it describes a process in which water is transferred
into concentrated CsCl solution. It includes therefore the unknown heat
of solution of water in CsCl solution.
TABLE I
l'alues of AH1 and ( br/bT)um
~
Case 1,
=
YDNA
AHI, cal./mole H,O
356
( b r / w u mg., H,O/
g. DNA "C.
YCsCl
-9 5
x
Case 2,
YDNA
=
o
?DNA
131
10-4
-3 5
x
Case 3,
5.0 x 10-4
=
394
10-4
-10
5
x
10-4
E’roin t,he produd of l/r and (dI‘/dT)(lu,i t is estiiiiated that orie-t.hird of
one per relit of the preferentially solvated wat,er is released per degree.
Correctmionof buoyant deiisit,y froiii any temperature to 25°C. may be
acconiplished with the esperinietital slopcs frotti l‘igure 1. If an apparciit
is determined it, should he correc3tc.d usiiig the slopc of t’hedashed line,
11.4 x
g./ml. “C. If t.he actual buoyant density pOn is detcriniried,
rorrections should be made 1vit.h the slope of t-he solid lines, 4.2 X
g./111I.-”C.
The authors wish to tliank I>r. T. \V.Thonipson :tiid Professor P. 0. P. Ts’o for the
gift of the DNA samples. One of 11s (It. (>.) WBS the recipient of a. siimnier scholarship
196%o f the California Heart Associatioii. This work was supported in part hy Itesearch Grants He 03394 and G?\llllSO from the National 1nstit.iites of Health, [ - . 8.
Piihlic Healt,h Service.
References
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58, 728 (1958).
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(1961).
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Received Oct,ober 21, 196-2