- Email: [email protected]
Nonaqueous solutions of DNA. Reversible and irreversible denaturation in methanol
ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
Nonaqueous
96,
Solutions
Irreversible
the
(1961)
of DNA.
Denaturation E. PETER
From
11k-129
Committee
Reversible
and
in Methanol’
GEIDUSCHEK
on Biophysics, The Chicago, Illinois
University
of Chicago,
AND
THEODORE From
the
Department
T. HERSKOVITS
of Chemistry, Lafayette, Indiana
Received
May
Purdue
University,
17, 1961
Deoxyribonucleic acid (DN,4) undergoes denaturation in methanol-water mixtures. The region of solvent composition in which this transition occurs depends on the ionic strength and temperature but is little influenced by the average nucleotide composition of the DNA. As judged by both macromolecular and optical criteria, the disruption of secondary structure is almost complete, yet methanol denaturation is rapidly and readily reversible by water. The structural basis of this reversibility differs from that controlling the reversibility of partial denaturation in aqueous solution. An irreversible thermal transition has also been found to occur in methanol-rich solvents. The products of this transition may be reconverted to native DNA by water. INTRODUCTION
In a preceding communication (1) we have presented evidence that two nonaqueous solvents, methanol and ethanol, alter the conformation of deoxyribonucleic acid (DNA). At this juncture we describe a systematic study of denaturation in methanolwater mixtures. The experiments reported here deal with the transition from the native, ordered, extended configuration present in aqueous solution to denatured, more or lessdisordered and altered, compact configurations as the solvent medium is changed. The ionic strength and temperature dependence of the transition and its reversibility have been studied with the aid of spectroscopic and macromolecular techniques. It is clear from these experiments that the phenomenon of reversibility in methanol denaturation is both qualitatively ‘Research supported, Health Service Research
in part, by U. Grant C 5697.
S. Public 114
and quantitatively different from any previously described. Results are presented in two parts. First, as a direct continuation of previously reported work, we describe the denaturation in electrolyte-free solvents, through measurements of sedimentation velocity, light scattering, conductance, and ultraviolet spectra. In a second section, experiments on the ionic strength dependence and reversibility of the denaturation in electrolytecontaining media are presented. Two brief sections on temperature effects and on denaturation in other alcohols conclude the presentation of experiments. I. EXPERIMENTAL A.
~UATERIALS
DNA samples and other materials have been desrribed in the preceding paper (1). Spectroscopic grade methanol (sets. IIB, C, and D) was used without further purification. Pseudomonas fluores-
NONAQUEOUS cc’nns DN;l was prepared by a method combining detergent, denaturation of proteins (2) and cetyltrimethylammonium bromide fractionation of DNA from ribonucleic acid (RNA) and polysaccharide (3). The same sample of DNA has been used in studies of thermal and acid denaturation (4,5).
B . METHODS Optical rotation was measured in a Rudolph polarimeter (model SOS), equipped with a thermostated cell compartment. Measurements were made at the 4358-A. line, selected from the emission of a mercury vapor lamp (AH-4), by a narrow band interference filter. Flow birefringence measurements were made on an instrument manufactured by the Rao Instrument Company. modeled on the design of Edsall et nl. (6). Birefringence was measured with the Henarmont compensator supplied by the manufacturer. The quantity A?~/cf, (which, for a single solute species, is proportional to the anisotropy factor, g1 - fi) was used as a measure of the amount of material oriented at any shear gradient. In is t,he amount of birefringcnce of the solution and f is the orientation factor. The latter quantity is calculated from the angle of isocline x, assuming infinite axial ratio, (7). For DNA, which is heterogeneous, an/cf is a function of gradient. In terms of what is now generally agreed upon regarding the structural properties of helical DNA, the anisotropy factor, g1 - pr, should be calculated from the limiting value of ~n/cf at high gradients. At lower shear the variation of 4r1/cj with gradient presumably reflects the distribution of molecular sizes and shapes (8). The light-scattering methods used in these experiments have bren described previously (1). Interpretation of light-scattering experiments on DSA-methanol-water mixtures has been made in terms of the theory of two-component solutions [Ref. (1) Eqs. 1 and 21. The justification for this simplification lies with the near identity of the refractive indices of water (n = 1.333) and methanol (n = 1.330). Ewart et al. (Cl), who first investigated such systems, showed that, as the refractive indices of the two solvents approach each other, multicomponent scattering effects disappear. The exact theories of light scattering from multicomponent systems confirm t,he soundness of this conclusion (10-12). Two types of kinetic spectroscopic experiments were performed. In one series of experiments DNA solutions and solvents were mixed in a stoppedflow rapid reaction spectrophotometer, built by Dr. J. M. Sturtevant (13): In the ot>her series, “To pitality
whom we extend and interest.
our
thanks
for
his
hos-
DNA
115
SOLUTIONS
solutions equilibrated at the desired temperature, were mixed by hand in a stoppered absorption cell and inserted as quickly as possible into the cell compartment of a thermostated Beckman DU spect,rophotometer, located in a constant-temperature room. Other measuring and analytical methods were used as described previously (1).
C. PREPARATION
OF
SOLUTIONS
Two methods were used in preparing DNA solutions in methanol-water mixtures: dialysis and direct mixing. The dialysis procedure has been described previously ( 1). Solutions made by direct mixing were prepared by adding constituents in sequencrs designed to minimize denaturation (e.g., methanol or water added to DNA-salt solutions). Detailed descriptions are included in the captions of Figs. 3,4, and 7. The procedure described in Fig. 4 is adequate for all experiments on the reversibility of methanol denaturation by water. The more cautious methods described in Figs. 3 and 7 were used to prepare solutions for experiments in which the irreversible thermal effects occurring in 80-100% methanol (sec. IIC) had to be avoided. Solvent compositions are expressed on a volume basis (v/v) unless otherwise stated. For convenience, a vol.%-weight %-mole % conversion table (at 15°C.) is given below. Methanol d.
(v/v)
Methanol
T,
10 20 30 40 50 60 70 80 90 9B
(w/w)
8.1 16.3 24.8 33.8 42.7 52.2 62.4 73.4 85.7 94.0
II. EXPERIMENTAL A. THE DENATURATION SALT-FREE
Methanol mole /raction
wt. %
0.047 0.099 0.157 0.2’11 0.295 0.381 0.483 O.GO9 0.771 0.897
RESULTS OF
DNA
IN
METHANOL-WATER MIXTURES
The original experiments on the behavior of DNA in ethanol-water and methanolwater mixtures showed a sharp change in average sedimentation velocity at finite concentration, in 80% methanol, and in 65% ethanol (1, 14). The methanol system, with its slightly greater tolerance for electrolyte and with its minimizing of multi-
116
c;EIikWfl~;K
.1ND HERSKO\‘ITS
component effects ih light scnttcring, pro- in the abscncc of salt is shown in Fig. I. A sudden change from slow Is approx. l-5) vitlcd the oljportlmit+ for ‘more dctailcd stkly. Sedimc’ntaGon, iight-scattering. spcc- to r:tl)i(l 1~sapprox. 30-50) wlimcntation trophotometric, tind conductance data arc’ occur’I_ at approximately 805 methanol prcscntcd here. These new results indicate I,IIIO~Cfr:\ction 0.61) .:’ that t.hc sedimentation cxpcriments. t.aken E’urthcvniorc~, solutions first dial~zcd into by themselves, give a quite distorted pic- 99.5“; mct.lianol, before cqmlibrating turc of t,he secondary structure t.rnnsition, against the appropriate solvent, show t.hc and that. their previous intcrprctat.ion is salnc behavior. It, is important to note that thr change in optical density 1259 rnp) incorrect. The sedimentation behavior of DNA tiow not coincide with the cllangc in sedi(H-11, c = 0.5-l mg./ml.), as a function of nwntation behavior, but owurs at low1 solvent composition at 3-8°C. and 23-27°C. alcohol content, (Fig. 3 cm-w (1) so that,, at 655‘; cthnnol and 80’;; methanol, the (‘xt,inc.tion is ;llrcndy high [Z uppros. 9000; cf. 501 Ref. ( 1 I, ‘l‘wblw I and II]:* T,ighb-scattering cxperiment5 in inctha40 nol-water mixtures arc‘ simple from a thcowtical point. of \licw bccauw of the nwrly 3 d 30 idcntic:il rcfr;ic.tivc indiws of tlic two wt2 writ conllwnrnts. At 80-1OOSC methanol s~icli mc~:lsurclllc~nt::arc also rclativcly sim20 l’h~. wvwc intc~rmolccul:ir intcrfcrciw cffwtb usually cntvring onlv al)ovc (: = 2-3 x IO-’ mg. ~ml. 11owvcr, In O-705 salt-free methanol, c5tvrii:il intcrfcrcncc cffrctti arv I I I I iiiiporkmt vvcn at. DK.4 concentrations of 100 0 20 40 60 60 0.01 mg. ,‘nil. (‘onscqucntly, cxpcrimcntal % Methanol (v/,1 rmcwtaintics in thr dctcrnjination of radii FIG. 1. Variation of sedimrntution velocity of gyration (p) aw wry large. Ncverthcwith solvent composition in methanol-water misless, thv InwisiO!i is sufficient to est.abli~h turrs (D?;A H-I, salmon ; no supporting c+ctrothat p tlocs not c~hangc Anrply at 80% lytcl; solutions prepared by dialysis at 4-6°C.). mcthaiiol (Fig., 2). Ilnthor, this solvent X: 3-8°C. or 2S28”C.; 0: 23-28°C.. rnwiously composition appears to corrtq~ontl, approxicxposcd t.o 99.5% mcrhanol at 4-6%. mntc)ly, with the vnd point 0i tlic change in avcragc radius of gyration. I I I I Clonducbtivity cqwrimc~nts at 0°C‘. ( 16 1 intlicatc thttt the nlost rapid changes in ion association of salt-frw I)SA solut.ions occur in methanol-poor media tO-IS?: m&hanal‘). 111tllc r:ingc of solvcrit coiiipositions :woci:~ttd wit11 t.he change in scdimcntat.ion bch:ivior, the degree of ion :woci:~t.ion clinngea rcl:ktivcly little. Increnacs of oj)tical density (259 rnp,I at 25°C. owur in t.hv O-50’;; inct.h:mol solwnt coniposit.ion range. ?rt 0.5”(.‘., such changes occur at liiglw
00
II
FIG. 2. Variation of (light-scattering average) radius of gyration with solvent composition at 5-6°C. in methanol-water mixtures (DNA H-II; no supporting electrolyte; solutions prepared by dialysis).
’ .A tliflcrent. interpretation of the nkol~l tlvnritllrat ion which has been presented rccvnt Iy (I.i), strm15 from 3 failure 10 apprcciniv this fnvt.
KONSQUEOUS
DNA SOLUTIONS
117
1
40
60
% Methanol
80
100
Cvjv)
FIG. 3. Methanol denaturation and ionic strength, at 0.5, and 25°C. c/eO, extinction coefficient relative to native DNA4 in aqueous salt solution. (a) no added salt, 0.5”C. : 0 ; ( i; ) 10e4 M NaCl, 0.5”C.: X ; (c) lo-’ J4 NaCl, lo-* M Tris pH 7.1,0.5”C.: 0 ; (d) 10e3 M l%e 1lIi 7.1, 5 X 10-j :Jf NaCl 25°C. (from Fig. 4, curve a) ; (e) lo-* M NaAc. 10m3M Trid, pH 6.7-i 5. 25°C.:
l .
DNA Sample: H-II (salmon). Preparation of solutions : curve a: DNA is dissolved in water at 0°C. at a concentration of 1 mg./ml. and diluted at 0°C. to 0.3 mg./ml. To 0.5-ml. aliquots of this stock solution, 4.5 ml. of the appropriate water-methanol mixtures are added dropwise with stirring. Reagents, and the final solution are maintained at -5 to O”C., and mixing is performed in a 0°C: roqm so that samples arc never exposed to temperatures above 05°C. curve b : as curve a, but DNA stock solution is lo-” M in NaCl. curve c: as curve a, but DNA stock solution, methanol, and water all contain lo-” -II NaCl, 10eJ M Tris pH 7.1. curues cl, e : as Fig. 4 curve a. content than at 25°C. (Fig. 3 curve a). Both the light-scattering (Fig. 2) and the extinction data show that the denaturation, in the absence of salt, occurs over a rather broad range of solvent. compositions. ,4 system may be judged to have undergone a sharp transition when a number of its properties change suddenly, and more or less in concert. Such is the case for the denaturation of DNA at extremes of pH,
methanol
or high temperature, in the presence of moderately high concentrations of electrolyte. It is clear that the methanol denat,uration in electrolyte-free solution does not, by any means, satisfy this criterion and t’hat the sharpness of the change in sedimentation velocity must be understood on some other basis. Most probably, the sedimentation change reflects an end point of the transition from extended to compact configurations. These are solutions of wry low
118
GEIDUSCHEK
AND
HERSKOVITS
attributed to other causes. For aqueous solutions of DNA, changes of optical density with salt concentration are correlated with denaturation. At O”C., we find the extinction coefficient of DNA to be independent of salt (NaCl) concentration, down to 10-4.5 M, a range of ionic strengths which causes great variation of the electrostatic free energy. (c) Change from one ordered periodic structure to another. If the spacing and orientation of chromophores, relative to each other or to the helix axis, is altered, then changes of absorption intensity may be expected (17). (d) Effect of solvent on the stacking and hydrogen-bonding ability of single (or disordered, or denatured) polynucleotide B. DENATURATION OF DNA IN chains such as found in tobacco mosaic WATER-METHANOL-ELECTROLYTE virus RNA (18-22), denatured DNA, and MIXTURES +X174 DNA (23).6 It is characteristic of DNA solutions in methanol-water mixthese configuration changes that they octures tolerate the presence of additional cur over wide ranges of environmental conelectrolyte without undergoing precipitaditions (ionic strength, temperature, pH). tion. This property has already been put to (e) Effect of solvent on the stability of use in studying the thermal denaturation the ordered “native” configuration of DNA, of DNA in methanol-water mixtures (1). relative to a disordered-denatured state. In the experiments to be described below For the purposes of our experiment, items we have used spectrophotometric measure- (a) and (b) are eliminated by the approments to permit a relatively simple survey priate control experiments cited above. We of properties. At suitable junctures, addi- have been able to find conditions under tional optical rotation and macromolecular which the partial ordering considered under experiments have been performed to insure (d) is almost completely unstable (e.g., that the correlations between optical and Fig. 7). Consequently, we may interpret macromolecular properties, which are so large extinction changes in terms of items consistently operative in aqueous media, (c) and (e), that is, the order + disorder also serve our purposes. It might be well, transition, and the change from one to anbefore proceeding, to summarize those fac- other ordered configuration. Both these tors which could cause changes in ultravioprocesseswould, in the most general sense, let light absorption by DNA: be termed ‘idenaturation.” They would be (a) Solvent effects on the spectra of con- readily distinguishable where one such stituent chromophores. However, control change occurred to the exclusion of the experiments show that the O.D.,,, of nu- other. However, a process in which part of cleoside mixtures does not depend on the each DNA molecule disordered, and part composition of methanol-water solvents.5 changed to another ordered configuration, (b) Effect of local electrostatic fields on would be difficult to distinguish from a absorption spectra. Two facts argue against simple denaturation. the existence of any such effect in DNA. With this discussion as background we The spectra of most nucleosides and their ‘Solvent effects of this type may arise from corresponding nucleotides are almost idenchanges in the intrinsic stabilities of hydrogen and tical. When difierences exist, they may be
ionic strength and very marked nonideality. The absence of rapidly sedimenting material at methanol concentrations below 80% is probably accounted for by the retardation of more mobile species in the medium through which they must move, and which contains the slower moving and presumably more extended molecules. In fact, Fig. le of Ref. (1) shows the presence of two slowly sedimenting components, one with a relatively broad boundary, This has frequently been observed both in ethanol- and methanol-water mixtures below the sedimentation transition. The presence of these two boundaries suggests that salmon DNA is heterogeneous in its resistance to denaturation in salt-free methanol.
“T.
T.
Herskovits,
manuscript
in preparation.
hydrophobic sion of
bonds,
or
from
coiled polynucleotide
electrostatic
chains.
expan-
WNAQUEOUS
DNA
proceed to describe experiments on the reversiblc DNA secondary structure transition in methanol-water-electrolyte mixtures. Figure 3 shows the ionic strength dependence of the denaturation of salmon DNA in methanol-water mixtures. At very low ionic strength, there is a marked influence of salt concentrat’ion on the location of t’he transition (cf. see. IIA). However, between 5 x lo-” and 5 X lo-” M salt, no further effect of salt concentration is dctectable.7 This is in keeping with the relatively great’ degree of ion association with DNA in methanol-rich solvents (16). The dependence of absorbance on solvent composition for native and heat-denatured DNA at 25°C. in methanol-water mixtures containing lo-” M Tris, pH 7 and 5 x lo-” ,U SaCl is shown in Fig. 4. At this low ionic strength the absorbance of denatured DKA is high, even in water (E/E,, = 1.35). For native DKA, on the other hand, the molar extinction coefficient is independent of solvent composition in the O-70% methanol composition range and unchanged from that observed in 0.2 M aqueous NaCl (Z = 6300 for DNA H-II). Between 80 and 90% methanol, E increases by 42 -+ 2%. As we show below, this increased absorbance parallels losses of viscosity and flow birefringence. We call such material solvent denatured. In 90% methanol containing 1OV” M Tris and 5 X lo-” M NaCl this solventdenatured DNA, and DNA denatured by heating in aqueous solution, have the same absorbance, yet they are distinguishable in a very dramatic way. The extinction change of the solvent-denatured DNA can be reversed when water is added back to the system (Fig. 4 curve n, data given by crosses) and 6 returns to its original value. On the other hand, when water is added back to heat,-denatured DISA in 90% methanol, z remains high (Fig. 4 curve b, data given by open squares). Spectra of native DNA in water, solvent-denatured DNA in 95% methanol, and water-heat-denatured DNA in 95y0 methanol, all at 25”C., lo-” M NaCl, lo-” 111Tris, pH 7 are shown in Fig. 5. i Above 0.05 M salt, aggregation and tion occur in 90-100% methanol, setting the range of this experiment.
precipitaa limit
to
119
SOLUTIONS
1.20 l.lOt-
Y I.003 n O n * O n 3x xx 0 x I 0
I
I
I
20
40
60
% Methanol
FIG. 4. Extinction coefficient position at 25°C. e/co, extinction tive to native DNb in aqueous
4
I
60
I
100
Cv/,)
and solvent coefficient salt solution.
comrela-
curve a: 0 : Untreated DNA; solutions prepared by adding methanol t,o aqueous DNA solutions. X: DNA previously exposed to 90 or 95% (v/v) methanol, 5 X lo-” ,lil NaCl; lo-” JI Tris, pH 7.1 25°C. CWVB h: l : DNA previously denatured by heating in aqueous 2 X lo-’ M Tris, pH 7.1. lo-” .II NaCl to 95°C. for 15 min. q : DNil heated to 95”C., 15 min. and exposed to 95% (v/v) mrthanol. Solvents: Methanol-water mixtures, IO-’ ~11 Tris pH 7,5 X 10-j ,VJ NaCI. DNA sample: H-II (salmon). Preparation of solutions : curve a, points shouva by circles: 0.50-l ml. DNA (0.3-0.6 mg./ml.) in 1-2 X 10e3 LV N&l, 1-2 X IO-’ M Tris, pH 7, diluted to 10 ml. with such quantities of water and methanol as will yield the methanol-water mixtures (at 10m3 11f Tris, 5 X lo-” iPl NaCl) shown. Prepared solutions are equilibrated 1 hr. at 25°C. before making absorbance measurements. CUTV~ a, points shop by CTOSSC.S: 0.50 ml. Dli-4 (0.6-1.2 mg./ml.) in lo-’ J4 NaCI 2 X lo-* ,V Tris, pH 7, diluted with 9.5 ml. methanol, and water, to 10 ml. Methanol may be added to DNA4 without, special care regarding mixing. This stock solution of DNA in 95% methanol is then kept at 25°C. for 1 hr. and retooled. It is now diluted with water-methanol-10m3 M Tris-5 X 10-j Jf NaCI solutions to yield the solvent compositions shoed. curvy b: DNB (0.3-1.2 mg./ml.) in 2 X lo-’ ,Zr Tris, pH 7, 1O-3 M NaCl serves as a stock solution. An aliquot is diluted and its absorbance measurpA so as to establish a reference from which E/E<, may be calculated. The remainder is heated to 95’C. for 15 min. and cooled rapidly in ice. From this stock, solutions are prcpnred as described abol-cx for curve a.
GEIDUSCHEK
120
AND HERSKOVITS
0.8 0.6 0.4 0.2 I
220
240
260
280
300
320
FIG. 5. Spectra of DNA (ST-l, salmon) at 25°C. in lo-’ M NaCl, lo-* M Tris, pH 7. fh/emax : relative extinction coefficient. 1: water (X,., = 258.5 mp) ; 2: 95% methanol (A,., = 260 mp) ; 3: 95% methanol. DNA denatured by heating aqueous solution to lOO”C., 15 min. (hmsr = 260 mp).
30(
0
20
40
60 % Methanol
80 (v/,)
FIG. 6. Comparison of absorbance, viscosity, and optical rotation of DNA in methanolwater mixtures. [a], : specific rotation at 436 rnq. [v]/[& : intrinsic viscosity relative to aqueous salt solution. Untreated DNA; solutions prepared by adding methanol to aqueous DNA solutions. e: 0; [q] : 0; [a] : @. DNA previously exposed to 9599% (v/v) methanol, 10m3M NaCl, 10d M Tris, pH7, pH7,25”C. [+j : W, [a] : 17. Solvents : Methanol-water, 1O-8 M NaCl, lOWaM Tris, pH 7,25”C. Preparation of solutions: as Fig. 4, curve a. Auxiliary experiments make it evident that this reversibility occurs despite the fact that DNA undergoes profound changes of macromolecular configuration between
80 and 90% methanol. The viscosity decreases drastically (Fig. 6) and no stream-
ing birefringence can be detected when 0.02% DNA solutions in 9&95% methanol containing 1O-3 M NaCl, 10W3 M Tris are
subjected to shears up to 6000 sec.-l. As the data of Fig. 6 also show, changes in intrinsic viscosity and extinction coefficient
NONAQUEOUS
DNA
occur in the same range of methanol composition. The specific rotation, [a], also changes in the transition region. However, we find that [a] varies with solvent composition even when no changes of viscosity or absorption (c = 259 mp) can be observed. Similar situations occur in other solvent mixtures and in concentrated aqueous salt solutions of DNA (4). Further evidence for the complete reversibility of methanol denaturation by addition of water is afforded by the following experiments : 1. The thermal denaturation transitions in 51% methanol, of native DNA and DNA recovered from 99% methanol containing 10P3 M NaCI, 10e3 M Tris, are compared in Fig. 7. In this solvent, heat-denatured DNA exhibits little hypochromicity. The
121
SOLUTIONS
identity of the two heating profiles indicates, therefore, that the methanol-treated DNA4 has recovered the long-range order characteristic of the native configuration. 2. The flow birefringence of native DN,4 and DNA recovered from 99% methanol, measured in 45% methanol, is essentially identical (Fig. 8). Not only is the gradient dependence of x the same for these two systems (curve a) but the fraction of solute orientable at each gradient is the same (curve 5). These two experiments constitute a stringent test of the re-establishment of the stiff, extended chain configurations characteristic of long-range order. 3. The intrinsic viscosity of native DNA, and DNA recovered from 99% methanol, measured in 47% methanol at 25”C., are virtually identical (Table I).
- so0 % N is a - .050
-0 I 25
35
45
I
55
T”C FIG. 7. Ultraviolet absorption thermal analysis of DNA (H-II) in 51% methanol (v/v), lo-’ M NaCI, lo-’ M Tris, pH 7. A : untreated; preparation of solutions: as Fig. 4, curve a. l , 0, n : Previously exposed to 95% (v/v) methanol, 10e8 M Nacl, lo-* M Tris, pH 7 at 0.5”C. ( 0 ), 25°C. (m), and 54.9”C. (01, respectively. Preparation of solution: To 1 ml. DNA (0.7 mg./ml.) in lo-* M NaCl, 10.’ M Tris, pH 7 add 20 ml. of 99% (v/v) methanol lo-’ M NaCl, 1O-3 M Tris, pH 7 dropwise, with stirring. Reagents and the final solution are maintained at -5 to 0°C. and mixing is performed in a 0°C. room. Aliquots of this solution are exposed to 0.5,25, and 54.9”C., respectively, for 1 hr. and retooled. Iced aqueous 10.’ M NaCl, IO-’ M Tris, pH 7 is then added dropwise, at O”C., to give a final methanol concentration of 51% methanol. 0: after one cycle of heating to 65°C. in 51% methanol. Data uncorrected for thermal expansion of solvent.
GEIDUSCHEK
122
1000
AND HERSKOVITS
2000
3000
4000
5000
G see-’ FIG. 8. Streaming birefringence in 44% (v/v) methanol lo-’ M NaCI, 10m3M Tris, pH 7, 20°C. x: extinction angle. An/cf: birefringence per unit DNA concentration, corrected for average orientation factor, 1, plotted in arbitrary units. 0: untreated; c = 0.119 mg./ml. l : previously exposed to 99% (v/v) methanol, (lOJ M NaCl, 10m3M Tris, pH 7, 25°C.). c = 0.102 mg./ml. Sample : H-II (salmon). Preparation of solutions : as Fig. 7.
TABLE I Sample
history
Solvent
[VI
Native (Sample HII, salmon)
0.2 M NaCl
68
Native
47.6T0 (v/v) methanol, 1OWM NaCl 1OWM Tris pH 7
70
Exposed t’o 99% (v/v) methanol, 10-a M NaCl, 10-a M Tris, pH 7, 25°C.
47.2% (v/v) methanol, 10-Z M NaCl, 10-s M Tris, PH 7
73
4. The effects of solvent composition on optical activity are reversible (Fig. 6). This complete reversibility of the methanol denaturation in a heterogeneous DNA at low ionic strength distinguishes it from the “renaturation” of thermal-aqueous denaturation described by Marmur, Doty, and co-workers (24, 25). The methanol denaturation is also distinguished from ‘(renaturation” of thermal aqueous denaturation on kinetic grounds. It has been found that the solvent-induced transition is fast in both directions. For example, the optical density reduction on mixing DNA-97%
methanol with 61% methanol (all solutions lo-” M NaCl, 10W3 M Tris, pH 7) to give 79.5% methanol is at least 90% complete within 5 sec. at 25°C. When DNA-95% methanol and water (same ionic composition) are mixed to give a 70% methanol solution, the absorbance reduction is more than 90% complete within 20 sec. at 25°C. No special procedure (such as gradual mixing or dialysis) is necessary to achieve reversibility. In fact, we have not been able to interpose any kinetic barrier to the reversal of methanol denaturation by water. As an example we cite the identity of optical density changes (“2%) in the following mixing procedures : (a) DNA in 94.5% methanol, O”C., added dropwise to rapidly stirred 20.5% methanol, at -18°C. (b) The same solutions poured into each other and mixed in volumetric flasks at 6 or 25°C. There is a small effect of DNA average composition on the denaturation. DNA with a high guanine-cytosine content denatures at higher methanol content (Fig. 9) and is, therefore, more resistant to solvent denaturation by methanol. Nevertheless, the relative invariance of denaturation to average base composition means that the rela-
NONAQUEOUS
DNA
SOLUTIONS
123
tively broad range of solvent composition through which the transition for a particular DNA sample occurs cannot be attributed to heterogeneity of base composition of that DNA.8 C. EFFECT OF TEMPERATURE METHANOL TRAXXTIOS
ON THE
Figure 10 shows the effect of temperature on the methanol transition. In these experiments, solutions are mixed at or near 0°C. Each experimental point in Fig. 10 represents a different solution, and only the mixed solution is exposed to the indicated temperature. At 0.5”C., 2 x lo-” M salts pH 7, the methanol transition is strongly suppressed. On warming a 92% methanol solution from 0.5”C., the transition occurs relatively sharply at 17-22°C. (1). In testing the reversibility of this LLthermal” denaturation one finds a striking situation. The effects of temperature on the 92% methanol DKA solution are totally irreversible. Data from a typical experiment are shown in Table II. Yet when 92% methanol solutions of DNA that have been exposed to 25’C. (e.g., solutions b and c, Table II) are returned t,o 51% methanol by t’hc addit,ion of aqueous lo-” M NaC1, 10W3 31 Tris, they cannot be distinguished, with regard to their thermal stability or absorbance, from native DIVA (Fig. 7). In other words, two forms of DNA can exist in methanol. They are connected by an irreversible transit,ion, but water converts them to identical products. The irreversible conversion, in methanol, of one DNA4 configuration to another also occurs upon lowering the electrolyte concentration. Figure 11 shows the results of sedimentation velocity experiments which demonstrate this. In one experiment (curve a) DNA is dialyzed into methanol, at 46”C,. in the presence of 10P3 M NaCl. In ‘One factor contributing to broadening of the transition is preferential binding of one solvent component. Even in so extended a macromolecular chain as DNA, preferential binding of one solvent, component ovrr the other will tend to cause variations of the solvent composition inside one macromolecular domain and thus favor the formation and persistence of partially denatured states in single molecules.
1.00 II 0
I
20
I
. e-----d’ 11
I
40
II
% Methanol FIG.
position, (67 mole 1O-4 M Fig. 4, mole % tin&ion aqueous
60
,I 1
I
SO
I1
100
(“Iv )
9. Methanol denatnration and DNA com25°C. l : Pseudomonas fhorescens DNA % guanine-cytosine) 10m3 M NaCl, 5 X Tris, pH 7.1. Preparation of solutions: as curve a. ---: salmon DNg (H-II ; 43 guaninc-cytosine; from Fig. 4). E/E,, , excoefficient relative to nat,i\-c, DSA in salt solution.
the other, DNA is dialyzed into methanol, at 4-6”C., using salt-free solvents, and NaCl is subsequently adjusted to lo-” M (curve b) . The sedimentation constants of the latter solutions are very much larger, arguing for the creation of a more compact configuration through extensive dcnaturation, which was not reversed by the added electrolyte. Yet the substantial reversibility of this dialysis process with respect to solvent change has already been demonstrated (1). The next extension of these experiments is to investigate the effect of heat on the solvent-denaturation reversibility, the test of reversibility being the optical density and thermal denaturation profile in 51% methanol. Results arc shown in Fig. 7. They indicate that in order to cause any decrease in the thermal stability of DNA secondary structure (tested at 51% methanol) 95% methanol solutions containing lo-:’ M NaCl, lo-” X Tris must be hcatcd above 55°C. D.
TRANSITIONS
IN OTHER ALCOHOLS
No transitions can be observed in ethanol, propanol, or tert-butyl alcohol at 25”C., lo-” M salt. Turbidity or precipitation sets in at 7&80% (v/v), before substantial changes in t/c0 occur.
124
GEIDUSCHEK
60
AND HERSKOVITS
70 % Methanol
FIG. 10. Effect of temperature on methanol Tris, pH 7.1). 05°C.: 0; 25°C.: n ; 40°C.: curve a; 25 and 4O”C., as Fig. 4, curve a.
Solvent: 0.001 92.3% methanol.
TABLE M NaCl,
II 0.001 M Tris, pH 7.1,
Thermal history
; C
and
0.5”C. 0.5 -+ 25”C.a 0.5 ++ 25” -+ 0.5%.
a Solutions 6 Uncorrected
equilibrated for thermal
0.876 1.123 1.136
80 (v/v)
I
90
denaturation 0; Preparation
100
(DNA H-II, 10e3 J4 NaCl, lo-* M of solutions: 0.5”C., as Fig. 3,
III.
DISCUSSION
The experiments which have just been described, provide information about two distinct types of denaturation processesoccurring in methanol-water mixtures, one reversible, and the other not. They are interrelated in the following manner:
1 hr. at 25°C. expansion of solvent..
01
methanol-denatured
DNA
I? native
DNA
B
However, it is possible to study the effect I i’ ‘\ (methanol) DNA’ of higher alcohols on secondary structure stability in mixtures with methanol and (N’) electrolyte (i.e., 0.6~s v/v alcohol + 0.4~7% “Methanol-denatured” DNA is the name v/v methanol + lo-” AL?Tris + [lo0 ~1% v/v water) at 25°C. It is then found stability in formamide and N,N’-dimethylformamthat the total mole fraction of alcohols at ide (DMF). The solvent denaturation occurs at the midpoint of the solvent denaturation a much Iower mole fraction DMF (0.23,5 X lo-* M (0.57, 5 X lo-* M decreases in the order methanol, ethanol, Tris acetate) than in formamide at 25°C. We consider this as addipropanol; i.e., the secondary structure sta- Tris acetate) tional evidence in favor of our earlier contention bility is lowered as the length of the alithat hydrophobic forces play an important role in phatic chain is increased.g, lo ‘A ing,
comparable is found in
effect, though much comparing secondary
more strikstructure
determining the stability of native, “These, and other solvent media ject of current experiments?
helical DNA. are the sub-
NONAQUEOUS
DNA
which we have given to the fully hyperchromic material found, say, in 95% methanol at 25”C., and stable over a consideraand ionic blc range of temperatures strengths. N’-DNA is the designation of the hypochronzic material observed in 90% methanol, lo-:$ X NaCl, 1O-:s M Tris at 0°C. (Fig. 3 curve c) . W-DNA and native DPI‘A are either identical or very similar. The major emphasis in this study is on reaction CL,the reversible interconversion of native and met’hanol-denatured DNA. Below, we shall discuss the effect of electrolyte on this transition, before commenting on its reversibility. Finally, we shall offer sonic brief remarks about reaction p, the irreversible, thermal conversion of N’-DNA to methanol-denatured DNA which occurs in methanol-rich solvents. It is clear [Fig. 3 this work, and Table II of Ref. (I)] that electrostatic factors do play a role in methanol denaturation at sufficiently low elect’rolyte concentration, in the sense t’hat removal of electrolyte makes the DKA helix more labile. On the other hand, the degree of ion pairing on DNA i 16) in salt-free methanol-water mixtures does not change markedly in the transition region. In fact, the relatively extensive ion association in methanol assures that electrical repulsions play a smaller stabilitydetermining role in methanol-rich solvent mixtures t,han in aqueous solution. The rather complex phenomena associated with t,he methanol denaturation in salt-free media [which had previously been misinterpreted by one of us (14)] are, instead, due to indirect effects of the low salt concentrat,ion. In these media one finds that rhanges of ultraviolet absorption, sedimentation velocity, and radius of gyration occur over a broad range of solvent mixtures. WC postulate that,, at the DNA concentrations used in t’he schlieren analysis of sedimcntation boundaries (0.5-l mg./ml.) , hydrodynamic interactions are sufficiently great, that the sudden increase of s at SOT0 methanol (Fig. 1) is merely an indication that the end point of a series of macromolecular changes has been reached. Moreowl’, I ow salt concentrations will tend to produce broad transitions, especially in the presence of trace contamination by bivalcnt
SOLUTIONS
60
01
I 20
I % Methanol
I 80
I loo (v/,)
FIG. 11. Sedimentation velocity in methanolwater, IO-” 111 NaCl at 4-6°C. 0 Solutions prepared by dialysis, at 4-6”C., in abscnca of electrolyte. NaCl added to DNA solution at final solvent
composition. Solutions prepared by dialysis, at 4-6”C.,
in the presence
of 10m3 M NaCl. In 90-98s and fast (0 ) component DNA sample: H-II; roncen-
methanol, a slow (0) could tration
be deteceted. 0.4-0.7 mg./ml.
cations.ll [We have previously commented on the fact that such contaminants can be scavenged by DNA during dialysis (I) .] Finally, in water-rich media, electrostatic repulsions on the DNA polyion are so great that even partly denatured DNA (as judged by optical density criteria) has a highly extended configuration.12 The experimental data on sedimentation velocity (Fig. I), radius of gyration (Fig. 2), and ultraviolet absorption (Fig. 3) are generally consistent with this interpretation. Ultraviolet absorption, as a measure of secondary structure ordering, shows changes in the O-7570 methanol composition range (at 0.5”C.). The light-scat’tering average radius of gyration (within the large limits of error imposed by the necessity of working with exceedingly dilute solutions, c = 0.004-0.02 mg./ml.) shows its greatest changes in the 45-75s methanol romposi‘I J%-. Dovr, I’. Brooks. and K. Davidson. in a private c*ommunicxtion (1960), proposed this interpretation for certain denaturation experiments performed by them. IL’ This has also been a source of complications in t,hc rsperimcnts on the flexibility of thr, DNA helix (26, 27).
126
GEIDUSCHEK
AND
tion range. Sedimentation velocity is measured at higher concentrations (c = 0.5-l mg./ml.), where DNA contributes a higher counterion concentration than in the lightscattering and absorption measurements, and consequently offers slight additional stabilization. One observes that the sharpest changes in sedimentation velocity occur at 80% methanol, that is, slightly higher than the end point of the changes in radius of gyration. In electrolyte-containing media, on the other hand, the definition by both optical and macromolecular criteria of that region of solvent composition in which denaturation occurs is simple: (a) Viscosity and optical density change in the same range of solvent composition (Fig. 6) . (b) When the hyperchromic shift is essentially complete, as in 92% methanol, 2 x lo-” M salts, 2O”C., one cannot produce detectable streaming birefringence of DNA. The viscosity, light-scattering, and birefringence experiments all show that methanol-denatured DNA assumes a very compact and/or flexible configuration. Methanol and ethanol denaturation, in the absence of salt, were previously shown to be substantially, but not completely, reversible. The experiments reported here not only demonstrate the complete reversibility of methanol denaturation, in the presence of 1O-3 M salt, by waterI (Fig. 4), but show that this reversibility is stable toward heating of DNA in methanol (Fig. 7). This complete reversibility of methanol denaturation by water immediately raises the question of exactly how disordered the DNA configuration in methanol really is, and how it differs from the structure of heataqueous-denatured DNA. In making this comparison we encounter two handicaps. I3 It is probable that the incomplete reversibility of the lengthy dialysis procedures employed previously results mainly from partial denaturation occurring in water and in water-rich solvent mixtures in the absence of added salt. It is entirely in keeping with this interpretation that the reversibility in the absence of added electrolyte was previously found to be less complete in methanol than in ethanol, since the latter solvent, by virture of its lower dielectric constant, provides greater ion-pairing.
HERSKOVITS
First, the ability to test the completeness of denaturation by optical criteria is limited both by the precision of measuring E/Q,, and by the need to consider a variety of possible causes for the hyperchromicity (cf. sec. IIB) . Secondly, macromolecular properties (p, [v], s) are not very sensitive to the final stages of secondary structure disruption. However, it is evident that methanol-denatured and heat-aqueous-denatured DNA are distinguished by the criterion of reversibility (e.g., Fig. 4). On the basis of the reversibility experiments we conclude that the strand dissociation proposed by Meselson and Stahl (29) and by Marmur et al. (24, 25) most probably does not occur and that, in the sense of dissociation of every secondary valence link between complernentary pol ynucleotide strands, methanol denaturation is not complete.‘” What does seemmost likely is that, even in 98% methanol, elements of secondary structure remain in each molecule, and that these provide nuclei for the reformation of long, ordered, helical sequences. When the environmental conditions are, once again, made thermodynamically favorable to the reformation of the native secondary structure, the kinetic path is established by these nuclei. On the other hand, DNA heat-denatured in water has lost these reference points. Whatever its subsequent treatment, it cannot undergo the rapid reassemblage of the double helix (Fig. 4) which characterizes the reversibility of methanol denaturation. This is assumed to be the structural basis for the difference in behavior of aqueous-heat-denatured DNA (Fig. 4, curve b) and methanol-denatured DNA (Fig. 4, curve a). To what property do the residual nuclei in methanol-denatured DNA owe their greater stability? The following alternatives must be considered: (a) Intramolecular heterogeneity of base composition. In thermal denaturation of aqueous DNA solutions, guanine-cytosine base pairs confer greater stability on DNA secondary structure, and the thermal denaturation temperature is therefore dependent on average base composition. The occur14Further proof of these sought through isotope-banding
statements will experiments.
be
r;ONAQUEOUS
DNA
rcnce of residual nuclei has also been demonstrated in thermal-aqueous denaturation (30). The extra thermal stability of these nuclei relative to their contiguous nucleotide sequences depends on the average DNA composition and on t,he solvent medium. The incremental stability is not greater than can be attributed to intramolecular heterogeneity of nucleotide composition and the firmer association of guanine-cytosine base pairs. In methanol denaturation, on the ot’her hand, the additional stability of these residual “nuclei” for reformation of secondary structure is extremely large. For instance, in 92% methanol, 2 X lo-” ~11 salt, the denaturation transition occurs rat’her sharply at 195°C. [Table III, Ref. (l)]. Yet the denaturation is not made irreversible toward water addition by heating to 55°C. (Fig. 7). Irreversibility of denaturation, as judged by this assay, sets in at least 30” above the transition temperature. At the same time, the met’hanol denaturation transition is not very sensitive to avercrge DSA composition (Fig. 9), so that it is not possible to ascribe this temperature spread to local heterogeneity of guaninecytosine content. However, there may be other combinations of base pairs which do confer such special stability to DIVA secondary structure in methanol and it would be interesting to pursue that question further. (5) Part of the secondary structure changes t’o a different ordered configuration, in which nucleotide pairs remain in register. Such regions, as has been not.ed above, need not be hypochromic. They would provide points of reference for the re-establishment of regular base stacking upon the addition of water. A transition of this type occurs in Na-DNA fibers at a thermodynamic activity of water slightly below 0.5 (31). Howcvcr, no example of such a transition has yet been identified in solution, and the difficulty of so doing has been commented upon in sec. IIH. In the experiments reported here, the macromolecular evidence points to a compact configuration in methanol-denatured DNA. Any structure containing stacked base pairs should be relatively inflexible. Consequently, the presumptive “new” ordered configuration can
SOLUTIOI’iS
127
only involve a fraction .of the nucleotide pairs of each molecule. In view of the superior stability of this hypothetical new secondary structure, there would have to be special steric factors which permit only a fraction of each molecule to be transformed to it,. (c) Aggregation. Sections of Dn’A molecules might be protected from solvent denaturation as a result of partial aggregation. However, auxiliary light-scattering experiments show that even when met,hanol solutions of DNA (10W3 M NaCl) are prepared by direct mixing, aggregation is slight. In addition, the methanol denaturation of DNA solut,ions (lo-” M NaCl, 1O-3 31 Tris) as dilute as 0.015 mg./ml. can be reversed by water. To extend these limits to lower concentrations, it is necessary to use a more sensit,ive assay of denaturation irreversibility, such as transforming ability. In the absence of such measurement’s we can only say that if an aggregation process is responsible for the observed reversibility, it is one which occurs at very low DNA concentrations, without, however, leading to precipitation at concentrations as high as 2 mg./ml. (d) A minor contaminant binds more avidly to helical than to denat,urcd DNA in methanol-water mixtures and is present in concentrations sufficient to stabilize only a small fraction of the secondary structure.ll However, reversibility of methanol denaturation is also observed in DNA isolated from Serratin mnrcescenu by an entirely different method (4). In addit’ion, extensive dialysis of aqueous DNA (HI1 in IO-’ JI 9aCl) in no way changes the reversibility of its methanol denaturation. 0f these four proposals, none can yet be rigorously excluded, although alternative (/I)-contamination-appears highly irnprobable. At this time me arc therefore unable to establish the structural basis of the reversibility of methanol denaturation. Xevertlielcss, the expcrimcnte presented here do establish t,he fact that this reversibility rests on different structural considerations from those applying to denaturation in aqueous solution. They permit the hope that new information about DNA secondary strurturc may be forthcoming from further
128
GEIDUSCHEK
AND
experiments on this and other nonaqueous solvent systems. One further comparison is appropriate. The reaction of DNA with mercuric chloride results in configuration changes which are, in some respects, similar to those described here. Upon binding mercuric ion, DNA assumes a more compact configuration, as shown by the light-scattering and viscosity measurements of Katz (32). Sequestration of mercuric ion by complexing agents results in a reversal of the configuration changes (33). Transforming DNA which has been put through such a mercury-binding cycle retains biological activity (34). However, Hg-DNA and methanol-denatured DNA differ in one important respect: Hg-DNA is hypochromic, and undergoes an irreversible thermal transition accompanied by a large increase in absorbance (33). Methanol-denatured DNA is hyperchromic. Its absorbance does not increase substantially upon further heating. In view of the remarkable reversibility of the methanol denaturation with respect to addition of water, it is, of bourse, surprising to find an irreversible transition (reaction p) in methanol-rich media, which can be caused to occur by heating (Table II) or by removal of electrolyte (Fig. 11). However, it is clear that this process differs from thermal denaturation in aqueous media in that its end product, methanol-denatured DNA, can be reconverted (reactivated) to native DNA by water. Evidently, water is necessary to provide the kinetic path for the re-establishment of long-range order on the one hand, and for its complete dismemberment, on the other. These observations suggest that in the kinetics, as well as in t,he equilibria of DNA secondary structure, water plays a special role. ACKNOWLEDGMENTS It is a pleasure to acknowledge our indebtedness to Dr. S. J. Singer for the advice and encouragement which he offered us throughout the course of this work and for his critical reading of the manuscript. We are grateful to Miss Susan Staves and Mr. Alan Daniels for their expert technical assistance. One of us (T. T. H.) held a U. S. Public Health Service predoctoral fellowship during a part of this investigation.
HERSKOVITS REFERENCES 1. HERSKOVITS, USCHEK,
T. T., SISGER, S. J., AND GEIDE. P., Arch. Biochem. Biophys. 94,
99 (1961). 2. SIMMONS, DUGGAX,
as quoted by STEVENS, V. L., AND E. L., J. Am. Chem. Sot. 79, 5703
Y.,
(1957). 3. JONES, A. S., Biochim. et Biophys. Acta 10, 607 (1953). 4. H~MAGuCHI, K., AND GEIDUSCHEK, E. P., Abstr. Biophys. Sot. (Feb. 1961). FBlO. 5. BUXVILLE, L. G., AND GEIDUSCHEK, E. P., Biochcm. Biophys. Research Commons. 2, 287 ( 1960). 6. EDSALL, J. T., RICH, -4., AND GOLDSTEIN, M. Rev. Sci. Instr. 23,695 (1952) 7. See, for instance, EDSALL, J. T., Advances in Colloid Sci. 1,269 (1942). 8. GOLDSTEIX, M., AND REICHMANN, M. E., J. Am. Chem. Sot. 76,3337 (1954). 9. EWART, R. H., ROE, C. P., DEBYE, P., AND MCCARTNEY, J. R., J. Chem. Phys. 14, 687 (1946). 10. BRINKMAR., H. C., AND HERMANS, J. J., J. Chem. Phys. 17,574 (1949). 11. KIRKWOOD, J. G., AND GOLDBERG, R. J., J. Chem. Phys. 18,54 (1950). 12. STOCKMAYER, W., J. Chem. Phys. 18, 58 (1950). 13. STURTEVANT, J. M., AND GUTFREUND, H., Proc. Natl. Acad. Sci. U. S. 42, 719 (1956). 14. GEIDUSCHEK, E. P., AND GRAY, I., J. Am. Chem.
Sot. 78,879 (1956). 15.
J. H., AND JORDAN, D. O., Biochim. et Acta 43,223 (1960). 16. HERSKOVITS, T. T., Ph.D. Thesis, Yale, 1959. 17. TINOCO, I., J. Am. Chem. Xoc. 82, 4785 (1960). 18. Cox, R. A., AND LITTAUER, U. Z., Nature 184, 818 (1959). 19. DOTY, P., BOEDTKER, H., FRESCO, J. R., HASELKORN, R. AND LITT, M., P~oc. Natl. Acad. Sci. U. S. 45,482 (1959). 20. HASCHEMEYER, R., SINGER, B., AND FRAENKELCONRAT, H., Proc. Natl. Acad. Sci. U. S. 45, 313 (1959). 21. LITTAUER, U. Z., AND EISENBERG, H., Biochem. et Biophys. Acta 32, 320 (1959). 22. SPIRIN, A. S., GAVRILOVA, L. P., BRESLER, S. E., AND MOSSEVITSKY, M. I., Biokhimiya 24, 938 (1959). 23. SINSHEIMER, R. L., J. Mol. Biol. 1,43 (1959). 24. M.~RMuR, J., AND LANE, D., Proc. Natl. Acad. Sci. U. S. 46,453 (1960). 25. DOTY, P., MARMUR, J., EIGNER, J., AND SCHILDICRAUT, C., Proc. Natl. Acad. Sci. U. S. 46, 461 (1960). 26. CONWAY, B. E., BUTLER, J. A. V., AND JAMES, D. F. W., Trans. Faraday Sot. 50,612 (1954). 27. SADRON, C., “Nucleoproteins,” p. 124. (Onzieme COATES,
Biophys.
NONAQUEOUS Conseil de Chimie Solvay, 1959 ; Interscience Publ., New York, 1960). 28. YANG, J. T., AND DOTY, P., J. Am. Chem. Sot. 79,761 (1957). 29. MESELSON, F. W., AND STAHL, F. W., P~oc. N&l. Acad. Sci. U. S. 44, 671 (1958). 30. GEIDUSCHEK, E. P., Federation Proc. 20, 353 (1961).
DNA
129
SOLUTIONS
31. LANGRIDGE,
WILSOS, H. R., HOOFER, C. W., M. F. H., AND HA~ILTOS, L. D., J. Mol. Biol. 2, 19 (1959). 32. KATZ, S., J. Am. Chem. Sot. 73,2238 (1952). 33. TAX~XE, T., AND DA~IDSOS, I’i., J. Am. Chem. Sot. in press. 34. DOVE. W. F., AND TA~SE, T., Biochem. Biophys. Research Communs. 3, 608 (1961). WILKINS,
R.,
OF BIOCHEMISTRY
AND
BIOPHYSICS
Nonaqueous
96,
Solutions
Irreversible
the
(1961)
of DNA.
Denaturation E. PETER
From
11k-129
Committee
Reversible
and
in Methanol’
GEIDUSCHEK
on Biophysics, The Chicago, Illinois
University
of Chicago,
AND
THEODORE From
the
Department
T. HERSKOVITS
of Chemistry, Lafayette, Indiana
Received
May
Purdue
University,
17, 1961
Deoxyribonucleic acid (DN,4) undergoes denaturation in methanol-water mixtures. The region of solvent composition in which this transition occurs depends on the ionic strength and temperature but is little influenced by the average nucleotide composition of the DNA. As judged by both macromolecular and optical criteria, the disruption of secondary structure is almost complete, yet methanol denaturation is rapidly and readily reversible by water. The structural basis of this reversibility differs from that controlling the reversibility of partial denaturation in aqueous solution. An irreversible thermal transition has also been found to occur in methanol-rich solvents. The products of this transition may be reconverted to native DNA by water. INTRODUCTION
In a preceding communication (1) we have presented evidence that two nonaqueous solvents, methanol and ethanol, alter the conformation of deoxyribonucleic acid (DNA). At this juncture we describe a systematic study of denaturation in methanolwater mixtures. The experiments reported here deal with the transition from the native, ordered, extended configuration present in aqueous solution to denatured, more or lessdisordered and altered, compact configurations as the solvent medium is changed. The ionic strength and temperature dependence of the transition and its reversibility have been studied with the aid of spectroscopic and macromolecular techniques. It is clear from these experiments that the phenomenon of reversibility in methanol denaturation is both qualitatively ‘Research supported, Health Service Research
in part, by U. Grant C 5697.
S. Public 114
and quantitatively different from any previously described. Results are presented in two parts. First, as a direct continuation of previously reported work, we describe the denaturation in electrolyte-free solvents, through measurements of sedimentation velocity, light scattering, conductance, and ultraviolet spectra. In a second section, experiments on the ionic strength dependence and reversibility of the denaturation in electrolytecontaining media are presented. Two brief sections on temperature effects and on denaturation in other alcohols conclude the presentation of experiments. I. EXPERIMENTAL A.
~UATERIALS
DNA samples and other materials have been desrribed in the preceding paper (1). Spectroscopic grade methanol (sets. IIB, C, and D) was used without further purification. Pseudomonas fluores-
NONAQUEOUS cc’nns DN;l was prepared by a method combining detergent, denaturation of proteins (2) and cetyltrimethylammonium bromide fractionation of DNA from ribonucleic acid (RNA) and polysaccharide (3). The same sample of DNA has been used in studies of thermal and acid denaturation (4,5).
B . METHODS Optical rotation was measured in a Rudolph polarimeter (model SOS), equipped with a thermostated cell compartment. Measurements were made at the 4358-A. line, selected from the emission of a mercury vapor lamp (AH-4), by a narrow band interference filter. Flow birefringence measurements were made on an instrument manufactured by the Rao Instrument Company. modeled on the design of Edsall et nl. (6). Birefringence was measured with the Henarmont compensator supplied by the manufacturer. The quantity A?~/cf, (which, for a single solute species, is proportional to the anisotropy factor, g1 - fi) was used as a measure of the amount of material oriented at any shear gradient. In is t,he amount of birefringcnce of the solution and f is the orientation factor. The latter quantity is calculated from the angle of isocline x, assuming infinite axial ratio, (7). For DNA, which is heterogeneous, an/cf is a function of gradient. In terms of what is now generally agreed upon regarding the structural properties of helical DNA, the anisotropy factor, g1 - pr, should be calculated from the limiting value of ~n/cf at high gradients. At lower shear the variation of 4r1/cj with gradient presumably reflects the distribution of molecular sizes and shapes (8). The light-scattering methods used in these experiments have bren described previously (1). Interpretation of light-scattering experiments on DSA-methanol-water mixtures has been made in terms of the theory of two-component solutions [Ref. (1) Eqs. 1 and 21. The justification for this simplification lies with the near identity of the refractive indices of water (n = 1.333) and methanol (n = 1.330). Ewart et al. (Cl), who first investigated such systems, showed that, as the refractive indices of the two solvents approach each other, multicomponent scattering effects disappear. The exact theories of light scattering from multicomponent systems confirm t,he soundness of this conclusion (10-12). Two types of kinetic spectroscopic experiments were performed. In one series of experiments DNA solutions and solvents were mixed in a stoppedflow rapid reaction spectrophotometer, built by Dr. J. M. Sturtevant (13): In the ot>her series, “To pitality
whom we extend and interest.
our
thanks
for
his
hos-
DNA
115
SOLUTIONS
solutions equilibrated at the desired temperature, were mixed by hand in a stoppered absorption cell and inserted as quickly as possible into the cell compartment of a thermostated Beckman DU spect,rophotometer, located in a constant-temperature room. Other measuring and analytical methods were used as described previously (1).
C. PREPARATION
OF
SOLUTIONS
Two methods were used in preparing DNA solutions in methanol-water mixtures: dialysis and direct mixing. The dialysis procedure has been described previously ( 1). Solutions made by direct mixing were prepared by adding constituents in sequencrs designed to minimize denaturation (e.g., methanol or water added to DNA-salt solutions). Detailed descriptions are included in the captions of Figs. 3,4, and 7. The procedure described in Fig. 4 is adequate for all experiments on the reversibility of methanol denaturation by water. The more cautious methods described in Figs. 3 and 7 were used to prepare solutions for experiments in which the irreversible thermal effects occurring in 80-100% methanol (sec. IIC) had to be avoided. Solvent compositions are expressed on a volume basis (v/v) unless otherwise stated. For convenience, a vol.%-weight %-mole % conversion table (at 15°C.) is given below. Methanol d.
(v/v)
Methanol
T,
10 20 30 40 50 60 70 80 90 9B
(w/w)
8.1 16.3 24.8 33.8 42.7 52.2 62.4 73.4 85.7 94.0
II. EXPERIMENTAL A. THE DENATURATION SALT-FREE
Methanol mole /raction
wt. %
0.047 0.099 0.157 0.2’11 0.295 0.381 0.483 O.GO9 0.771 0.897
RESULTS OF
DNA
IN
METHANOL-WATER MIXTURES
The original experiments on the behavior of DNA in ethanol-water and methanolwater mixtures showed a sharp change in average sedimentation velocity at finite concentration, in 80% methanol, and in 65% ethanol (1, 14). The methanol system, with its slightly greater tolerance for electrolyte and with its minimizing of multi-
116
c;EIikWfl~;K
.1ND HERSKO\‘ITS
component effects ih light scnttcring, pro- in the abscncc of salt is shown in Fig. I. A sudden change from slow Is approx. l-5) vitlcd the oljportlmit+ for ‘more dctailcd stkly. Sedimc’ntaGon, iight-scattering. spcc- to r:tl)i(l 1~sapprox. 30-50) wlimcntation trophotometric, tind conductance data arc’ occur’I_ at approximately 805 methanol prcscntcd here. These new results indicate I,IIIO~Cfr:\ction 0.61) .:’ that t.hc sedimentation cxpcriments. t.aken E’urthcvniorc~, solutions first dial~zcd into by themselves, give a quite distorted pic- 99.5“; mct.lianol, before cqmlibrating turc of t,he secondary structure t.rnnsition, against the appropriate solvent, show t.hc and that. their previous intcrprctat.ion is salnc behavior. It, is important to note that thr change in optical density 1259 rnp) incorrect. The sedimentation behavior of DNA tiow not coincide with the cllangc in sedi(H-11, c = 0.5-l mg./ml.), as a function of nwntation behavior, but owurs at low1 solvent composition at 3-8°C. and 23-27°C. alcohol content, (Fig. 3 cm-w (1) so that,, at 655‘; cthnnol and 80’;; methanol, the (‘xt,inc.tion is ;llrcndy high [Z uppros. 9000; cf. 501 Ref. ( 1 I, ‘l‘wblw I and II]:* T,ighb-scattering cxperiment5 in inctha40 nol-water mixtures arc‘ simple from a thcowtical point. of \licw bccauw of the nwrly 3 d 30 idcntic:il rcfr;ic.tivc indiws of tlic two wt2 writ conllwnrnts. At 80-1OOSC methanol s~icli mc~:lsurclllc~nt::arc also rclativcly sim20 l’h~. wvwc intc~rmolccul:ir intcrfcrciw cffwtb usually cntvring onlv al)ovc (: = 2-3 x IO-’ mg. ~ml. 11owvcr, In O-705 salt-free methanol, c5tvrii:il intcrfcrcncc cffrctti arv I I I I iiiiporkmt vvcn at. DK.4 concentrations of 100 0 20 40 60 60 0.01 mg. ,‘nil. (‘onscqucntly, cxpcrimcntal % Methanol (v/,1 rmcwtaintics in thr dctcrnjination of radii FIG. 1. Variation of sedimrntution velocity of gyration (p) aw wry large. Ncverthcwith solvent composition in methanol-water misless, thv InwisiO!i is sufficient to est.abli~h turrs (D?;A H-I, salmon ; no supporting c+ctrothat p tlocs not c~hangc Anrply at 80% lytcl; solutions prepared by dialysis at 4-6°C.). mcthaiiol (Fig., 2). Ilnthor, this solvent X: 3-8°C. or 2S28”C.; 0: 23-28°C.. rnwiously composition appears to corrtq~ontl, approxicxposcd t.o 99.5% mcrhanol at 4-6%. mntc)ly, with the vnd point 0i tlic change in avcragc radius of gyration. I I I I Clonducbtivity cqwrimc~nts at 0°C‘. ( 16 1 intlicatc thttt the nlost rapid changes in ion association of salt-frw I)SA solut.ions occur in methanol-poor media tO-IS?: m&hanal‘). 111tllc r:ingc of solvcrit coiiipositions :woci:~ttd wit11 t.he change in scdimcntat.ion bch:ivior, the degree of ion :woci:~t.ion clinngea rcl:ktivcly little. Increnacs of oj)tical density (259 rnp,I at 25°C. owur in t.hv O-50’;; inct.h:mol solwnt coniposit.ion range. ?rt 0.5”(.‘., such changes occur at liiglw
00
II
FIG. 2. Variation of (light-scattering average) radius of gyration with solvent composition at 5-6°C. in methanol-water mixtures (DNA H-II; no supporting electrolyte; solutions prepared by dialysis).
’ .A tliflcrent. interpretation of the nkol~l tlvnritllrat ion which has been presented rccvnt Iy (I.i), strm15 from 3 failure 10 apprcciniv this fnvt.
KONSQUEOUS
DNA SOLUTIONS
117
1
40
60
% Methanol
80
100
Cvjv)
FIG. 3. Methanol denaturation and ionic strength, at 0.5, and 25°C. c/eO, extinction coefficient relative to native DNA4 in aqueous salt solution. (a) no added salt, 0.5”C. : 0 ; ( i; ) 10e4 M NaCl, 0.5”C.: X ; (c) lo-’ J4 NaCl, lo-* M Tris pH 7.1,0.5”C.: 0 ; (d) 10e3 M l%e 1lIi 7.1, 5 X 10-j :Jf NaCl 25°C. (from Fig. 4, curve a) ; (e) lo-* M NaAc. 10m3M Trid, pH 6.7-i 5. 25°C.:
l .
DNA Sample: H-II (salmon). Preparation of solutions : curve a: DNA is dissolved in water at 0°C. at a concentration of 1 mg./ml. and diluted at 0°C. to 0.3 mg./ml. To 0.5-ml. aliquots of this stock solution, 4.5 ml. of the appropriate water-methanol mixtures are added dropwise with stirring. Reagents, and the final solution are maintained at -5 to O”C., and mixing is performed in a 0°C: roqm so that samples arc never exposed to temperatures above 05°C. curve b : as curve a, but DNA stock solution is lo-” M in NaCl. curve c: as curve a, but DNA stock solution, methanol, and water all contain lo-” -II NaCl, 10eJ M Tris pH 7.1. curues cl, e : as Fig. 4 curve a. content than at 25°C. (Fig. 3 curve a). Both the light-scattering (Fig. 2) and the extinction data show that the denaturation, in the absence of salt, occurs over a rather broad range of solvent. compositions. ,4 system may be judged to have undergone a sharp transition when a number of its properties change suddenly, and more or less in concert. Such is the case for the denaturation of DNA at extremes of pH,
methanol
or high temperature, in the presence of moderately high concentrations of electrolyte. It is clear that the methanol denat,uration in electrolyte-free solution does not, by any means, satisfy this criterion and t’hat the sharpness of the change in sedimentation velocity must be understood on some other basis. Most probably, the sedimentation change reflects an end point of the transition from extended to compact configurations. These are solutions of wry low
118
GEIDUSCHEK
AND
HERSKOVITS
attributed to other causes. For aqueous solutions of DNA, changes of optical density with salt concentration are correlated with denaturation. At O”C., we find the extinction coefficient of DNA to be independent of salt (NaCl) concentration, down to 10-4.5 M, a range of ionic strengths which causes great variation of the electrostatic free energy. (c) Change from one ordered periodic structure to another. If the spacing and orientation of chromophores, relative to each other or to the helix axis, is altered, then changes of absorption intensity may be expected (17). (d) Effect of solvent on the stacking and hydrogen-bonding ability of single (or disordered, or denatured) polynucleotide B. DENATURATION OF DNA IN chains such as found in tobacco mosaic WATER-METHANOL-ELECTROLYTE virus RNA (18-22), denatured DNA, and MIXTURES +X174 DNA (23).6 It is characteristic of DNA solutions in methanol-water mixthese configuration changes that they octures tolerate the presence of additional cur over wide ranges of environmental conelectrolyte without undergoing precipitaditions (ionic strength, temperature, pH). tion. This property has already been put to (e) Effect of solvent on the stability of use in studying the thermal denaturation the ordered “native” configuration of DNA, of DNA in methanol-water mixtures (1). relative to a disordered-denatured state. In the experiments to be described below For the purposes of our experiment, items we have used spectrophotometric measure- (a) and (b) are eliminated by the approments to permit a relatively simple survey priate control experiments cited above. We of properties. At suitable junctures, addi- have been able to find conditions under tional optical rotation and macromolecular which the partial ordering considered under experiments have been performed to insure (d) is almost completely unstable (e.g., that the correlations between optical and Fig. 7). Consequently, we may interpret macromolecular properties, which are so large extinction changes in terms of items consistently operative in aqueous media, (c) and (e), that is, the order + disorder also serve our purposes. It might be well, transition, and the change from one to anbefore proceeding, to summarize those fac- other ordered configuration. Both these tors which could cause changes in ultravioprocesseswould, in the most general sense, let light absorption by DNA: be termed ‘idenaturation.” They would be (a) Solvent effects on the spectra of con- readily distinguishable where one such stituent chromophores. However, control change occurred to the exclusion of the experiments show that the O.D.,,, of nu- other. However, a process in which part of cleoside mixtures does not depend on the each DNA molecule disordered, and part composition of methanol-water solvents.5 changed to another ordered configuration, (b) Effect of local electrostatic fields on would be difficult to distinguish from a absorption spectra. Two facts argue against simple denaturation. the existence of any such effect in DNA. With this discussion as background we The spectra of most nucleosides and their ‘Solvent effects of this type may arise from corresponding nucleotides are almost idenchanges in the intrinsic stabilities of hydrogen and tical. When difierences exist, they may be
ionic strength and very marked nonideality. The absence of rapidly sedimenting material at methanol concentrations below 80% is probably accounted for by the retardation of more mobile species in the medium through which they must move, and which contains the slower moving and presumably more extended molecules. In fact, Fig. le of Ref. (1) shows the presence of two slowly sedimenting components, one with a relatively broad boundary, This has frequently been observed both in ethanol- and methanol-water mixtures below the sedimentation transition. The presence of these two boundaries suggests that salmon DNA is heterogeneous in its resistance to denaturation in salt-free methanol.
“T.
T.
Herskovits,
manuscript
in preparation.
hydrophobic sion of
bonds,
or
from
coiled polynucleotide
electrostatic
chains.
expan-
WNAQUEOUS
DNA
proceed to describe experiments on the reversiblc DNA secondary structure transition in methanol-water-electrolyte mixtures. Figure 3 shows the ionic strength dependence of the denaturation of salmon DNA in methanol-water mixtures. At very low ionic strength, there is a marked influence of salt concentrat’ion on the location of t’he transition (cf. see. IIA). However, between 5 x lo-” and 5 X lo-” M salt, no further effect of salt concentration is dctectable.7 This is in keeping with the relatively great’ degree of ion association with DNA in methanol-rich solvents (16). The dependence of absorbance on solvent composition for native and heat-denatured DNA at 25°C. in methanol-water mixtures containing lo-” M Tris, pH 7 and 5 x lo-” ,U SaCl is shown in Fig. 4. At this low ionic strength the absorbance of denatured DKA is high, even in water (E/E,, = 1.35). For native DKA, on the other hand, the molar extinction coefficient is independent of solvent composition in the O-70% methanol composition range and unchanged from that observed in 0.2 M aqueous NaCl (Z = 6300 for DNA H-II). Between 80 and 90% methanol, E increases by 42 -+ 2%. As we show below, this increased absorbance parallels losses of viscosity and flow birefringence. We call such material solvent denatured. In 90% methanol containing 1OV” M Tris and 5 X lo-” M NaCl this solventdenatured DNA, and DNA denatured by heating in aqueous solution, have the same absorbance, yet they are distinguishable in a very dramatic way. The extinction change of the solvent-denatured DNA can be reversed when water is added back to the system (Fig. 4 curve n, data given by crosses) and 6 returns to its original value. On the other hand, when water is added back to heat,-denatured DISA in 90% methanol, z remains high (Fig. 4 curve b, data given by open squares). Spectra of native DNA in water, solvent-denatured DNA in 95% methanol, and water-heat-denatured DNA in 95y0 methanol, all at 25”C., lo-” M NaCl, lo-” 111Tris, pH 7 are shown in Fig. 5. i Above 0.05 M salt, aggregation and tion occur in 90-100% methanol, setting the range of this experiment.
precipitaa limit
to
119
SOLUTIONS
1.20 l.lOt-
Y I.003 n O n * O n 3x xx 0 x I 0
I
I
I
20
40
60
% Methanol
FIG. 4. Extinction coefficient position at 25°C. e/co, extinction tive to native DNb in aqueous
4
I
60
I
100
Cv/,)
and solvent coefficient salt solution.
comrela-
curve a: 0 : Untreated DNA; solutions prepared by adding methanol t,o aqueous DNA solutions. X: DNA previously exposed to 90 or 95% (v/v) methanol, 5 X lo-” ,lil NaCl; lo-” JI Tris, pH 7.1 25°C. CWVB h: l : DNA previously denatured by heating in aqueous 2 X lo-’ M Tris, pH 7.1. lo-” .II NaCl to 95°C. for 15 min. q : DNil heated to 95”C., 15 min. and exposed to 95% (v/v) mrthanol. Solvents: Methanol-water mixtures, IO-’ ~11 Tris pH 7,5 X 10-j ,VJ NaCI. DNA sample: H-II (salmon). Preparation of solutions : curve a, points shouva by circles: 0.50-l ml. DNA (0.3-0.6 mg./ml.) in 1-2 X 10e3 LV N&l, 1-2 X IO-’ M Tris, pH 7, diluted to 10 ml. with such quantities of water and methanol as will yield the methanol-water mixtures (at 10m3 11f Tris, 5 X lo-” iPl NaCl) shown. Prepared solutions are equilibrated 1 hr. at 25°C. before making absorbance measurements. CUTV~ a, points shop by CTOSSC.S: 0.50 ml. Dli-4 (0.6-1.2 mg./ml.) in lo-’ J4 NaCI 2 X lo-* ,V Tris, pH 7, diluted with 9.5 ml. methanol, and water, to 10 ml. Methanol may be added to DNA4 without, special care regarding mixing. This stock solution of DNA in 95% methanol is then kept at 25°C. for 1 hr. and retooled. It is now diluted with water-methanol-10m3 M Tris-5 X 10-j Jf NaCI solutions to yield the solvent compositions shoed. curvy b: DNB (0.3-1.2 mg./ml.) in 2 X lo-’ ,Zr Tris, pH 7, 1O-3 M NaCl serves as a stock solution. An aliquot is diluted and its absorbance measurpA so as to establish a reference from which E/E<, may be calculated. The remainder is heated to 95’C. for 15 min. and cooled rapidly in ice. From this stock, solutions are prcpnred as described abol-cx for curve a.
GEIDUSCHEK
120
AND HERSKOVITS
0.8 0.6 0.4 0.2 I
220
240
260
280
300
320
FIG. 5. Spectra of DNA (ST-l, salmon) at 25°C. in lo-’ M NaCl, lo-* M Tris, pH 7. fh/emax : relative extinction coefficient. 1: water (X,., = 258.5 mp) ; 2: 95% methanol (A,., = 260 mp) ; 3: 95% methanol. DNA denatured by heating aqueous solution to lOO”C., 15 min. (hmsr = 260 mp).
30(
0
20
40
60 % Methanol
80 (v/,)
FIG. 6. Comparison of absorbance, viscosity, and optical rotation of DNA in methanolwater mixtures. [a], : specific rotation at 436 rnq. [v]/[& : intrinsic viscosity relative to aqueous salt solution. Untreated DNA; solutions prepared by adding methanol to aqueous DNA solutions. e: 0; [q] : 0; [a] : @. DNA previously exposed to 9599% (v/v) methanol, 10m3M NaCl, 10d M Tris, pH7, pH7,25”C. [+j : W, [a] : 17. Solvents : Methanol-water, 1O-8 M NaCl, lOWaM Tris, pH 7,25”C. Preparation of solutions: as Fig. 4, curve a. Auxiliary experiments make it evident that this reversibility occurs despite the fact that DNA undergoes profound changes of macromolecular configuration between
80 and 90% methanol. The viscosity decreases drastically (Fig. 6) and no stream-
ing birefringence can be detected when 0.02% DNA solutions in 9&95% methanol containing 1O-3 M NaCl, 10W3 M Tris are
subjected to shears up to 6000 sec.-l. As the data of Fig. 6 also show, changes in intrinsic viscosity and extinction coefficient
NONAQUEOUS
DNA
occur in the same range of methanol composition. The specific rotation, [a], also changes in the transition region. However, we find that [a] varies with solvent composition even when no changes of viscosity or absorption (c = 259 mp) can be observed. Similar situations occur in other solvent mixtures and in concentrated aqueous salt solutions of DNA (4). Further evidence for the complete reversibility of methanol denaturation by addition of water is afforded by the following experiments : 1. The thermal denaturation transitions in 51% methanol, of native DNA and DNA recovered from 99% methanol containing 10P3 M NaCI, 10e3 M Tris, are compared in Fig. 7. In this solvent, heat-denatured DNA exhibits little hypochromicity. The
121
SOLUTIONS
identity of the two heating profiles indicates, therefore, that the methanol-treated DNA4 has recovered the long-range order characteristic of the native configuration. 2. The flow birefringence of native DN,4 and DNA recovered from 99% methanol, measured in 45% methanol, is essentially identical (Fig. 8). Not only is the gradient dependence of x the same for these two systems (curve a) but the fraction of solute orientable at each gradient is the same (curve 5). These two experiments constitute a stringent test of the re-establishment of the stiff, extended chain configurations characteristic of long-range order. 3. The intrinsic viscosity of native DNA, and DNA recovered from 99% methanol, measured in 47% methanol at 25”C., are virtually identical (Table I).
- so0 % N is a - .050
-0 I 25
35
45
I
55
T”C FIG. 7. Ultraviolet absorption thermal analysis of DNA (H-II) in 51% methanol (v/v), lo-’ M NaCI, lo-’ M Tris, pH 7. A : untreated; preparation of solutions: as Fig. 4, curve a. l , 0, n : Previously exposed to 95% (v/v) methanol, 10e8 M Nacl, lo-* M Tris, pH 7 at 0.5”C. ( 0 ), 25°C. (m), and 54.9”C. (01, respectively. Preparation of solution: To 1 ml. DNA (0.7 mg./ml.) in lo-* M NaCl, 10.’ M Tris, pH 7 add 20 ml. of 99% (v/v) methanol lo-’ M NaCl, 1O-3 M Tris, pH 7 dropwise, with stirring. Reagents and the final solution are maintained at -5 to 0°C. and mixing is performed in a 0°C. room. Aliquots of this solution are exposed to 0.5,25, and 54.9”C., respectively, for 1 hr. and retooled. Iced aqueous 10.’ M NaCl, IO-’ M Tris, pH 7 is then added dropwise, at O”C., to give a final methanol concentration of 51% methanol. 0: after one cycle of heating to 65°C. in 51% methanol. Data uncorrected for thermal expansion of solvent.
GEIDUSCHEK
122
1000
AND HERSKOVITS
2000
3000
4000
5000
G see-’ FIG. 8. Streaming birefringence in 44% (v/v) methanol lo-’ M NaCI, 10m3M Tris, pH 7, 20°C. x: extinction angle. An/cf: birefringence per unit DNA concentration, corrected for average orientation factor, 1, plotted in arbitrary units. 0: untreated; c = 0.119 mg./ml. l : previously exposed to 99% (v/v) methanol, (lOJ M NaCl, 10m3M Tris, pH 7, 25°C.). c = 0.102 mg./ml. Sample : H-II (salmon). Preparation of solutions : as Fig. 7.
TABLE I Sample
history
Solvent
[VI
Native (Sample HII, salmon)
0.2 M NaCl
68
Native
47.6T0 (v/v) methanol, 1OWM NaCl 1OWM Tris pH 7
70
Exposed t’o 99% (v/v) methanol, 10-a M NaCl, 10-a M Tris, pH 7, 25°C.
47.2% (v/v) methanol, 10-Z M NaCl, 10-s M Tris, PH 7
73
4. The effects of solvent composition on optical activity are reversible (Fig. 6). This complete reversibility of the methanol denaturation in a heterogeneous DNA at low ionic strength distinguishes it from the “renaturation” of thermal-aqueous denaturation described by Marmur, Doty, and co-workers (24, 25). The methanol denaturation is also distinguished from ‘(renaturation” of thermal aqueous denaturation on kinetic grounds. It has been found that the solvent-induced transition is fast in both directions. For example, the optical density reduction on mixing DNA-97%
methanol with 61% methanol (all solutions lo-” M NaCl, 10W3 M Tris, pH 7) to give 79.5% methanol is at least 90% complete within 5 sec. at 25°C. When DNA-95% methanol and water (same ionic composition) are mixed to give a 70% methanol solution, the absorbance reduction is more than 90% complete within 20 sec. at 25°C. No special procedure (such as gradual mixing or dialysis) is necessary to achieve reversibility. In fact, we have not been able to interpose any kinetic barrier to the reversal of methanol denaturation by water. As an example we cite the identity of optical density changes (“2%) in the following mixing procedures : (a) DNA in 94.5% methanol, O”C., added dropwise to rapidly stirred 20.5% methanol, at -18°C. (b) The same solutions poured into each other and mixed in volumetric flasks at 6 or 25°C. There is a small effect of DNA average composition on the denaturation. DNA with a high guanine-cytosine content denatures at higher methanol content (Fig. 9) and is, therefore, more resistant to solvent denaturation by methanol. Nevertheless, the relative invariance of denaturation to average base composition means that the rela-
NONAQUEOUS
DNA
SOLUTIONS
123
tively broad range of solvent composition through which the transition for a particular DNA sample occurs cannot be attributed to heterogeneity of base composition of that DNA.8 C. EFFECT OF TEMPERATURE METHANOL TRAXXTIOS
ON THE
Figure 10 shows the effect of temperature on the methanol transition. In these experiments, solutions are mixed at or near 0°C. Each experimental point in Fig. 10 represents a different solution, and only the mixed solution is exposed to the indicated temperature. At 0.5”C., 2 x lo-” M salts pH 7, the methanol transition is strongly suppressed. On warming a 92% methanol solution from 0.5”C., the transition occurs relatively sharply at 17-22°C. (1). In testing the reversibility of this LLthermal” denaturation one finds a striking situation. The effects of temperature on the 92% methanol DKA solution are totally irreversible. Data from a typical experiment are shown in Table II. Yet when 92% methanol solutions of DNA that have been exposed to 25’C. (e.g., solutions b and c, Table II) are returned t,o 51% methanol by t’hc addit,ion of aqueous lo-” M NaC1, 10W3 31 Tris, they cannot be distinguished, with regard to their thermal stability or absorbance, from native DIVA (Fig. 7). In other words, two forms of DNA can exist in methanol. They are connected by an irreversible transit,ion, but water converts them to identical products. The irreversible conversion, in methanol, of one DNA4 configuration to another also occurs upon lowering the electrolyte concentration. Figure 11 shows the results of sedimentation velocity experiments which demonstrate this. In one experiment (curve a) DNA is dialyzed into methanol, at 46”C,. in the presence of 10P3 M NaCl. In ‘One factor contributing to broadening of the transition is preferential binding of one solvent component. Even in so extended a macromolecular chain as DNA, preferential binding of one solvent, component ovrr the other will tend to cause variations of the solvent composition inside one macromolecular domain and thus favor the formation and persistence of partially denatured states in single molecules.
1.00 II 0
I
20
I
. e-----d’ 11
I
40
II
% Methanol FIG.
position, (67 mole 1O-4 M Fig. 4, mole % tin&ion aqueous
60
,I 1
I
SO
I1
100
(“Iv )
9. Methanol denatnration and DNA com25°C. l : Pseudomonas fhorescens DNA % guanine-cytosine) 10m3 M NaCl, 5 X Tris, pH 7.1. Preparation of solutions: as curve a. ---: salmon DNg (H-II ; 43 guaninc-cytosine; from Fig. 4). E/E,, , excoefficient relative to nat,i\-c, DSA in salt solution.
the other, DNA is dialyzed into methanol, at 4-6”C., using salt-free solvents, and NaCl is subsequently adjusted to lo-” M (curve b) . The sedimentation constants of the latter solutions are very much larger, arguing for the creation of a more compact configuration through extensive dcnaturation, which was not reversed by the added electrolyte. Yet the substantial reversibility of this dialysis process with respect to solvent change has already been demonstrated (1). The next extension of these experiments is to investigate the effect of heat on the solvent-denaturation reversibility, the test of reversibility being the optical density and thermal denaturation profile in 51% methanol. Results arc shown in Fig. 7. They indicate that in order to cause any decrease in the thermal stability of DNA secondary structure (tested at 51% methanol) 95% methanol solutions containing lo-:’ M NaCl, lo-” X Tris must be hcatcd above 55°C. D.
TRANSITIONS
IN OTHER ALCOHOLS
No transitions can be observed in ethanol, propanol, or tert-butyl alcohol at 25”C., lo-” M salt. Turbidity or precipitation sets in at 7&80% (v/v), before substantial changes in t/c0 occur.
124
GEIDUSCHEK
60
AND HERSKOVITS
70 % Methanol
FIG. 10. Effect of temperature on methanol Tris, pH 7.1). 05°C.: 0; 25°C.: n ; 40°C.: curve a; 25 and 4O”C., as Fig. 4, curve a.
Solvent: 0.001 92.3% methanol.
TABLE M NaCl,
II 0.001 M Tris, pH 7.1,
Thermal history
; C
and
0.5”C. 0.5 -+ 25”C.a 0.5 ++ 25” -+ 0.5%.
a Solutions 6 Uncorrected
equilibrated for thermal
0.876 1.123 1.136
80 (v/v)
I
90
denaturation 0; Preparation
100
(DNA H-II, 10e3 J4 NaCl, lo-* M of solutions: 0.5”C., as Fig. 3,
III.
DISCUSSION
The experiments which have just been described, provide information about two distinct types of denaturation processesoccurring in methanol-water mixtures, one reversible, and the other not. They are interrelated in the following manner:
1 hr. at 25°C. expansion of solvent..
01
methanol-denatured
DNA
I? native
DNA
B
However, it is possible to study the effect I i’ ‘\ (methanol) DNA’ of higher alcohols on secondary structure stability in mixtures with methanol and (N’) electrolyte (i.e., 0.6~s v/v alcohol + 0.4~7% “Methanol-denatured” DNA is the name v/v methanol + lo-” AL?Tris + [lo0 ~1% v/v water) at 25°C. It is then found stability in formamide and N,N’-dimethylformamthat the total mole fraction of alcohols at ide (DMF). The solvent denaturation occurs at the midpoint of the solvent denaturation a much Iower mole fraction DMF (0.23,5 X lo-* M (0.57, 5 X lo-* M decreases in the order methanol, ethanol, Tris acetate) than in formamide at 25°C. We consider this as addipropanol; i.e., the secondary structure sta- Tris acetate) tional evidence in favor of our earlier contention bility is lowered as the length of the alithat hydrophobic forces play an important role in phatic chain is increased.g, lo ‘A ing,
comparable is found in
effect, though much comparing secondary
more strikstructure
determining the stability of native, “These, and other solvent media ject of current experiments?
helical DNA. are the sub-
NONAQUEOUS
DNA
which we have given to the fully hyperchromic material found, say, in 95% methanol at 25”C., and stable over a consideraand ionic blc range of temperatures strengths. N’-DNA is the designation of the hypochronzic material observed in 90% methanol, lo-:$ X NaCl, 1O-:s M Tris at 0°C. (Fig. 3 curve c) . W-DNA and native DPI‘A are either identical or very similar. The major emphasis in this study is on reaction CL,the reversible interconversion of native and met’hanol-denatured DNA. Below, we shall discuss the effect of electrolyte on this transition, before commenting on its reversibility. Finally, we shall offer sonic brief remarks about reaction p, the irreversible, thermal conversion of N’-DNA to methanol-denatured DNA which occurs in methanol-rich solvents. It is clear [Fig. 3 this work, and Table II of Ref. (I)] that electrostatic factors do play a role in methanol denaturation at sufficiently low elect’rolyte concentration, in the sense t’hat removal of electrolyte makes the DKA helix more labile. On the other hand, the degree of ion pairing on DNA i 16) in salt-free methanol-water mixtures does not change markedly in the transition region. In fact, the relatively extensive ion association in methanol assures that electrical repulsions play a smaller stabilitydetermining role in methanol-rich solvent mixtures t,han in aqueous solution. The rather complex phenomena associated with t,he methanol denaturation in salt-free media [which had previously been misinterpreted by one of us (14)] are, instead, due to indirect effects of the low salt concentrat,ion. In these media one finds that rhanges of ultraviolet absorption, sedimentation velocity, and radius of gyration occur over a broad range of solvent mixtures. WC postulate that,, at the DNA concentrations used in t’he schlieren analysis of sedimcntation boundaries (0.5-l mg./ml.) , hydrodynamic interactions are sufficiently great, that the sudden increase of s at SOT0 methanol (Fig. 1) is merely an indication that the end point of a series of macromolecular changes has been reached. Moreowl’, I ow salt concentrations will tend to produce broad transitions, especially in the presence of trace contamination by bivalcnt
SOLUTIONS
60
01
I 20
I % Methanol
I 80
I loo (v/,)
FIG. 11. Sedimentation velocity in methanolwater, IO-” 111 NaCl at 4-6°C. 0 Solutions prepared by dialysis, at 4-6”C., in abscnca of electrolyte. NaCl added to DNA solution at final solvent
composition. Solutions prepared by dialysis, at 4-6”C.,
in the presence
of 10m3 M NaCl. In 90-98s and fast (0 ) component DNA sample: H-II; roncen-
methanol, a slow (0) could tration
be deteceted. 0.4-0.7 mg./ml.
cations.ll [We have previously commented on the fact that such contaminants can be scavenged by DNA during dialysis (I) .] Finally, in water-rich media, electrostatic repulsions on the DNA polyion are so great that even partly denatured DNA (as judged by optical density criteria) has a highly extended configuration.12 The experimental data on sedimentation velocity (Fig. I), radius of gyration (Fig. 2), and ultraviolet absorption (Fig. 3) are generally consistent with this interpretation. Ultraviolet absorption, as a measure of secondary structure ordering, shows changes in the O-7570 methanol composition range (at 0.5”C.). The light-scat’tering average radius of gyration (within the large limits of error imposed by the necessity of working with exceedingly dilute solutions, c = 0.004-0.02 mg./ml.) shows its greatest changes in the 45-75s methanol romposi‘I J%-. Dovr, I’. Brooks. and K. Davidson. in a private c*ommunicxtion (1960), proposed this interpretation for certain denaturation experiments performed by them. IL’ This has also been a source of complications in t,hc rsperimcnts on the flexibility of thr, DNA helix (26, 27).
126
GEIDUSCHEK
AND
tion range. Sedimentation velocity is measured at higher concentrations (c = 0.5-l mg./ml.), where DNA contributes a higher counterion concentration than in the lightscattering and absorption measurements, and consequently offers slight additional stabilization. One observes that the sharpest changes in sedimentation velocity occur at 80% methanol, that is, slightly higher than the end point of the changes in radius of gyration. In electrolyte-containing media, on the other hand, the definition by both optical and macromolecular criteria of that region of solvent composition in which denaturation occurs is simple: (a) Viscosity and optical density change in the same range of solvent composition (Fig. 6) . (b) When the hyperchromic shift is essentially complete, as in 92% methanol, 2 x lo-” M salts, 2O”C., one cannot produce detectable streaming birefringence of DNA. The viscosity, light-scattering, and birefringence experiments all show that methanol-denatured DNA assumes a very compact and/or flexible configuration. Methanol and ethanol denaturation, in the absence of salt, were previously shown to be substantially, but not completely, reversible. The experiments reported here not only demonstrate the complete reversibility of methanol denaturation, in the presence of 1O-3 M salt, by waterI (Fig. 4), but show that this reversibility is stable toward heating of DNA in methanol (Fig. 7). This complete reversibility of methanol denaturation by water immediately raises the question of exactly how disordered the DNA configuration in methanol really is, and how it differs from the structure of heataqueous-denatured DNA. In making this comparison we encounter two handicaps. I3 It is probable that the incomplete reversibility of the lengthy dialysis procedures employed previously results mainly from partial denaturation occurring in water and in water-rich solvent mixtures in the absence of added salt. It is entirely in keeping with this interpretation that the reversibility in the absence of added electrolyte was previously found to be less complete in methanol than in ethanol, since the latter solvent, by virture of its lower dielectric constant, provides greater ion-pairing.
HERSKOVITS
First, the ability to test the completeness of denaturation by optical criteria is limited both by the precision of measuring E/Q,, and by the need to consider a variety of possible causes for the hyperchromicity (cf. sec. IIB) . Secondly, macromolecular properties (p, [v], s) are not very sensitive to the final stages of secondary structure disruption. However, it is evident that methanol-denatured and heat-aqueous-denatured DNA are distinguished by the criterion of reversibility (e.g., Fig. 4). On the basis of the reversibility experiments we conclude that the strand dissociation proposed by Meselson and Stahl (29) and by Marmur et al. (24, 25) most probably does not occur and that, in the sense of dissociation of every secondary valence link between complernentary pol ynucleotide strands, methanol denaturation is not complete.‘” What does seemmost likely is that, even in 98% methanol, elements of secondary structure remain in each molecule, and that these provide nuclei for the reformation of long, ordered, helical sequences. When the environmental conditions are, once again, made thermodynamically favorable to the reformation of the native secondary structure, the kinetic path is established by these nuclei. On the other hand, DNA heat-denatured in water has lost these reference points. Whatever its subsequent treatment, it cannot undergo the rapid reassemblage of the double helix (Fig. 4) which characterizes the reversibility of methanol denaturation. This is assumed to be the structural basis for the difference in behavior of aqueous-heat-denatured DNA (Fig. 4, curve b) and methanol-denatured DNA (Fig. 4, curve a). To what property do the residual nuclei in methanol-denatured DNA owe their greater stability? The following alternatives must be considered: (a) Intramolecular heterogeneity of base composition. In thermal denaturation of aqueous DNA solutions, guanine-cytosine base pairs confer greater stability on DNA secondary structure, and the thermal denaturation temperature is therefore dependent on average base composition. The occur14Further proof of these sought through isotope-banding
statements will experiments.
be
r;ONAQUEOUS
DNA
rcnce of residual nuclei has also been demonstrated in thermal-aqueous denaturation (30). The extra thermal stability of these nuclei relative to their contiguous nucleotide sequences depends on the average DNA composition and on t,he solvent medium. The incremental stability is not greater than can be attributed to intramolecular heterogeneity of nucleotide composition and the firmer association of guanine-cytosine base pairs. In methanol denaturation, on the ot’her hand, the additional stability of these residual “nuclei” for reformation of secondary structure is extremely large. For instance, in 92% methanol, 2 X lo-” ~11 salt, the denaturation transition occurs rat’her sharply at 195°C. [Table III, Ref. (l)]. Yet the denaturation is not made irreversible toward water addition by heating to 55°C. (Fig. 7). Irreversibility of denaturation, as judged by this assay, sets in at least 30” above the transition temperature. At the same time, the met’hanol denaturation transition is not very sensitive to avercrge DSA composition (Fig. 9), so that it is not possible to ascribe this temperature spread to local heterogeneity of guaninecytosine content. However, there may be other combinations of base pairs which do confer such special stability to DIVA secondary structure in methanol and it would be interesting to pursue that question further. (5) Part of the secondary structure changes t’o a different ordered configuration, in which nucleotide pairs remain in register. Such regions, as has been not.ed above, need not be hypochromic. They would provide points of reference for the re-establishment of regular base stacking upon the addition of water. A transition of this type occurs in Na-DNA fibers at a thermodynamic activity of water slightly below 0.5 (31). Howcvcr, no example of such a transition has yet been identified in solution, and the difficulty of so doing has been commented upon in sec. IIH. In the experiments reported here, the macromolecular evidence points to a compact configuration in methanol-denatured DNA. Any structure containing stacked base pairs should be relatively inflexible. Consequently, the presumptive “new” ordered configuration can
SOLUTIOI’iS
127
only involve a fraction .of the nucleotide pairs of each molecule. In view of the superior stability of this hypothetical new secondary structure, there would have to be special steric factors which permit only a fraction of each molecule to be transformed to it,. (c) Aggregation. Sections of Dn’A molecules might be protected from solvent denaturation as a result of partial aggregation. However, auxiliary light-scattering experiments show that even when met,hanol solutions of DNA (10W3 M NaCl) are prepared by direct mixing, aggregation is slight. In addition, the methanol denaturation of DNA solut,ions (lo-” M NaCl, 1O-3 31 Tris) as dilute as 0.015 mg./ml. can be reversed by water. To extend these limits to lower concentrations, it is necessary to use a more sensit,ive assay of denaturation irreversibility, such as transforming ability. In the absence of such measurement’s we can only say that if an aggregation process is responsible for the observed reversibility, it is one which occurs at very low DNA concentrations, without, however, leading to precipitation at concentrations as high as 2 mg./ml. (d) A minor contaminant binds more avidly to helical than to denat,urcd DNA in methanol-water mixtures and is present in concentrations sufficient to stabilize only a small fraction of the secondary structure.ll However, reversibility of methanol denaturation is also observed in DNA isolated from Serratin mnrcescenu by an entirely different method (4). In addit’ion, extensive dialysis of aqueous DNA (HI1 in IO-’ JI 9aCl) in no way changes the reversibility of its methanol denaturation. 0f these four proposals, none can yet be rigorously excluded, although alternative (/I)-contamination-appears highly irnprobable. At this time me arc therefore unable to establish the structural basis of the reversibility of methanol denaturation. Xevertlielcss, the expcrimcnte presented here do establish t,he fact that this reversibility rests on different structural considerations from those applying to denaturation in aqueous solution. They permit the hope that new information about DNA secondary strurturc may be forthcoming from further
128
GEIDUSCHEK
AND
experiments on this and other nonaqueous solvent systems. One further comparison is appropriate. The reaction of DNA with mercuric chloride results in configuration changes which are, in some respects, similar to those described here. Upon binding mercuric ion, DNA assumes a more compact configuration, as shown by the light-scattering and viscosity measurements of Katz (32). Sequestration of mercuric ion by complexing agents results in a reversal of the configuration changes (33). Transforming DNA which has been put through such a mercury-binding cycle retains biological activity (34). However, Hg-DNA and methanol-denatured DNA differ in one important respect: Hg-DNA is hypochromic, and undergoes an irreversible thermal transition accompanied by a large increase in absorbance (33). Methanol-denatured DNA is hyperchromic. Its absorbance does not increase substantially upon further heating. In view of the remarkable reversibility of the methanol denaturation with respect to addition of water, it is, of bourse, surprising to find an irreversible transition (reaction p) in methanol-rich media, which can be caused to occur by heating (Table II) or by removal of electrolyte (Fig. 11). However, it is clear that this process differs from thermal denaturation in aqueous media in that its end product, methanol-denatured DNA, can be reconverted (reactivated) to native DNA by water. Evidently, water is necessary to provide the kinetic path for the re-establishment of long-range order on the one hand, and for its complete dismemberment, on the other. These observations suggest that in the kinetics, as well as in t,he equilibria of DNA secondary structure, water plays a special role. ACKNOWLEDGMENTS It is a pleasure to acknowledge our indebtedness to Dr. S. J. Singer for the advice and encouragement which he offered us throughout the course of this work and for his critical reading of the manuscript. We are grateful to Miss Susan Staves and Mr. Alan Daniels for their expert technical assistance. One of us (T. T. H.) held a U. S. Public Health Service predoctoral fellowship during a part of this investigation.
HERSKOVITS REFERENCES 1. HERSKOVITS, USCHEK,
T. T., SISGER, S. J., AND GEIDE. P., Arch. Biochem. Biophys. 94,
99 (1961). 2. SIMMONS, DUGGAX,
as quoted by STEVENS, V. L., AND E. L., J. Am. Chem. Sot. 79, 5703
Y.,
(1957). 3. JONES, A. S., Biochim. et Biophys. Acta 10, 607 (1953). 4. H~MAGuCHI, K., AND GEIDUSCHEK, E. P., Abstr. Biophys. Sot. (Feb. 1961). FBlO. 5. BUXVILLE, L. G., AND GEIDUSCHEK, E. P., Biochcm. Biophys. Research Commons. 2, 287 ( 1960). 6. EDSALL, J. T., RICH, -4., AND GOLDSTEIN, M. Rev. Sci. Instr. 23,695 (1952) 7. See, for instance, EDSALL, J. T., Advances in Colloid Sci. 1,269 (1942). 8. GOLDSTEIX, M., AND REICHMANN, M. E., J. Am. Chem. Sot. 76,3337 (1954). 9. EWART, R. H., ROE, C. P., DEBYE, P., AND MCCARTNEY, J. R., J. Chem. Phys. 14, 687 (1946). 10. BRINKMAR., H. C., AND HERMANS, J. J., J. Chem. Phys. 17,574 (1949). 11. KIRKWOOD, J. G., AND GOLDBERG, R. J., J. Chem. Phys. 18,54 (1950). 12. STOCKMAYER, W., J. Chem. Phys. 18, 58 (1950). 13. STURTEVANT, J. M., AND GUTFREUND, H., Proc. Natl. Acad. Sci. U. S. 42, 719 (1956). 14. GEIDUSCHEK, E. P., AND GRAY, I., J. Am. Chem.
Sot. 78,879 (1956). 15.
J. H., AND JORDAN, D. O., Biochim. et Acta 43,223 (1960). 16. HERSKOVITS, T. T., Ph.D. Thesis, Yale, 1959. 17. TINOCO, I., J. Am. Chem. Xoc. 82, 4785 (1960). 18. Cox, R. A., AND LITTAUER, U. Z., Nature 184, 818 (1959). 19. DOTY, P., BOEDTKER, H., FRESCO, J. R., HASELKORN, R. AND LITT, M., P~oc. Natl. Acad. Sci. U. S. 45,482 (1959). 20. HASCHEMEYER, R., SINGER, B., AND FRAENKELCONRAT, H., Proc. Natl. Acad. Sci. U. S. 45, 313 (1959). 21. LITTAUER, U. Z., AND EISENBERG, H., Biochem. et Biophys. Acta 32, 320 (1959). 22. SPIRIN, A. S., GAVRILOVA, L. P., BRESLER, S. E., AND MOSSEVITSKY, M. I., Biokhimiya 24, 938 (1959). 23. SINSHEIMER, R. L., J. Mol. Biol. 1,43 (1959). 24. M.~RMuR, J., AND LANE, D., Proc. Natl. Acad. Sci. U. S. 46,453 (1960). 25. DOTY, P., MARMUR, J., EIGNER, J., AND SCHILDICRAUT, C., Proc. Natl. Acad. Sci. U. S. 46, 461 (1960). 26. CONWAY, B. E., BUTLER, J. A. V., AND JAMES, D. F. W., Trans. Faraday Sot. 50,612 (1954). 27. SADRON, C., “Nucleoproteins,” p. 124. (Onzieme COATES,
Biophys.
NONAQUEOUS Conseil de Chimie Solvay, 1959 ; Interscience Publ., New York, 1960). 28. YANG, J. T., AND DOTY, P., J. Am. Chem. Sot. 79,761 (1957). 29. MESELSON, F. W., AND STAHL, F. W., P~oc. N&l. Acad. Sci. U. S. 44, 671 (1958). 30. GEIDUSCHEK, E. P., Federation Proc. 20, 353 (1961).
DNA
129
SOLUTIONS
31. LANGRIDGE,
WILSOS, H. R., HOOFER, C. W., M. F. H., AND HA~ILTOS, L. D., J. Mol. Biol. 2, 19 (1959). 32. KATZ, S., J. Am. Chem. Sot. 73,2238 (1952). 33. TAX~XE, T., AND DA~IDSOS, I’i., J. Am. Chem. Sot. in press. 34. DOVE. W. F., AND TA~SE, T., Biochem. Biophys. Research Communs. 3, 608 (1961). WILKINS,
R.,