J. Mol. Biol. (1962) 5, 550-559

The Thermal Denaturation of Sodium DeoxyribonucleateI. The Limits

A. R. PEACOCKE AND 1. O. WALKER

Department of Biochemistry, University of Oxford, England

(Received 7 March 1962, and in revised form 21 August 1962)

The thermal denaturation of calf thymus and herring sperm sodiumdeoxyribonucleate (DNA) has been followed by means of the displacement in thetitration curves and by the changes in optical rotation and ultravioletabsorption. All three methods measured quantitatively the transition from ahelical to a more random configuration. This process of denaturation proceededto a maximum, limiting extent which increased with temperature and wasirreversible. The maximum extent of denaturation at anyone temperature wasindependent of the nucleate concentration except at very low concentration. It isconcluded that the assembly of DNA molecules contains different regions each ofwhich denatures only above a certain critical temperature. The distributionof stability with respect to temperature could be deduced from the experimentalobservations. The critical denaturation temperature increased with ionic strengthand the thermodynamic basis of this effect is discussed.

1. Introduction

When solutions of DNA are heated to high temperatures changes occur in theultraviolet absorption spectrum, optical rotation and viscosity, the titration anomalydisappears and the dimensions of the molecule decrease (for reviews see: Jordan,1960; Peacocke, 1960). These changes have been attributed to the breakdown of thehydrogen bonds holding the molecule in the ordered, helical configuration. Thisprocess is called denaturation. Complete denaturation may result in separation ofthe two polynucleotide strands depending on the ionic strength, the source of theDNA and its concentration in solution (Marmur & Lane, 1960; Doty, Marmur, Eigner& Schildkraut, 1960). It has been shown that the limiting extent of the changes inviscosity and optical density which occurred when solutions of DNA were heatedincreased with temperature (Goldstein & Stern, 1950; Rice & Doty, 1957; Pouyet& Weill, 1957). Attempts have been made to deduce values for the activation energyof the reaction from the temperature coefficient of the rate of change in viscosity andto calculate the "heat of denaturation" for the over-all process by assuming that thefinal extent of the change in viscosity was a measure of an equilibrium constant forthe denaturation reaction (Rice & Doty, 1957). However, it is not clear that thedecrease in viscosity which occurs on denaturation can be used without qualificationas a quantitative measure of the extent of the reaction, nor that the final extent of thechange can be properly regarded as at equilibrium, unless the process can be shownto be reversible at the temperature in question.

From these earlier studies many problems arise concerned with the reversibility ofthe reaction, the meaning of the final extents ofdenaturation at different temperatures,

550

THE THERMAL DENATURATION OF D~A. I 551

the rate of the process at different temperatures and whether this is first-order withrespect to time and concentration of DNA. Furthermore, it has not yet beenestablished whether DNA that has not been completely denatured consists of amixture of partially denatured molecules, or a mixture of completely denatured andcompletely undenatured molecules, or a complex combination of these two types ofsystem.

This study is an attempt to establish a method for the quantitative measurement ofthe extent of denaturation in partly denatured DNA and thence to examine theeffect of temperature, concentration and ionic strength on the final extent of denatura­tion. The titration method (Cox & Peacocke, 1956a) together with the changes inoptical density at 259 m,.,. and the changes in optical rotation (Doty, Boedtker,Fresco, Haselkorn & Litt, H159) have been used to follow the process.

2. Materials and Methods(a) Preparation of DNA. The preparation of herring sperm DNA has been described

(Cox & Peacocke, 1956a). Calf t.hymus DNA was prepared from nucleoprotein isolated bythe method of Zubay & Doty (1959). The nucleoprotein was dissolved in 1·0 M-NaOI andprotein was precipitated by the addition of sodium dodecylsulphate. Final traces of proteinwere removed by shaking the solution ofD~Awith chloroform and oetano!. The DXA wasprecipitated with ethyl alcohol, redissolved in distilled water, dialysed and then dried inthe frozen state. All preparations showed no reaction when tested for protein and all con­tained less than I % RNA. The characteristics of the four preparations are given inTable I.

TABLE 1

DNA characteristics

Extinctioncoefficient, £ Percentage Mol. wt. Radius

Sample (per mole of increase (x 10-8 ) ofphosphorus in .+ gyrationat 259 miL) A

Herring spermDNA 6650 15·9 5·6 2100Calf thymus DNATD~A·l 6760 14·5 7·0 :WOOCalf thymus DNATDNA-2 6580 14·9 8·3 2100Calf thymus DNATDXA-3 6580 16·9 7·4 3200

t Percentage increases in £ on heating at 100 DC for 20 min in 0·05 M-NaCI.

(b) Heating procedure. Solutions of D~A were heated in stoppered Pyrex flasks or tubesin a thermostat maintained at the required temperature to within ± 0·01 DC. Selectedvessels exhibited no chango in weight after heating and a correction was made in determiningthe period of heating to allow for the time needed for the contents of the tubes to reachthe required temperature. Except where otherwise stated, solutions were cooled to O°Cin an ice-water bath in a few seconds.

(c) Titration experiments. Solutions of D~A (2 mg/ml. in 0·05 ~-NaCl) were titratedfrom neutral pH with 0·1 M-HCl to pH 2·75 and then titrated with 0·1 M-NaOH to pH 7(Cox & Peacocke, 1956a,b). Titrations were carried out in a thermostat at 25·0°C using acell with a liquid junction and a calomel-glass electrode system to detect changes in pH.

552 A. R. PEACOCKE AND 1. O. WALKER

A Cambridge pH meter was used to follow changes in pH. This was calibrated with thefollowing standard buffers at 25'0°C: 0·20 M-potassium hydrogen phthalate, pH 4,01,and 0·20 M-sodium borate decahydrate, pH 9·18.

(d) Ultraviolet absorption measurements. The heated DNA solutions were diluted withsolvent after cooling and the absorption at 259 mfl-, relative to the solvent, was measuredat room temperature (18 to 20°C), in a Unicam S.P.500 spectrophotometer.

(e) Optical rotation measurements. The optical rotations of solutions of DNA weremeasured at 589 mfl- in a Hilger polarimeter at 20°C using a cell with a pathlength of 20 em.

3. Results

(a) The effect of temperature of heating and the measurement of denaturation

The titration curves of a series of calf thymus DNA solutions (2 mgjml. in 0·05M-NaOI) which had been heated for periods of 20 minutes at increasing temperaturesare shown in Fig. 1. The difference between the forward- and back-titration curvesdecreased progressively as the temperature of heating increased. From the differencecurves shown in the inset, the mean relative displacement of the titration curves(p) was calculated (Oox & Peacocke, 1956b). As previously noted, p increased withincrease in temperature.

~o

~a>

~ 1-0~Q.

-g::Jo

.D

"U's 2-0'­o

~'""6>-:;CT

W l~ j3-0 4·0

pH

5-0 6-0 7-0

FIG. 1. The titration curves of calf thymus DNA (TDNA-l) in 0-05 M-NaCl: (a) forward­titration curve of unheated DNA; (b)-(f) forward-titration curves of DNA heated for 20 min at83-0, 85,0, 87,0, 91·0 and 100·0°C, respectively; (f) back-titration curve for all samples. Thedifference curves are shown in the inset.

It was observed that at anyone temperature of heating, Papproached a maximum,limiting value as the time of heating was increased. The limiting value was reached atmost temperatures after heating both calf thymus and herring sperm DNA for 20minutes, although a longer period of time was required when heating a herring spermDNA at temperatures below 77'5°0. The increase with temperature of this limitingvalue (Pmax) is shown in Fig. 2. These curves were reproducible for different, carefullyprepared samples of thymus DNA and the temperature at which half the total dis­placement in the titration curves had occurred (Pmax = 0,5) was reproducible towithin± 0'5°0.

THE THERMAL DENATURATION OF DNA. I 553

A linear relation (Fig. 3) was observed between pand !f1,which is the relative increasein optical density at 259 m«, obtained at different times and temperatures andexpressed as a fraction of the maximum increase which is obtained on heating at100°0 for 20 minutes in 0·05 l\I-NaOI. A characteristic limiting value (!f1max) wasapproached at each temperature as the time of heating was increased and thisconfirms the results ofPouyet & Weill (1957). The variation of!f1max with temperaturecorresponded closely with the variation of fJmax obtained by titration. It was notpossible to estimate p (with the present accuracy of the titration curves) to betterthan 5 to 7%, and the error in measuring e is probably about 5%.

1·0

0·8

0·6

J0·4

0·2

o70 80 90 100

Temperature (QCl

FIG. 2. The denaturation profiles for calf thymus and a herring sperm DNA in 0·05 l\I-NaCI:()-herring sperm DNA; .-calf thymus DNA (TDNA-l); Q-ealf thymus DNA (TDNA-2).

0-8

0·6I<Q.,

0·4

0·2

0·2 0·4 0'6 0'8yr

•

FIG. '3. The relation between p, the displacement of the titration curves, and .p, the fractionalincrease 'in optical density, obtained on heating DNA solutions in 0·05 M·NaCI: Q-herring spermDNA; .-calfthymus DNA (TDNA. l ).

The changes in the optical density and in the specific optical rotation [a:]~O (alsoexpressed as a fraction of the maximum change and denoted by'), on heating calfthymus DNA (2 mgjml. in 0·05 M-NaOI) for periods of20 minutes, were almost parallel(Fig. 4). The specific optical rotation decreased from 130° to a minimum value of 91°on heating at 100°0. A single curve may be drawn through both sets of experimentalresults over most of the temperature range although a small but real deviationoccurred below 80°0. These results should be compared with those of Doty etal. (1959) ,who, while they observed a close parallel between changes in optical rotation and

36

554 A. R. PEACOCKE AXD I. O. WALKER

optical density, also reported much larger actual changes in these quantities. Thisdifference probably arises because their measurements were made at the temperatureof heating and the solutions were not cooled to room temperature as in this study.

1·0

of1·0

0·8 o • 0·8

0-6 I ' 0·6

~ ""(}4 004

0·2 d 0·2r'.' CJ •

0 ..;, 0

20 I 80 90 100

Temperature roFIG. 4. The relation between the fractional increase in optical density, r/s, and the fractional

decrease in optical rotation, " on heating calf thymus DXA(TDKA·3) for 20 min at a series oftemperatures: O-increases in r/s; .-decrllases in ,.

(b) The effect of DNA concentration

The maximum increase in optical density on heating calf thymus DNA in 0·05M-NaCI at a given temperature remained constant over a wide range of DNA concen­trations. At 82·5°C with concentrations of 2'0, 1,0, 0·68 and 0·05 mgjml., fmn hadvalues of 0,29, 0,32, 0·24 and 0,29, respectively. However, at a concentration of0·02 mgjrnl. the value of fmax increased to 0·59 and remained constant at this valuefor only about 7 minutes, after which the optical density again increased steadily withtime, probably because of hydrolysis. 1t appears, therefore, that, except at very lowvalues, the concentration of DNA has no effect on the final extent of denaturation.

(c) Reversibility of the reaction

A solution of calf thymus DNA (2 mg/m!. in 0·05 M-NaCI) was heated for 40 minutesat temperatures of 82,5, 85·0 or 87·0°C and cooled quickly to O°C over a period of afew seconds. The solutions were then titrated at 25·0°C when the values of Pma.in the three cases were 0,24, 0·39 and 0·70 respectively. Part of the solution whichhad been heated to 82·5°C and cooled was then heated for a further 10 minutes at85·0°C, cooled quickly to O°C and titrated; another part of this same solution washeated for 10 minutes at 87·0°C, cooled to O°C and titrated. The values of Pma.x were0·44 and 0,68, respectively, compared with those of 0·39 and 0·70 obtained bydirect heating at 85·0 and 87·0°C in the first instance. Thus, the same extent ofdenaturation was obtained whether solutions were heated at a given temperaturedirectly (e.g. 85 0 or 87°C) or were first heated at a lower temperature (82'5°C), cooledand then heated for a further period at the given higher temperature. (10 minutes'heating at each stage was sufficient to eause the maximum effect appropriate to thesetemperatures.)

A portion ofthe undenatured DNA solution was heated for 10 minutes at 87·0°C anddivided into three parts which were then subjected to the following operations beforetitration.

THE THERMAL DENATURATION OF DNA. I 555

(i) Cooled quickly to O°C; Pm"", = 0·6l.(ii) Cooled slowly to 85·0°C over 15 minutes, maintained at this temperature for

30 minutes, cooled in a few seconds to O°C;Pmax = 0·66.(iii) Cooled slowly to 82·5°C over 28 minutes, maintained at this temperature for

30 minutes, cooled in a few seconds to O°C; Pmar. = 0·66. Thus, within the experimentalerror of the method, the same value of Pmar. is obtained whether a solution is cooledquickly to O°C or whether it is cooled slowly via intermediate temperatures. The rateof cooling therefore has no influence on the final value of Pmax which is governedonly by the highest temperature to which the solution has been heated. These resultswere supported by a parallel series of optical density measurements. It may beconcluded that for calfthymus DNA, heating produces a reaction which is irreversiblewith respect to titration behaviour and changes in optical density, thus confirming anearlier observation of Doty et al. (1960).

(d) The effect of ionic strength

The curves showing the variation of if;mar. with temperature for solutions ofincreasingionic strength were displaced to higher temperature ranges (Fig. 5). This result is inagreement with many others showing the effect of salt on the stability of DNA

0·25

.6: ~ 0·20o-Ol""19'"til:! 0·/5

.s .?;-_"Vio c: 0·108{l

";:i_uo~.~ 0·05LL.8-

0

w m 00 ~ ~

Temperoture roFIO. 5. The denaturation profiles of calf thymus D~A at different ionic strengths: (a) 0·7

rnx-phosphate buffer, pH7; (b) 0·05 M.KaCI; (c) 0·60 M·~aCI; (d) 1·50 )1·XaCI.

solutions towards denaturation (see Jordan, 1960). The range of temperature overwhich if;me.x increased at anyone value of the ionic strength remained much the same(about lOOC), although the value of if;me.x which corresponded to complete denaturationdecreased with increasing ionic strength.

4. Discussion(a) The extent of denaturation

The denaturation of two samples of DNA has been followed by measuring, after thesolutions had been cooled to room temperature, the displacement of the titrationcurves, the increase in optical density and the decrease in the optical rotation as afunction of time and temperature. Parallel changes were observed in the paramctersP and if; (Fig. 3), and in if; and ~ (Fig. 4). The displacement of the forward-titrationcurves of heated DNA solutions may be attributed to the change of dissociationconstant consequent upon the rupture of the specific hydrogen bonds of the double

556 A. R. PEACOCKE AXD 1. O. WALKER

helix, for the groups involved in titration over the pH range 7 to 2·75 are just thoseinvolved in the hydrogen bonds of the helix (Cox & Peacocke, 1956a,b). Secondly, ithas been proposed that in the helical molecule the well-known hypochromicity iscaused by interaction of the bases (Laland, Lee, Overend & Peacocke, 1954; Thomas,1954; Tinoco, 1960). If the polynucleotide chains are no longer constrained and areable to take up a more random configuration which results in a decreased interactionbetween the bases then the optical density increases. Finally, Yang & Doty (1957)have shown that the changes in optical rotation of protein solutions may be directlycorrelated with loss of helical configuration within the molecules. Similarly it isprobable that changes in optical rotation of DNA solutions reflect changes in thenumber of helical regions within the molecules. The parallelism of the experimentalresults (Figs. 3 and 4) shows that the three parameters ~, if and ~ reflect the samefundamental change in the DNA molecule. It is eoncluded that anyone of the threeparameters ~, if or~ may be used as a quantitative measure of the extent of denatura­tion, a process which results in changes in the dissociation constants of the titratablegroups, changes in the interactions between the bases and changes in helical content.The "denaturation profiles", the curves representing the variation of ~max or ifmax withtemperature, were reproducible for different preparations of calf thymus DNA, andmay be characterized by their "denaturation temperature", TD , the point at whichhalf the maximum change has occurred (i.e. ~max = ifmax = 0,5).

The denaturation process for calf thymus DNA was irreversible, even when solu­tions were cooled slowly. The same result has also been reported by Doty et al. (1960)for thymus DNA, but these authors also concluded that the denaturation of certainbacterial DNA was, under suitable circumstances, partly reversible ("renaturation").They suggested that renaturation was not observed with thymus DNA because therewere few separate single strands within the denatured DNA which had complementarycompositions and which could reform double helices. However, the molecular weightof a partly denatured solution of calf thymus DNA was unaltered (paper III of thisseries, Peacocke & Walker, 1962). The constancy of the molecular weight up to~ = 0·77 shows that the single strands in denatured regions, or molecules, remainentangled sufficiently to continue to act thermodynamically as one component withrespect to the scattering of light by which the molecular weight was determined. Thepossibility of two entangled, complementary strands reuniting through hydrogenbonds to form helices is more fully discussed in paper III of this series. Thus, the valuesof ~max cannot be regarded as representing a reversible equilibrium between undena­tured helical regions and denatured disordered regions in the molecules. It has beenproposed that regions of different stability may exist within the DNA throughthe various arrangements of the base-pairs adenine-thymine (A-T) and guanine­cytosine (G-C), which themselves have differing thermal stabilities (Rice & Doty, 1957;Cox & Peacocke, 1956b). So, at any temperature at which denaturation occurs,different sequences of bases, which may comprise whole molecules or parts of amolecule, could have different stabilities and some could denature while othersremain unaffected. When the temperature is increased other regions, with differentsequences, should become unstable and then denature in their turn.

The conditions for denaturation may be tentativelyexpressedin thermodynamic termsin the following way. Consider a particular sequence of n base-pairs which denatures onlyat temperatures equal to or greater than T a• If the change in free energy which occurswhen the sequence denatures is !1G (and!1H and!1S are the corresponding enthalpy and

THE THERMAL DENATl;RATIO~ OF D~A. I 557

entropy changes) then, for the process to occur spontaneously, b.G must be zero ornegative. The lowest temperature at which this may occur is T. = b.HIb.S. The valueof b.S should be determined largely by the configurational change which accompaniesdenaturation and not by the chemical nature of the base-pairs. The value of b.Hwould be expected to depend on the relative proportions of G-C and A-T base-pairswithin the sequence since there is evidence to suggest that !1HG-C >!1HA-T (Marrnur

& Doty, 1959) and also possibly on the arrangement of these base-pairs within thesequence. Thus the denaturation temperature of a sequence should depend on thesesame factors. Alternatively, a denaturation sequence may be defined by a constantvalue of !1H. The denatured sequence would then contain a variable number (n) ofbase-pairs depending on its base composition and the denaturation temperature wouldrise as n decreased. These considerations show that the variation of Pmax and t/Jmaxwith temperature may be explained in terms of the variation in base composition ofthe sequences within the DNA.

0·2

0·1

100

FIG. 6. The relation between (dPrru..Jd T) and temperature. This curve is the frequency distri­bution curve showing the fractional extont of denaturation at a given temperature. Unbroken line:0. herring spenn D~A; broken line : calf thymus DKA.

However, from the symmetrical nature of the displacement of the titration curves,as shown by the shape of the difference curves (Fig . 1 inset), it is concluded that thefinal result of heating is to produce a form of the DNA in which the hydrogen bondsbetwecn adenine and thymine and between guanine and cytosine have been broken ina random manner. This conclusion is based on the observation (Peacocke, 1957)that adenine titrates over a different pH range from cytosine and a skew displacementwould be expected for non-random rupture. The error involved in estimating therandomness of the breakage of hydrogen bonds is ± 10%, so that small deviationsfrom random behaviour would not be detected by this method. Sequences whichdenature at lower temperatures should have a comparatively higher content ofA--T base-pairs than those which denature at higher temperatures. Once the helicalconfiguration in which a sequence is located has been destroyed the spontaneousdenaturation of helical regions on either side of the sequence may follow. The ratio ofsevered G-C base-pairs to severed A-T base-pairs in the finally denatured region maythen be indistinguishable from the G-C/A-T ratio prevailing in the DNA preparationas a whole, in spite of the higher A-T content. of the sequence, the opening of whichinitiates the denaturation at the lower temperatures. A similar conclusion has beenreached by Ginoza & Zimm (1961) from studies on the heat inactivation of genetic

558 A. R. PEACOCKE AND 1. O. WALKER

markers in bacterial DNA. On the other hand, the results of density gradient ultra­centrifugation experiments suggest that the DNA fraction which is first to bedenatured is comparatively richer in A-T base-pairs (Marmur, Schildkraut & Doty,1961).

The curves showing the variation of ~max with temperature, the denaturation profiles,are the cumulative distribution curves showing the total number of hydrogen bondswhich may be severed at a particular temperature. From them may be derived(d~m&J./dT) which is the increment in the fraction of bonds broken per unit incrementof temperature. The distribution curves for the calf thymus preparations arereproducible and differ markedly in shape from that of the one sample of herringsperm DNA studied (Fig. 6). The former curves are single-peaked with the maximumat the denaturation temperature, TD' while the latter is bimodal. The difference indistribution may be due to the herring sperm DNA having been partly denaturedduring its preparation. Independent viscosity measurements on this same samplehave indicated this (Eisenberg, 1957).

(b) The effect of ionic 8trength

The decrease in the values of fmu' which correspond to complete denaturation,with increasing ionic strength ofthe heated DNA solutions (Fig. 5) can probably be ex­plained by the greater ease with which DNA strands separated by heating re-associateon cooling at higher ionic strengths (Doty et al., 1960). At low ionic strengthsthe strands associate less readily with consequently less interaction between thebases and a lower hypochromicity, i.e. a greater actual optical density of the cooledsolution of denatured DNA. The. effect of varying the ionic strength, J, on thedenaturation may be considered in terms of its effect on the difference in free energy,/),.G, between the helical and denatured forms of a sequence. If /),.G is a function ofboth temperature and ionic strength,

Hence,

where /)"8r is the entropy difference between the two forms at ionic strength, I.Let T. be the denaturation temperature of the sequence. Then /),.G = 0 when T = T.and

(aT.) I (O/)"G)oJ M=O = Mr' -aT T.

Preliminary experiments show that the difference between the intrinsic viscosityof native and denatured DNA does not vary between ionic strengths of 0·05 to 1·50.This indicates that /),.8r, the entropy of denaturation of a sequence, will be approxi­mately constant over this range of ionic strength. The dependence of denaturationtemperature on ionic strength, in this range, is then only a function of (o/)"G/oJh•.A relation between ionic strength and denaturation temperature should thereforeultimately be derivable by considering the effect of ionic strength on the free energiesof helical and coiled sequences.

THE THERMAL DENATURATION O F D);A. I 559

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Sci ., Wash. 45, 482.Do ty, P., Marmur, J ., E igner , J . & Schildkraut , C. L . (1960) . P roc, N at. A cad. Sci ., W ash.

46,461.Ei senberg, H . (1957). J . P olymer Sci . 25, 257.Ginoza, W. & Zimm, B. H . (1961) . Proc , N at . Acad. Sci., Wash. 47, 639.Goldstein , G. & Stern, K . G. (1950). J . Polymer Sci. 5, 687.Jordan, D . O. (1960). The ChemiBtry of the N ucleic A cids. London: But terworth & Co.Laland, S . G., Lee, W . A., Overend, W . G. & Peacocke, A. R . (1954). B iochim. biophys.

A cta, 14, 356 .Marrnur, J . & DOLY, P . (1959) . N ature, 183, 1427.Marmur, J. & Lane, D. (1960) . Pro c, Na.t. Acad. Sci., Wash . 46, 453.Marrnur, J., Schildkraut, C. L . & Doty, P. (1961) . •J . chim. Phys. 58, 945.Peacocke, A. R. (1957). Chem. Soc ., Special Pub!. 8, 139.P eacocke, A. R. (1960). Pro gress in Biophysics, 10, 55.P eacocke, A. R. & Walkcr, I. O. (1962). J. Mol. Biol. 5,564.Pouyet, J. & Weill, G. (1957) . J . P olymer Sci. 23, 739.Rice, S. A. & Doty, P. (1957) . J . Amer. Chem. Soc . 79, 3937.Thomas, R. (1954) . Biochim. biophys. A cta, 14, 231.Tinoco , I. (1960) . .1. Amer. Ohern; S oc. 82 , 4785.Yang, J. T . & Do ty, P . (1957). J . A mer. Ohem, Soc. 79, 761.Zubay, G. & Doty, P . (1959). J . M ol. Biol. 1, 1.