Download Solubilization and binding ofDNA-CTAB complex with SDS

Indian Journal of Biochemistry & Biophysics
Vol. 36, August 1999, pp. 233-239
Solubilization and binding ofDNA-CTAB complex with SDS in aqueous media
SA Gani, D K Chattoraj* and D C Mukherjee t
Department of Food Technology and Biochemical Engineering, Jadavpur University, Calcutta 700 032
Received 5 August /998; revised /9 March /999
Extent of binding (r~) of sodium dodecyl sulphate (SDS) to the binary complex fonned between calfthymus DNA. and cetyltrimethylammonium bromide (CTAB) has been measured in mole per mole of
nucleotide in the complex as function of concentration of SDS by using equilibrium dialysis technique at
different temperatures and pH. Binding of SDS to thennally denatured DNA-CTAB complex has also been
studied. The most interesting aspect to be noted in this experiment is that the water insoluble DNA-CTAB
binary complex gets solubilized in the ternary mixture in presence of SDS but when DNA is thennally
denatured, the ternary system DNA-CTAB-SDS remains insoluble. Significant change in the extent of binding
has been noted with the variation of the relative composition of DNA and CT AB in their binary mixture. The
data of binding of SDS to DNA-CTAB complex are compared more precisely in terms of the standard Gibbs '
free energy decrease (-MJ") for the saturation of the binding sites in the complex with the change of SDS
activity from zero to unity in the rational mole fraction scale.
It i~ well-known that anionic surfactant sodium
dodecylsulphate (SDS) has been frequently used for
the lysis of living cell whereby intact high molecular
DNA on separation from cell becomes solubilized in
SDS solution along with some basic protein
histone1.2. It has also been reported that the
dissociation of chromatin to form histone and DNA is
enhanced in the presence of non-ionic surfactane
Triton-x I 00. Chatterjee and Chattora/ have earlier
shown from equilibrium dialysis experiments that in
the aqueous media DNA polyanion is totally unable
to bind SDS anion in aqueous solvent because of high
electrostatic repulsion effect which apparently
indicates that there is no direct relation between high
solubility of cellular DNA to its gross binding with
SDS dissolved in aqueous media.
Chatterjee and Chattora/, however, observed from
equilibrium dialysis experiments that cationic
surfactant cetyltrimethylammonium bromide (CTAB)
in aqueous solvent bind to dissolved DNA
exteQsively forming saturated insoluble DNA-CTAB
complex at neutral pH. In the saturated state, it has
*To whom all correspondence regarding this paper be made.
lUniversity College of Science and Technology, Department of
Pure Chemistry, Calcutta University, 92, A.P.C. Road,
Calcutta 700 009 .
been noted that one negatively charged nucleotide ion
is in ion-pairing interaction with one surfactant cation
under suitable physicochemical condition. Further
hydrophobic groups of bound CTAB are associated in
the complex as a result of strong hydrophobic effect.
From a recent studl of the kinetics of binding of
CTAB to DNA, it has been observed that
electrostatic, hydrophobic and conforrnatiomil change
effects of biopolymers have significant role in the
interaction of CTAB to DNA. Earlier from spectroscopic study 7.8, hyp 'chromic shift of DNA 7,8 and
DNA-CTAB comple . .9 and the melting temperatures
were observed to be close to each other which
indicated the presence of large fraction of doublehelical DNA in DNA-CTAB complex,
Recently, Kunjappu and Nair lO from the analysis of
optical data reported that DNA-CTAB complex
solubilizes in SDS micelles by a mechanism of some
complex interactions, In the light of solubilization of
cellular DNA in presence of SDS and in the absence
of any interaction of pure DNA with SDS earlier
reported 4 by us, further analysis of inertness of SDS
against binding to pure DNA in aqueous solution'and
strong affinity of SDS for solubilization of DNACTAB complex have been made in the present
investigation.
234
INDIAN J. BIOCHEM. BIOPHYS., VOL. 36, AUGUST 1999
Materials and Methods
Calf-thymus ONA (Lot No. 67F-9725) completely
free from protein (confirmed by comparing the
optical density values of a solution of ONA in 0.1 M
NaCI at 260 nm and 230 nm as well as from a
negative folin-reagent test)II.12 and surfactants cetyl
trimethylammonium bromide (CTAB) and sodium
dodecyl sulphate (SOS) of Sigma Chemicals
Company, USA were used. Other chemicals like
NaCI, CH 3 COONa, NaH 2P0 4 • Na 2HP04 • methylene
blue, disulphine blue and chloroform of standard
grade were used. The cellophane sacks (lot No. 22H615.6, Cat No. 250-7U) capable of retaining proteins
of mol ecular weight greater than 12000 (purchased
from Sigma Diagnostics, St. Louis M063 178, USA)
were used. Oouble distilled water was used
throughout for the preparation of solutions and
washing purpose. The di alysing casings were washed,
dri ed and made ready for the experiment in the usual
procedure IS, cutting into pieces of three inches length.
Stock solution s of acetate (PH 5.0) and phosphate
(pH 7.2) buffer were prepared and the ionic strength
was maintained by the addition of suitable quanti ties
of NaCI solution . The pH of the solutions were
measured in a digital pH-meter (LI20, Elico, India)
with an accuracy of ±O.O I pH. Certain volumes each
of 0.05 g % (w/v) ONA solution (having 1.5 x 10-3 M
nucleotide concentration ' \ 1.2 x 10.3 M CT AB solution and 0.0 I M SOS solution were prepared in a
buffe r o f definite pH and ionic strength. The heatdenatured DNA solution was prepared from the stock
so lution foll owing sta ndard procedure '4 . The stock
solution orONA was also diluted to prepare 0.02 g %
and 0.0 I g % of native ONA solutions. Further, stock
solution of CT AB was diluted to prepare 6.0 x 10-4 M
and 4.0 x 10-4 M solutions.
In the equi libri um dialysis experiment' s.'6, one end
of each dialysing casing was knotted by thread and 1
ml DNA, I ml CTAB and 3 ml of SOS solution were
taken in it, knotted the other end, the mixture was
shaken very well to solubilize the precipitate of
DNA-CT AB complex in SOS and the bag was
dropped in to a well stoppered standard-joint conical
flask containing 20 ml buffer solution of same pH
and ionic strength. The flasks were then placed on a
mechanical shaker kept in a temperature-controlled
incubator for 48 hrs to attain the dialysis equilibrium.
The temperature was maintained at 28.0±O.0 1°C. The
equilibrium concentration C 2 of SOS in the dialysate
was determined by dye-partition technique using
methylene blue dye ' 7.l8 . Since ratio of added moles of
CT AB (n~TAB) to moles of nucleotide (n~uc1eJis 1.0
in most cases, almost all CTAB molecules are bound
to ONA which should lead to complete precipitation
of the ONA-CTAB complex. It has been earlier
shown s that one mole of nucleotide may li>ind nearly
one mole of CTAB forming saturated complex.
Concentration of CT AB in the solution within the bag
should be negligibly small indeed during dialysis. But
due to the addition of 3 ml of SOS ~olution (diluted
25/3 times at dialysis equilibrium) the precipitate of
ONA-CTAB complex was observed to dissolve
completely in solution due to the formation of CTABDNA-SOS ternary complex. Absence of CTAB in the
dialysate solution at equilibrium was confirmed by
testing the outside solution with dye-partition
technique ' using disulphine blue IS. Moreover, if
CTAB would come out from the dialysis bag some
precipitate would have been observed in the dialysate
solution . due to the formation of SOS-CTAB
complex 19. But no such precipitate was observed in
the dialysate at equilibrium. Since the solution inside
the dialysis bag containing the ternary complex is
also optically clear, there is no formation of
precipitate made of CT AB-SOS complex also in the
inner compartment.
From the known initia{ and the measured
equilibrium concentrations of SOS in each set, the
number of moles of SOS (r~) bound per mole of
nucleotide in the DNA-CTAB complex was
calculated using equation (I)
rl =
2
(C~ -CJ:~ X330
W
1000
. .. (1)
where C~ and C 2 are the molar concentrations of SOS
in the total volume yt (equal to 25 ml) of the
dialysing system before and after dialysis equilibrium
respectively. W is the amount of ONA (in gram)
taken in the dialysing casing. The average number of
moles of nucleotide present per kg of ON A was found
by Falk 13 to be 3.02 and thus one mole of nucleotide
weighs 330 g. The solution of SOS being very dilute,
the equilibrium molar concentration (C 2) of SOS was
converted to mole fraction (X2) using equation (2)
x ~~
2 -
55.5
... (2)
235
GANI et al.: SOLUBILIZATION AND BINDING OF DNA-CTAB COMPLEX WITH SDS
28° and 37°C and at pH. 5.0 and ionic strength 0.05
have been plotted against concentration (C 2) of SDS
remaining free in the system. In all these
By varying C~ in the system, values of r~ for
different values of C2 have been determined
experimentally at constant ratio r equal to
I
/
experiments, n~TAB / n~ucleo before binding was unity
to ensure formation of DNA-CTAB saturation
complex. Trace of CTAB in the solution may
combine with SDS and the binary complex also
combines with DNA-CTAB complex so that solution
becomes optically clear inside the bag. From the
isotherms presented in Fig. 1, we note that with
I
nCTAB nnUcleOlide '
Results and Discussion
Like many observations made by earlier workers 5•IO
we have also noted that at a suitable pH, addition of
cationic surfactant to DNA leads to the precipitation
of saturated DNA-CTAB complex when r is close to
unity. In agreement with recent observations of
Kunjappu et al. \0 we have further noted that DNACTAB complex is solubilized by gradual addition of
SDS solution. But the system becomes optically clear
when SDS concentration in·the system at equilibrium
is considerably lower than its cmc. This solubilization
is in all probability due to the formation of CTABDNA-SDS ternary complex since in the presence of
SDS we observe that there is no dissociation of DNACTAB complex leading to the precipitation of CTABSDS complex. Moulik et al. '9 and others have earlier
shown that free CT AB may interact with free SDS in
aqueous medium due to the formation and
precipitation of ion-paired binary complexes.
increase of C2 • the magnitude of r~ increases from
zero and at critical concentration
3.0x 10-4 M,
r~ reaches maximum value
given value of
SDS.
3.6
C~
considerably lower than cmc of
10.8
14.4
6.0
8.0
'"
0
.!!
u
:>
Z
a 2·0
II
0
~
~
0
~
c
""
:>
0
.Jl
•
""
V>
0
V>
(r;,). This
from Table 1 that values of r 2m 1.8, 1.6 and 1.3
moles of SDS are respectively bound per mole of
nucleotide of DNA-CTAB 'complex at 18°, 28° and
37°C. This indicates physical nature of SDS binding
to the complex. Further the binding process leading to
solubilization becomes complete in all cases at a
'5
--
nearly equal to
value of r 2m at different temperatures may be
regarded as apparent maximum amount of SDS in
moles required to saturate CTAB-DNA complex thus
forming the ternary complex. It may also be noted
In Fig . 1, moles of SDS bound (r~) per mole of
nucleotide present in the DNA-CTAB complex at 18°,
0·0
C~
1.0
.
1)
II
"0
~
-L'"
Fig. I- Plot of r~ against C2 or X2 of SDS at pH 5.0 and 1-1=0.05 [(.), 18°C; (0), 28°C; (1'.), 37°q
236
INDIAN J. BIOCHEM. BIOPHYS., VOL. 36, AUGUST 1999
In Fig. 2, the isothenns for binding of SDS at 28°C
and ionic str~ngth 0.05 to saturated DNA-CTAB
complex at three different pH values 5.0, 7.2 and 9.0
helix structure in the complex remains intact. Further
although the ternary complex of DNA-CTAB-SDS is
able to fonn optically clear aqueous solution, the
solution in the dialysis bag at equilibrium at pH 7.2
and 9.0 becomes turbid since alkali denaturation of
the DNA present in the teranary complex leads to the
insolubility of DNA-CTAB-SDS component in
aqueous solvent used. In the same figure the isothenn
for binding SDS to heat-denatured DNA-CTAB
have been compared. With increase of pH, r 2ffi is
observed to decrease systematically (vide Table 1).
DNA is known to denature with increase of pH from
5.0. The denatured complex has lower capacity for
binding SDS . Maximum binding sites for saturated
DNA-CTAB complex are available when the douhle-
X2 xlO
10.8
7.2
3.6
0·0
6
14.4
I
II
I
~
.
o
II
v
::>
Z
'0
~
~
~
c
::>
/
o
/
.D
.
I
o
-..
- -----------.
0
/
VI
VI
.
./"
/
-v
2·0
o
-
I'
I
I
I
1·0
I
o
I
/
I
-0
•
I.
~
2.0
4.
8.0
6.0
4
C2x10 (M)
Fig.2-Plot of r~ against Cz or Xl of SDS at 28°C and ~ = 0.05 [Native DNA-CTAB complex: (-----), pH 5.0; (0), pH 7.2;
(~) , pH 9.0. Heat-denatured DNA-CTAB complex: (e), pH 5.0]
Table I-Binding parameters for SDS binding to DNA-CTAB complex at
Composition
Temp.
pH
c
2
mx
lO4
(K)
~
= 0.05
Moles
SDS/mole
nucleotide
-6(i.ox 10.2
kJlMole of
nucleotide
-(6G~i xl 0.2
f2m
kJlMole of
nucleotide
30 :
291
)10
301
301
5.0
5.0
5.0
7.2
9.0
3.4
3.2
3.6
3.4
3.0
1.6
1.8
1.3
1.4
1.1
0.61
0.68
0.50
0.53
0.42
4.77
2.72
3.27
4.04
5.42
Heat-denatured DNA (0.02 g%)
+CTAB (O.6xlO·) M)
301
5.0
3.2
0.78
0.29
5.67
Native DNA (0.01 g%)
+CT AB (l.2x 10'" M)
301
5.0
4.4
2.6
1.01
5.39
i.e., n ~"TAB / n~ucl<o =4.0
Native DNA (0.05 g%)
+ CT AB (0.4x I O·~ M)
301
5.0
3.0
0.6
0.23
3.97
Native DNA (0.02 g%)
+ CrAB (0.6x I0··' M)
i.e., n~AB/n~ucleo = 1.0
i.e., n~AB / n~ucleo =0.27
237
GANI el af.: SOLUBILIZATION AND BINDING OF DNA-CTAB COMPLEX WITH SDS
complex has been compared with that of native DNA-
r=0.27, 1.0 and 4.0, DNA-CTAB complexes form
m
optically clear solution. The variation of [2m with r
has been shown in Fig. 4.
Values of the standard free energy change (t1CO) in
CTAB complex at pH 5.0 and 28°C. The value of [2
has been found to be reduced to half due to heat
denaturation of DNA. Further ternary complex
containing denatured DNA remains insoluble in
solvent used .
In Fig. 3, isotherms for binding SDS to DNACTAB complexes prepared in the ratio r (i.e.
kJ per mole of nucleotide for the transfer of [ ;
moles of SDS to DNA-CTAB complex due to change
of SDS concentration in the bulk from zero to unity in
the mole fraction scale have been calculated using the
integrated form of our derived equation I5 ,2o.22.
n ~TAB I n:1UCleO) equal to 0.27, 1.0 and 4.0 respe~tively
at pH 5.0 and at 28°C have been compared under
identical physico-chemical condition. With increase
t1G'<>= -RT
of r from 0.27 to 4.0 [;1 increases from 0.60 to 2.60.
This means that there is excess binding of CTABSDS complex on saturated DNA-CTAB complex
when r is eqllal to 4.0 leading to the formation of
DNA-CT AB-SDS ternary complex. When r is as low
as 0.27,
m
[2
[Ill
[fX
dX + [m I n 2
2
Xm
_ 2
... (3)
0 2 2
Here X~ is equal to C~ 155 .5 since the solution is
dilute,
These values are included in Table 1. t1CO for
is as low as 0.6. It may be pointed out
different systems are found to vary linearly with
here that previously [ 2m has been found to be zero,
when CTAB is absent in DNA solution (i.e. when
r=0) . It may further be pointed out here that for
z
3.6
6
7.2
10.8
14.4
•
-0
0
.! 3.0
v
:>
Z
/
/
"0
/
•
-
I
/
0
I
~
-0
c
/
/
2.0
I
:>
0
I
..Q
/
/
."
"
0
."
.
"0
•
'0
~
"2"
).0
o
6.
o
0
o
2.0
m
[2
and the slope of the curve equal to 11 COl [ 2m are found
to be -38.0±0.6 kJ per mole of SDS transferred from
bulk to the ternary complex, This value represents the
X x 10
0.0
X~
4.0
6.0
S.O
4
CzxlO (M)
fig . 3-Plot of r~ against Cz or X z of SDS at 28°C. pH 5.0 and J.1 = 0.05 [(~), (CT A B)/(N ucl eo )= 4.0; (----), (CTAB)/(Nucleo)= 1.0;
(0), (CT AB)/(Nucleo)=0.271
238
INDIAN J. BIOCHEM. BIOPHYS., VOL. 36, AUGUST 1999
3·0r-----------------------~
o
.!!
region of concentration can be calculated from
derived thermodynamic equation2l
/)"G o =
2.5
ap
u
::J
Z
o
Q)
(5
'U
C
::J
o
.D
2
.. . (4)
2
From the linear plot of L'lG ~p against 11
'.0
VI
Q)
o
~
2
2
estimated from graphical integration 20•21.
1.5
o
(/)
o
o
1 ]
X
dx +r'ln-
equal to unity so that AG ~p for each event can be
(/)
-
rl
_ 2
[
Here it is assumed that value of r~ at experimental
value of X2 remains hypothetically constant up to
2.0
~
"
fX
X2
-RT
0·5
EN
t....
O·OL-____
____ ____ ____
2.0
1.0
4·0
3·0
0·0
~
nf
~
{ CT AS
Fig.4- Pl ot o f
r;'
~
~~
jntnucleol
aga in st n ~TAB I n ~ucleo for binding SDS to
nati ve DNA -CTAB compl ex at 28°C, pH 5.0 and Il = 0.05
standard free energy change for the transfer of one
mole of SDS from bulk solution to the complex as a
result of change of bulk mole fraction of SDS from
zero to unity.
From Table I we find that values of /),,(f for
binding SDS to DNA-CT AB complex at pH 5.0 and
at 18°, 28° and 37°C are - 68 .0, -61.0 and -50 .0 kJ
per mole of nucleotide respectively. Using GibbsHelmholtz equation2 1 values of enthalpy change
/),,}{ ~v at average temperatures 296 and 305 K are 271 and -428 kJ per mole of nucleotide and
corresponding values of entropy (Tav LlS'av) contributions are -206 and -372 kJ per mole of nucleotide
respectively. It thus appears that binding process is
both enthalpy and entropy controlled.
In all the binding isotherms presented in Figs. 1 to
3 r~ increases sharply from the apparently maximum
value r~" when C z » C ~' . Such increase in r~ near
and above C 2 equal to 6.0x 10.4 M or so is sharp and
binding of SDS in this region appears to be cooperative in nature although C 2 is still far below emc
of SDS . Apparent values of standard free energy
change L'lG ~J1 for each value of C2 in this higher
JX:
as
shown in Fig. 5, the standard free energy change
.(/),,(f)hi at unit mole fraction for each system at
relatively high range of concentration of SDS has
been evaluated (vide Table 1). Difference between (M;<»hi and -/),,(f is negative and large in all cases.
Further this difference increases without limit or C2 is
brought closer to cmc of SDS. Large number of SDS
molecules thus bound to the DNA-CTAB complex
undergo co-operative interaction with each other
wher-eby solubility of DNA is increased to a large
extent. Interestingly, solubility of DNA-CTAB
complex in SDS micelles depends strictly on the
maintenance of double helical structure of DNA.
Using the Gibbs-Helmholtz equation for different
values of /),,(f at different temperatures, values of
~J-/ :,
at average temperatures 296 and 305K are
found to be 5690 and -5490 kJ/mole respectively
whereas corresponding values of T.v L'l S ~v at these
two average tl;:mperatures are 6060 and -5090
kJ/mole respectively. All these results indicate that
the complex inte:raction or DNA-CTAB complex with
SDS both at 'high and lower ranges of surfactant
concentrations
are
guided
by
co-operative
interactions involving enthalpy-entropy compensation
effect.
As far as solubilization of DNA-CTAB complex in
SDS is concerned, only at pH 5.0 and at three
different temperatures the DNA-CTAB-SDS complex
in aqueous media form optically clear solution at low
and high concentration of C 2 below erne of SDS. For
DNA, denatured! by heat or alkali addition, SDS binds
to CTAB-DNA complex but the mixture becomes
turbid in aqueous solution of SDS even though excess
binding of SDS to insoluble CTAB-DNA complex
occurs to significant extent.
Therefore for complete solubilization of CTABDNA complex by SDS confonnation of DNA must be
239
GANI et al.: S.OLUBILIZATION AND BINDING OF DNA-CTAB COMPLEX WITH SDS
2.0r------------------------,
N
'0
... ...
)(
:2'1.0
....,
---::t:
a.
00
19
<J
"~--_o_-
_
____.&
e-
,
O. O_~------~------__".L"..__-------J
2.5
3.0
3.5
4.0
1
-2
-x10
fi2
Fig.5- Plot of -!l G ~p against
1/.JX:
for binding SDS to native DNA-CTAB complex at 37°C, pH 5.0 and
native and double helical structure is maintained and
even tertiary folding of DNA in the complex takes
place. Thus although SDS does not bind to pure
DNA, the DNA-protein complex within the cell
similar to DNA-CT AB complex can be completely
solubilize,d in SDS solution. Intact and possibly very
high molecular weight DNA along with bound
protein from the cell can thus be extracted by the use
of moderate as well as high concentration of SDS.
Further, recent experiment with X-ral 2 has indicated
the position of various basic proteins bound with
DNA in the nucleosome. SDS solution of different
concentrations may be used for partial or complete
solubilisation of various types of protein-DNA
complexes from the nucleosome.
6
Acknowledgement
The financial assistance of Indian National Science
Academy, New Delhi to one of us (DKC) is
acknowledged with thanks.
16
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2
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