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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 References 19 I 2 3 4 5 Davidson J N (1972) in The Biochemistry of Nucleic Acids 7th edition, pp.132, Academic Press, New York Marmur J ( 1961) J Mol Bioi 3, 208-213 T'oczko K & Kalinski A (1974) Bull Acid Pol Sci, Ser Sci Bioi. 22 (3), 163-169 Chatterjee R, Mitra S P & Chattoraj D K (1979) Indian J Biochem Biophys, 16, 22-27 Chatterjee R & Chattoraj D K (1979) Biopolymers, 18,147166 7 8 9 10 11 12 13 14 15 17 18 20 21 22 ~= 0.05 Moulik S, Chattoraj D K & Moulik S P (1998) Colloids and Surfaces (in press) Marmur J & Doty M (1962) J Mol Bioi 5, 109-114 Stryer L (1995) Biochemistry (4th edn.) pp. 86, W H Freeman and Co. New York Chatterjee R & Chattoraj D K ( 1979) Indian J. Biochem. 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