STUDIES ON THE STRUCTURE OF NUCLEICACIDS
Dec. 20, 195,j [CONTRIBUTION FROM
THE
6499
LABORATORIES OF THE SLOAN-KETTERING DIVISIOXOF CORNELL UNIVERSITY MEDICALCOLLEGE]
Studies on the Structure of Nucleic Acids. VIII. Apparent Dissociation Constants of Deoxypentose Nucleic Acid1 BY LIEBE F. CAVALIERI AND A. L. STONE RECEIVEDJUWE 22, 1955 The ultraviolet spectrum of DNA is discussed a n d a basis is developed for calculating the apparent dissociation constants (pK,’) of the nuclear hydroxyl and XH3+groups of the deoxycytidylic and deoxyguanylic acid moieties. From the effect of salt on the pK,’s it is concluded that there are charged phosphate groups near the various amino groups, possibly resulting from the intertwining of two or more double helices with one another. The effect of PH changes on the macromolecular structure of DNA is also discussed.
component nucleotides and the spectra of the individual nucleotides as a function of pH in order to be able to assign apparent dissociation constants to the component nucleotides. The difficulty apt to be encountered is t h a t macromolecular interactions alter the optical density changes. In the case of DNA i t appears t h a t these interactions can be assessed and corrections can therefore be made. Advantage was taken of the fact that a t 260 mp the optical density of the individual nucleotides does not change with p H in the acid r e g i ~ n . ~ For the DNA macromolecule a t 260 mp, however, changes are observed5 and these have been attributed to alterations in macro-structure. In the present investigation the increments with p H a t The sample of calf thymus DKA (sodium salt) was prepared according to the method of Schwander and Signer.* 260 mp have been taken as a blank and subtracted from those occurring a t 250, 280 and 290 mp. I t s composition is as follows: adenine 8.8%, guanine 7.6%, I n Fig. 1, curves A, B and C represent titrations cytosine 5.3%, thymine 7.7%, N 13.42%, P 7.97%, water 21.3%, E:;+ 201 (HzO, pH 6.8, 260 mp), molecular weight carried out in water; A‘, B’ and C’ were carried 5.8 million (light scattering). Crystalline beef pancreatic out in 0.1M salt. In the acid range the increments deoxyribonuclease was purchased from the Worthington a t 280 mp (curves B and B’) are due principally to Biochemical Laboratory. Method of Measurements.-Stock solutions (1 mg./ml.) the deoxycytidylic acid moiety with only minor were made as follows: ten mg. of DNA was allowed t o swell contributions from the other component nucleoovernight in 1 ml. of water. Sine ml. of water was then tides. The mid-points of these curves have thereadded gradually, while stirring. Appropriate amounts of fore been assigned as the $KL values of deoxycytithe stock solution were delivered by means of a microburet dylic acid in the DNA molecule. At 250 and 290 into 3 ml. of solvent contained in 1 cm. silica cuvettes. Measurements were made with a Model DU Beckman mp both deoxycytidylic and deoxyguanylic acids Spectrophotometer with an accuracy of 1 0 . 0 0 2 unit on the contribute to the increments. The contribution optical density scale. Cell corrections were determined immediately before adding the DNA% to the cells. The addi- of each was calculated from the concentration of DNA, its nucleotide analysis and the known intion of salt, sodium hydroxide and hydrochloric acid was also accomplished by means of a microburet, using incre- crements of the mononucleotides as a function of ments of about 0.001 ml.; solutions were stirred with a stream of nitrogen. The concentrations were: sodium pH, using the assigned pKL value. pKL values in the alkaline range were calculated similarly. chloride, 5 M; hydrochloric acid, 0.1 and 1.0 M; sodium hydroxide 0.1 M . pH measurements were carried out with a Since the spectrum of deoxyadenylic acid remains Model G Beckman pH meter. nearly unchanged with pH, its pKL in DNA could not be calculated. Results and Discussion The open circles in Fig. 1 are the corrected experiIn general, a plot of optical density change mental points, while the unbroken lines are theoversus $H yields a typical sigmoid curve whose mid-point is the pK. The procedure is repeated retical curves based on the PKL’s given in Table I. a t a number of wave lengths and under optimal It will be noted t h a t these pKL’s are about 2 units conditions the pK will be identical for all wave higher than those of the individual nucleotides and lengths. For a molecule such as DNA, however, that the values calculated for these three wave it is necessary to know the concentrations of the lengths are in good agreement both in the presence and absence of salt. These calculations include the (1) This investigation was supported by grants from the National implicit assumption t h a t the entire wave band is Cancer Institute, National Institutes ot Health, Public Health Service, uniformly effected by the alterations in macroGrant No. C-471, and from the Atomic Energy Commission, Contract No. AT(30-1)-910. structure. This seems plausible since i t is highly (2) E. Hammarsten, Biochem. Z . , 144, 383 (1924). improbable t h a t pKL’s calculated for three wave (3) J . M . Gulland, D. 0. Jordan and H. F. W. Taylor, J. Chem. Soc., lengths would agree if this were not so. It should 1131 (1947); D. J , Cosgrove and D . 0. Jordan, ibid., 1413 (1949). (4) R. Signer and H . Schwander, Helo. Chim. Acta, 32, 863 (1919). be noted t h a t any given pK determined in this ( 5 ) L. F. Cavalieri, THIS JOWRSAL, 74, 1242 (1952). manner may not represent all the groups present; (6) J. Shack and J. M. Thompsett, J . BioL Chem., 197, 17 (1952).
The dissociation constants of calf thymus deoxypentose nucleic acid (DNA) isolated in a mild manner were first determined by Hammarsten2 and since t h a t time a number of other investigat o r ~ ~have - ~ obtained essentially similar results. Recently, Lee and Peacocke’ have shown t h a t the values of the dissociation constants are greatly affected by salt, although they made no attempt to calculate these values. The present paper reports the results of a spectrophotometric titration which permit a possible correlation of the apparent dissociation constants with specific groups in the DNA molecule. Experimental
(7) W. A. Lee and A. R. Peacocke, Res. Su$$l., [6] 2, 155 (1953). (8) H. Schwander and R. Signer, H e h . Chim. Acta, 33, 1521 (1950).
(9) See Beavan, Holiday and Johnson, “Nucleic Acids,” Chargaff and Davidson, Academic Press, New York, N. Y . , 1955, p. 513.
LIEREF.C.IY~~I,IERI .ZND
6.500
-0.I O L
12
II
IO
7
6
5
4
3
2
PH. Fig. 1.-Determination of the apparent dissociation cons t a n t s . The increments shown a t 250 (curve A ) , 280 (curve B ) and 290 rnfi (curve C ) iwre obtained (for various PH values) by subtracting the increments at 260 mp from those observed at 250, 280 and 290 m p . Curves A, B and C represent titrations in water; il', B' and C' represent those carried out in 0.1 Jl salt.
hence potentiometric data need not necessarily agree. Since our curves are clear-cut we feel t h a t any differences between our interpretations and those that have been drawn from potentiometric data may be due t o differences in the DNA concentrations employed in the determinations. It can be seen from Table I t h a t 0.1 II salt lowers the apparent dissociation constants by about 2 units, a situation which is indicative of a field effect; a similar salt effect has been observed potentiometrically.' In DKA the singly charged phosphate groups may serve as field sources. T h a t such a field effect is capable of operating in a polynucleotide system has been demonstrated in the following manner. X solution of DNA was treated with DS.iase and magnesium chloride. The resulting mixture of mono-, di- and oligonucleotides was titrated spectrophotometrically; the PK; of the deoxycytidylic acid moiety was found io be 4.7, and the pKA of the deoxyguanylic acid moiety was 2 9. I n 0.1 IZI salt the pKAs were 4.3 and 2.5, respectively. The oligonucleotides contain significant amounts of tri-, tetra- and pentanucleotides and the phosphate groups in these derivatives are capable of virtually touching the various amino groups. It appears reasonably certain, therefore, that field effects can account for the fact t h a t the p K i values are higher than in the respective mononucleotides. It should be emphasized t h a t in DX-1 a field effect mal; occur either within a single chain or be-
L. STOW
1-01, 77
tween chains, depending on the rigidity of the structure. The field should operate most effectively through the DNA molecule, where the dielectric constant is lower than t h a t of the solvent. However, in the double helical structurelo the smallest distance between a phosphateogroup and a n amino or hydroxyl group is about 7 A.ll and the molecule would have to be greatly kinked in order to permit a closer approach of a phosphate from one part of the chain to an amino or hydroxyl group in another part. Light scattering measurements12 show t h a t the molecule behaves like a relatively stiff coil. Under these circumstances it is improbable t h a t a phosphate group can exert a field effect on a purine or pyrimidine within the same molecule. X simple modification suffices to make the proposed inodel'O consistent with the existing data. We suggest that there exist in solution packets of double helices arranged in such a manner as to permit close approach of the charged phosphate groups t o the various amino groups. Further, this structure cannot be one resulting from mere association, or it would dissociate a t the high dilutions used for the determinations. T h e packets must therefore represent a structure with integrity, not readily alterable with environment; for example, one involving double helices entwined about each other, providing thereby an opportunity for intimate interaction. Such a structure would also provide the heterogeneity of phosphate groups which is required by dye binding data*3z1.'b u t unaccounted for by the simple helix model, where the phosphates are symmetrically disposed about the long axis and provide no opportunity for electrostatic heterogeneity. The interrupted double strand model proposed by Dekker and Schachman l5 cannot account for our results, but would be consistent with them if intertwinning were also postulated. DKA was found to possess an apparent stability a t low p H values. Titrations were carried out by adding very small increments of acid or alkali. Back titration from about pH 2.3 to neutrality gives p S L ' s for deoxycytidylic and deoxyguanylic acids of 5 . 3 and 3.7, respectively (Table I ) . The decrease over the values obtained in the forward titration may be accounted for on the basis of the salt effect arising from salt formed on neutralization of the hydrochloric acid. This suggests that the structure has not changed materially from an electrostatic point of view as a result of the gradual decrease in pH in the forward titration. However, the macro-structure has been altered since the backtitration curve a t 260 mp (Fig. 2, curve B) is markedly different from the forward.16 Similarly, the back-titration curve differed from the forward in (10) J . D. \Vatson and I; H. Crick, S u l m " , 171. 739 i l O . i 3 ) (11) L f . H F \Vilkin;, A. R . Stokes and H. P. \\-ilwn, ibicf., 171,729
(1953) (12) 3 I E . Reichmann. S .4. Rice, C A . Thomas a n d P. D o t y , THIS J O U R X A L , 76, T D I i ( 1 9 X ) . (13) I-, I; Ca\.alieri and -4. Angelos, ibid., 72, 4686 (1950). (14) 1,. 1' Cavalieri. A. .\ngelos and XI. E. Balis, i b i L , 73, 4902 (1951). (1.5) C. A . Dekker and H. K. Schachman, Pror. Y u l L . A c a d . Sci., 40, 894 1193-1) fl(i) Cleavage of hydrogen bonds due t o high local concentrations of acid is minimal since increments of 0.001 ml. were added to 3 ml. uf DNA sdution.
Dec. 30, 19.7.5
STUDIES ON THE STRUCTURE OF
TABLE I APPARENTDISSOCIATION CONSTANTS DETERMINED SPECTROPHOTOMETRICALLY A, m p
Water
0.01 0.1 BackM M titraNaCl NaCl tiona
Nucleotide moieties in DNA Deoxycytidylic acid 250 6.0 5.2 (SH3') 280 6.2 5.1 290 6.3 5.1 Deoxyguanylic acid 250 4.0 3.7 "(3 +) 280 290 4.0 3.6 Deoxyguanylic acid 250 11.6b (OH) 280 11.6 290 11.6 250 11.6 Thymidylic acid ( O H ) 280 11.6 11.6 290
3.8 4.0 4.0 2.7
5.2 5.3 5.3 3.7
2.7 3.7
Mononucleotides Cytidylic acid ("8') 4.2 Guanylic acid (NH,+)C 2 . 4 Adenylic acid (XH3+) 3 . 7 Guanylic acid (0H)C 9.3 Thymidine (OHY 9.4 a The salt concentration as the result of forward (water) and back-titration was 0.008 M . b Shack and ThompsettG observed a sharp break a t p H 11.8 for 230 and 260 m r . However, those results include both ionization and macrostructure effects. Potentiometric (cf. D . 0. Jordan "Nucleic Acids," Chargaff and Davidson, Academic Press Inc., S e w York, S . Y . , 1955, p . 459).
NUCLEICL4CIDS
6501
-
shoulder a t pH 6 appears to be due to changes in macro-structure involving the amino groups of deoxycytidylic acid while the sharp increase a t PH 4 is probably associated with the amino groups of deoxyguanylic acid, based on the pKL values given in Table I. The fact t h a t the increase $H 4 occurs a t pH 3 in 0.1 M salt supports at the view t h a t deoxyguanylic acid is involved since the saIt effect on the pK,l of this component is similar (Table I). The steepness a t pH 4 in curve A, Fig. 2 , indicates an abnormal titration process. It would appear t h a t as neutralization begins, with the attendant cleavage of hydrogen bonds, the molecule collapses before titration is actually completed. If this is so, it may be inferred t h a t the amino groups of deoxyguanylic acid play a critical role in the macro-structure of DNA.
-
-
-
-
/---
I
+ .440
t cn
5 .420
the presence of 0.1 M salt. It has been shown17 Q that the size and shape a t pH 6.5 remain unchanged J ,400 as a result of exposure (by dialysis) to acid (pH 2.6) in 0.2 M salt. Altogether, these results tend to suggest t h a t the changes in macro-structure are ,380 minute. In terms of the double helix it appears t h a t small changes in the relative positions of the 7 6 5 4 3 chromophores within the helix may occur without PH. grossly affecting the outer contours of the molecule, Fig. 2.-Effect of PH on optical density at 260 mp: It is pertinent to consider the origin of the optical density increments a t 260 m,u. Curve A of Fig. 2 A, typical changes as function of p H ; B, typical back-titrashows the forward titration a t 260 mp.lY The tion curve; C, back-titration curve a t low D S A concentra-
-
cLnnlnlT
g
(17) M. E. Reichmann, B. Bunce and P. D o t y , J. Poly. Sci , 10, 109 (1953). (18) Similar studies have been carried out by E. R . Blout and A. Asadourian, Biochem. Biophys. A r i a , 13, 161 (1954); R . Thomas, ibid., 14, 231 (1954); and G . Frick, ibid.,8, 628 (195.)). Although there is essential agreement in some areas, there are also discrepancies. T h e latter must be attributed to the different methods of isolation of the D N A . It cannot he statrd with certainty as t o which if any, of the materials are degraded.
tions (optical density -0.1).
Acknowledgment.-The authors wish to thank Dr. George Bosworth Brown for continued interest and helpful discussions. KEWYORK,S. Y .
