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The helix-coil transition in deuterated deoxyribonucleic acids
789
PRELIMINARY NOTES
Many salts are known to decrease the uptake of streptomycinn and the bactericidal action of streptomycinL 3, kanamycin, and neomycin12. However, we found that phosphate (added as sodium phosphate buffer) had an apparently specific effect in antagonizing the protection against RNA breakdown afforded by the polybasic antibiotics. Sodium salts of other anions (chloride, citrate, and sulfate) at equal molarity had little effect, though citrate and sulfate were effective in antagonizing the bactericidal action. These observations suggest that a phosphate-stimulated nuclease might be involved in the observed RNA degradation. A similar paradoxical effect on nucleic acid breakdown has been reported for low and high concentrations of the polypeptide antibiotic, polymyxinTM. While this polybasic compound may protect through the same mechanism as the polybasic amino sugar anti.biotics its bactericidal action must be quite different, since prevention of protein synthesis by chloramphenicol protects against killing by the amino sugar group 3 but did not protect against polymyxin. While evidence from this laboratory3, ~ indicates that the lethal action of streptomycin is associated with membrane damage, a direct interaction with ribosomes best explains the RNA-stabilizing effect of streptomycin at high concentrations.
Department o/ Bacteriology and Immunology, Harvard Medical School, Boston, Mass. (U.S.A.) 1 i a 4 s 6 s 9 10 ix i2 18
DAVID S. FEINGOLD BERNARD D. DAVIS
R. DONOVlCK, A. P. BAYAN, P. CANALES, AND F. PANSY, J. Bacteriol., 56 (1948) 125. S. R. GREEN AND S. A. WAKSMAN, P~'0C. SOC. Exptl. Biol. Med., 67 (1948) 28I. N. ANAND ANn B. D. DAVIS, l~amre, 185 (196o) 22. H. 1ROTH, H. AMOS, AND B. D. DAVIS, Biochim. Bioph~s. Acta, 37 (196o) 398. ]3. D. DAVIS, u n p u b l i s h e d data. D. T. DUBIN AND B. D. DAVIS, Biochim. Biophys. Acta, 55 (1962) 793. D. T, DUBIN AND B. D. DAVIS, Biochim. Biophys. Acta, 52 (1961) 4oo. S. S. COHEN AND J. LICHTENSTEIN, J. Biol. Chem., 235 (196o) 2112. A. HERSHKO, S. AMOZ, AND J. MAGER, Biochem. Biophys. Research Communs., 5 (1961) 46. j . MAGER, Biochim. Biophys. Acta, 36 (1959) 529. p. H. PLOTZ, D. T. DUBIN, AND B. D. DAVIS, Nature, 19i (1961) I324. D. S. FEINGOLD, u n p u b l i s h e d d a t a . B. A. NEWTON, J . Gen. Microbiol., 9 (1953) 54-
Received February 26th, I962 Biochim. Biophys. Aaa, 55 (196~) 787-789
The helix-coil transition in deuterated deoxyribonucleic acids* The introduction of deuterium for hydrogen in bioorganic macromolecules containing internal hydrogen bonds, such as proteins and nucleic acids, is of considerable intrinsic interest and, in the case of proteins and polypeptides, has also been used as a means for assessing the contribution of hydrogen bonding to helical content and stability1-4. Specifically SC~ERAGA8 has interpreted the pronounced effects exerted by deuterium substitution on the thermal helix-coil transition with poly-y-benzyl-L-glutamate (~Tm** in an organic solvent, - - I I °) o r ribonuclease (~Tm in water, 4.2 °) as * Publication No. 1o4o f r o m t h e s e L a b o r a t o r i e s . ** ~Tm is defined as t h e a b s o l u t e difference b e t w e e n t h e m i d - p o i n t of the t h e r m a l t r a n s i t i o n of the d e u t e r a t e d p o l y m e r in a d e u t e r a t e d s o l v e n t a n d t h a t of the p r o t o n a t e d p o l y m e r in a p r o t o n a t e d solvent. Similarly for a n y q u a n t i t y or function, ~Q -----QD-~H.
Biochim. Biophys. Acta, 55 (1962) 789-792
79 °
PRELIMINARY NOTES
showing that the helical structures of these molecules in solution are stabilized by hydrogen bonding. In spite of the great importance of hydrogen-bonded helical conformations 5, e and helix-coil transitions e-9 to the field of nucleic acid chemistry, analogous experiments in this area have not as yet been reported. We have now measured the thermal hyperchromicity vin H20-and D20-containing buffers of the DNA's of three microorganisms (Eseherichia coli, Pseudomonas aeruginosa and Bacteriophage T2). In brief we find (Fig. I) that in all three cases, under I00 90
8O 70 II
E.coli B
5O
II
i11-
40 30 20 I0 0 81
I 83
I 85
I I I 87 89 91 TernpePatur'e (°C)
I 93
I 95
I 97
99
Fig. i. Thermal denaturation of protonated and deuterated DNA in H20 and D20. The data are A ~ e 0 (t) --A 2s0(25 °) plotted as °~l.~rel against temperature. The former is defined as ioo ×A 26o(Tm -{-I o ) - A ~80(25 °)" A 2 8 o for all the DNA samples used was sensibly constant between 22 ° and 75 °. Tm and d T are the midpoint of the transition and the dispersion as described by DOTY el al. 7 Z]Arel has been called the transition fraction,/to by HAMAGUCHIAND GEIDUSCHEK12;it is a measure of the extent of the transition helix---> coil at any one temperature. Bacterial samples were prepared by the method of MARMURIs, T2 DNA by that of MANDELL AND HERSHEY14. Mole °/o (Cr--C)% t° were for T2 35.2; for E. coli B, 52.4, for P. aeruginosa (strain 10145), 65. 9. All experiments i n o . i 5 2~/NaCl-o.oI5M sodiumcitrate prepared in H20 or D~O () 99.5 o~), respectively; DNA samples were first diluted into, then dialyzed against the appropriate medium; this was followed by thermal equilibration at a temperature approx, equal to Tm for 3 h and slow cooling. Total hyperchromicity and Tm after t r e a t m e n t were found to be essentially unchanged compared to untreated controls. All experiments were run in triplicate; all values rerorted are the mean ! standard deviation. c o n d i t i o n s t h a t a s s u r e c o m p l e t e e x c h a n g e 9-11, t h e p r o f i l e s for t h e r m a l d e n a t u r a t i o n of t h e d e u t e r i u m - c o n t a i n i n g s p e c i e s in a d e u t e r i u m - c o n t a i n i n g m e d i u m a r e v i r t u a l l y i d e n t i c a l w i t h t h e c o r r e s p o n d i n g p r o f i l e s for t h e h y d r o g e n - c o n t a i n i n g s p e c i e s in a h y d r o g e n - c o n t a i n i n g m e d i u m . ( 6 T m ~ 0 . 4 °, b A T ~ 0 . 2 ° ) . O n e p o s s i b l e i n t e r p r e t a t i o n of t h i s , t o us, r a t h e r s u r p r i s i n g r e s u l t is t h a t t h e h y d r o g e n b o n d i n g of i n d i v i d u a l b a s e p a i r s w i t h i n t h e D N A d o u b l e h e l i x , a l t h o u g h u n q u e s t i o n a b l y a m a j o r f a c t o r in i t s s t r u c t u r e 5,e, is n o t n e c e s s a r i l y t h e o n l y c o n t r i b u t o r t o i t s stability. T h e a r g u m e n t s g i v i n g r i s e t o t h i s c o n c l u s i o n a r e as follows: t h e v i r t u a l i d e n t i t y Biochim. Biophys. dcla, 55 (I962) 789-792
PRELIMINARY NOTES
791
of the melting-point profiles in the H- and the D-containing case shows that for the unfolding reaction 3 helix ~- 2 coils
B y analogy with the protein and polypeptide cases ls,le we then assume that for a very long polymer any of the thermodynamic functions can be approximated b y appropriate summation over the individual residues, i.e., that A Funt, A Hum and zt Sun f Aun f ~
(2n+3m)Aav
(2)
where n = number of A - T pairs (with 2 hydrogen bonds each) 5, m = G-C pairs (with 3 hydrogen bonds each) 17 and, to a first approximation, AFar, zlHav, ASav are taken as equivalent for all hydrogen bonds in these pairs. It then follows from (I) and (2) that (3) also holds. 3Fav ~ ~Hiv ~.~ 6Say ~___ o
(3)
This might occur if (a) the "deuterium" bond is indeed no stronger than the "hydrogen" bondlS; b) AFar and AHav are so small as to be virtually equal to zero", or, the somewhat more likely alternative (c) that the initial assumption inherent in Eqn. 2 is incorrect and that we must write instead Aunf = ( 2 n + 3 m ) A a v + A x
(4)
where AFx and AHx and ASx refer to some unspecified co-operative process present and important to polymer stability, but less stringent or absent for individual base pairs. Since at the midpoint of the transition, at the melting temperature Tin, AF = 0 it follows that AFx here is equal to and opposite in sign to (2n+3m)AFav and that at T < Tm the helix is stabilized not only by hydrogen bonds but also by the x-process. As contributory evidence we m a y cite that, for approximately equal chain length Tm itself is a function of base composition 7,1° while A Hunf. = R d l n K u m / d T - l ( i . e . , a function of the slope at the midpoints 1~ of the curves in Fig. i) is not. Yet equation (2) predicts such a functional dependence, since we can write AHu~t = 2n'AHav + / t o o l . % (G+C)]n'zlHav
(5)
where n' now represents the total number of base pairs. As to the nature of the stabilizing forces little can be said at this moment, but it is perhaps suggestive that (a) it is now believed that a major contributory factor to conformational stability in globular proteins is provided b y " a p o l a r " or "hydrophobic" bonds 2°-z~ related to an increase in order within the water structure surrounding the polymer; (b) that rupture of similar bonds by a variety of agents leads to the denaturation (i.e., helix -~ coil transition) of double-stranded DNA2S,24; (c) the explicit suggestion by HAMAGUCHIAND GEIDUSCHEKlz that the denaturing effect on DNA of anions at high concentration is due to their action as hydrophobic bond breaking agents, i.e., their ability to disrupt ordered water structures responsible for helix stability and (d) our demonstration by means of nuclear magnetic resonance techni* This presents a distinct possibility since for each individual base pair we must take into account three different types of hydrogen bonds broken and/or made: those between bases, those between bases and water and those of water itself. Some of these combinations may well be such as to lead to no net change in dF and/lt-I (c[. footnote 3 of ref. 19 and ref. 3). It is also of interest that exchange of deuterium for protons on films of DNA salts is exceedingly rapid it.
Biochim. Biophys. Acta, 55 (1962) 789-792
792
PRELIMINARY NOTES
ques that a considerable proportion of the solvent molecules around native DNA is immobilized in a highly ordered form and that the effect disappears with denaturation ~5, 2e. We are indebted to Professor W. J. MOORE and Professor V. J. SHINER, Jr., for many helpful and illuminating discussions, and to the National Science Foundation (Grant G 8959) and the U. S. Public Health Service (Grant E 1854) for supporting this research.
Chemical Laboratories, Indiana University, Bloomington, Ind. (U.S.A.)
H. R. MAHLER B. D. MEHROTRA
x K. LINDERSTROM-LANG, Syrup. on Peptide Chemistry, Chem. Soc., (London), Spec. Publ., 2 (I955) I. 2 H. SCHERAGA, Broohhaven Symposia in Biol., 13 (196o) 71. 3 H. SCHERAGA, Ann. N . Y . Acad. Sci., 84 (196o) 604. 4 L. G. AUGENSTINE, C. A. GHIRON, K. L. GRIST AND R. MASON, Proc. Natl. Acad. Sci. U.S., 47 (1961) 1733. b j . D. WATSON AND F. H. C. CRICK, Nature, 171 (1953) 737; Cold Spring Harbor Symposia QuaDs. Biol., 18 (1953) 126. s Review in D. O. JORDAN, The Chemistry o/Nucleic Acids, 156 (196o) 241. P. DOTY, J. MARMUR AND •. SUEOKA, Brookhaven Symposia in Biol., 12 (I959) i. s p. DOTY, J. MARMUR, J. EIGNER AND C. SCHILDKRAUT, Proc, Natl. Acad. Sci. U.S., 46 (196o) 461. s j . MARMUR AND P. DOTY, J. Mol. Biol., 3 (1961) 585 • x0 j . MARMUR, C. L. SCHILDKRAUT AND P. DOT'/', in The Molecular Basis o/Neoplasia, The Univ e r s i t y of Texas, H o u s t o n , Texas, 1962, in the press. n /~. M. BRADBURY, W. C. PRICE AND G. R. WILKINSON, J. Mol. Biol., (1961) 3, 3 °1. 12 K. HAMAGUCHI AND E. P. GEIDUSCI-IEK, J . Am. Chem. Soc., (1962) in the press; see also E. P. GEIDUSCHEK AND T. T. HERSKOVITS, Arch. Biochem. Biophys., 95 (1961) 114. x8 j . MARMUR, J . Mol. Biol., 3 (1961) 208. 14 j . D. MANDELL AND A. D. HERSHEY, Anal. Biochem., I (196o) 66. 15 j . A. SCHELLMAN, CompS. rend. tray. lab. Carlberg Sdr. chim., 29 (1955) 230. le W. F. HARRINGTON AND J. A. SCHELLMAN, CompS. rend. tray. lab. Carlsberg Ser. chim., 3 ° (1956) 21. 1T L. PAtILING AND R. B. COREY, Arch. Biochem. Biophys., 65 (1956) I64. is G. C. PIMENTEL AND A. L. MCCLELLAN, in The Hydrogen Bond, W. H. F r e e m a n , San F r a n cisco, Calif,. 196o, p. 21o. 10 C. TANFORD AND P. K. DE, J. Biol. Chem., 236 (1961) 1711. 20 I. M. ]5~LOTZ, Broohhaven Symposia in Biol., 13 (196o) 25. 21 W. KAUZMANN, Advances in Protein Chem., 14 (1959) I. 22 C. TANFORD, in The Molecular Basis o/Neoplasia, The University o5 Texas, H o u s t o n , Texas, 1962, in t h e press. ms ~/V. P. JENCKS, personal c o m m u n i c a t i o n 34 j . GORDON, L. LEVINE AND W. P. JENCKS, Abstracts, Biophysical Society Meeting, Washington, D. C. (1962). 25 M. K. CAMPBELL, u n p u b l i s h e d observations. 26 i . K. CAMPBELL, H. R. MAHLER AND W. J. MOORE, Abstracts, Div. o] Biochem., I4ISt. Natl. Meeting, Am. Chem. Soc., Washington, D.C., (1962).
Received February 9th, I962 Biochim. Biophys. Acta, 55 (1962) 789-792
PRELIMINARY NOTES
Many salts are known to decrease the uptake of streptomycinn and the bactericidal action of streptomycinL 3, kanamycin, and neomycin12. However, we found that phosphate (added as sodium phosphate buffer) had an apparently specific effect in antagonizing the protection against RNA breakdown afforded by the polybasic antibiotics. Sodium salts of other anions (chloride, citrate, and sulfate) at equal molarity had little effect, though citrate and sulfate were effective in antagonizing the bactericidal action. These observations suggest that a phosphate-stimulated nuclease might be involved in the observed RNA degradation. A similar paradoxical effect on nucleic acid breakdown has been reported for low and high concentrations of the polypeptide antibiotic, polymyxinTM. While this polybasic compound may protect through the same mechanism as the polybasic amino sugar anti.biotics its bactericidal action must be quite different, since prevention of protein synthesis by chloramphenicol protects against killing by the amino sugar group 3 but did not protect against polymyxin. While evidence from this laboratory3, ~ indicates that the lethal action of streptomycin is associated with membrane damage, a direct interaction with ribosomes best explains the RNA-stabilizing effect of streptomycin at high concentrations.
Department o/ Bacteriology and Immunology, Harvard Medical School, Boston, Mass. (U.S.A.) 1 i a 4 s 6 s 9 10 ix i2 18
DAVID S. FEINGOLD BERNARD D. DAVIS
R. DONOVlCK, A. P. BAYAN, P. CANALES, AND F. PANSY, J. Bacteriol., 56 (1948) 125. S. R. GREEN AND S. A. WAKSMAN, P~'0C. SOC. Exptl. Biol. Med., 67 (1948) 28I. N. ANAND ANn B. D. DAVIS, l~amre, 185 (196o) 22. H. 1ROTH, H. AMOS, AND B. D. DAVIS, Biochim. Bioph~s. Acta, 37 (196o) 398. ]3. D. DAVIS, u n p u b l i s h e d data. D. T. DUBIN AND B. D. DAVIS, Biochim. Biophys. Acta, 55 (1962) 793. D. T, DUBIN AND B. D. DAVIS, Biochim. Biophys. Acta, 52 (1961) 4oo. S. S. COHEN AND J. LICHTENSTEIN, J. Biol. Chem., 235 (196o) 2112. A. HERSHKO, S. AMOZ, AND J. MAGER, Biochem. Biophys. Research Communs., 5 (1961) 46. j . MAGER, Biochim. Biophys. Acta, 36 (1959) 529. p. H. PLOTZ, D. T. DUBIN, AND B. D. DAVIS, Nature, 19i (1961) I324. D. S. FEINGOLD, u n p u b l i s h e d d a t a . B. A. NEWTON, J . Gen. Microbiol., 9 (1953) 54-
Received February 26th, I962 Biochim. Biophys. Aaa, 55 (196~) 787-789
The helix-coil transition in deuterated deoxyribonucleic acids* The introduction of deuterium for hydrogen in bioorganic macromolecules containing internal hydrogen bonds, such as proteins and nucleic acids, is of considerable intrinsic interest and, in the case of proteins and polypeptides, has also been used as a means for assessing the contribution of hydrogen bonding to helical content and stability1-4. Specifically SC~ERAGA8 has interpreted the pronounced effects exerted by deuterium substitution on the thermal helix-coil transition with poly-y-benzyl-L-glutamate (~Tm** in an organic solvent, - - I I °) o r ribonuclease (~Tm in water, 4.2 °) as * Publication No. 1o4o f r o m t h e s e L a b o r a t o r i e s . ** ~Tm is defined as t h e a b s o l u t e difference b e t w e e n t h e m i d - p o i n t of the t h e r m a l t r a n s i t i o n of the d e u t e r a t e d p o l y m e r in a d e u t e r a t e d s o l v e n t a n d t h a t of the p r o t o n a t e d p o l y m e r in a p r o t o n a t e d solvent. Similarly for a n y q u a n t i t y or function, ~Q -----QD-~H.
Biochim. Biophys. Acta, 55 (1962) 789-792
79 °
PRELIMINARY NOTES
showing that the helical structures of these molecules in solution are stabilized by hydrogen bonding. In spite of the great importance of hydrogen-bonded helical conformations 5, e and helix-coil transitions e-9 to the field of nucleic acid chemistry, analogous experiments in this area have not as yet been reported. We have now measured the thermal hyperchromicity vin H20-and D20-containing buffers of the DNA's of three microorganisms (Eseherichia coli, Pseudomonas aeruginosa and Bacteriophage T2). In brief we find (Fig. I) that in all three cases, under I00 90
8O 70 II
E.coli B
5O
II
i11-
40 30 20 I0 0 81
I 83
I 85
I I I 87 89 91 TernpePatur'e (°C)
I 93
I 95
I 97
99
Fig. i. Thermal denaturation of protonated and deuterated DNA in H20 and D20. The data are A ~ e 0 (t) --A 2s0(25 °) plotted as °~l.~rel against temperature. The former is defined as ioo ×A 26o(Tm -{-I o ) - A ~80(25 °)" A 2 8 o for all the DNA samples used was sensibly constant between 22 ° and 75 °. Tm and d T are the midpoint of the transition and the dispersion as described by DOTY el al. 7 Z]Arel has been called the transition fraction,/to by HAMAGUCHIAND GEIDUSCHEK12;it is a measure of the extent of the transition helix---> coil at any one temperature. Bacterial samples were prepared by the method of MARMURIs, T2 DNA by that of MANDELL AND HERSHEY14. Mole °/o (Cr--C)% t° were for T2 35.2; for E. coli B, 52.4, for P. aeruginosa (strain 10145), 65. 9. All experiments i n o . i 5 2~/NaCl-o.oI5M sodiumcitrate prepared in H20 or D~O () 99.5 o~), respectively; DNA samples were first diluted into, then dialyzed against the appropriate medium; this was followed by thermal equilibration at a temperature approx, equal to Tm for 3 h and slow cooling. Total hyperchromicity and Tm after t r e a t m e n t were found to be essentially unchanged compared to untreated controls. All experiments were run in triplicate; all values rerorted are the mean ! standard deviation. c o n d i t i o n s t h a t a s s u r e c o m p l e t e e x c h a n g e 9-11, t h e p r o f i l e s for t h e r m a l d e n a t u r a t i o n of t h e d e u t e r i u m - c o n t a i n i n g s p e c i e s in a d e u t e r i u m - c o n t a i n i n g m e d i u m a r e v i r t u a l l y i d e n t i c a l w i t h t h e c o r r e s p o n d i n g p r o f i l e s for t h e h y d r o g e n - c o n t a i n i n g s p e c i e s in a h y d r o g e n - c o n t a i n i n g m e d i u m . ( 6 T m ~ 0 . 4 °, b A T ~ 0 . 2 ° ) . O n e p o s s i b l e i n t e r p r e t a t i o n of t h i s , t o us, r a t h e r s u r p r i s i n g r e s u l t is t h a t t h e h y d r o g e n b o n d i n g of i n d i v i d u a l b a s e p a i r s w i t h i n t h e D N A d o u b l e h e l i x , a l t h o u g h u n q u e s t i o n a b l y a m a j o r f a c t o r in i t s s t r u c t u r e 5,e, is n o t n e c e s s a r i l y t h e o n l y c o n t r i b u t o r t o i t s stability. T h e a r g u m e n t s g i v i n g r i s e t o t h i s c o n c l u s i o n a r e as follows: t h e v i r t u a l i d e n t i t y Biochim. Biophys. dcla, 55 (I962) 789-792
PRELIMINARY NOTES
791
of the melting-point profiles in the H- and the D-containing case shows that for the unfolding reaction 3 helix ~- 2 coils
B y analogy with the protein and polypeptide cases ls,le we then assume that for a very long polymer any of the thermodynamic functions can be approximated b y appropriate summation over the individual residues, i.e., that A Funt, A Hum and zt Sun f Aun f ~
(2n+3m)Aav
(2)
where n = number of A - T pairs (with 2 hydrogen bonds each) 5, m = G-C pairs (with 3 hydrogen bonds each) 17 and, to a first approximation, AFar, zlHav, ASav are taken as equivalent for all hydrogen bonds in these pairs. It then follows from (I) and (2) that (3) also holds. 3Fav ~ ~Hiv ~.~ 6Say ~___ o
(3)
This might occur if (a) the "deuterium" bond is indeed no stronger than the "hydrogen" bondlS; b) AFar and AHav are so small as to be virtually equal to zero", or, the somewhat more likely alternative (c) that the initial assumption inherent in Eqn. 2 is incorrect and that we must write instead Aunf = ( 2 n + 3 m ) A a v + A x
(4)
where AFx and AHx and ASx refer to some unspecified co-operative process present and important to polymer stability, but less stringent or absent for individual base pairs. Since at the midpoint of the transition, at the melting temperature Tin, AF = 0 it follows that AFx here is equal to and opposite in sign to (2n+3m)AFav and that at T < Tm the helix is stabilized not only by hydrogen bonds but also by the x-process. As contributory evidence we m a y cite that, for approximately equal chain length Tm itself is a function of base composition 7,1° while A Hunf. = R d l n K u m / d T - l ( i . e . , a function of the slope at the midpoints 1~ of the curves in Fig. i) is not. Yet equation (2) predicts such a functional dependence, since we can write AHu~t = 2n'AHav + / t o o l . % (G+C)]n'zlHav
(5)
where n' now represents the total number of base pairs. As to the nature of the stabilizing forces little can be said at this moment, but it is perhaps suggestive that (a) it is now believed that a major contributory factor to conformational stability in globular proteins is provided b y " a p o l a r " or "hydrophobic" bonds 2°-z~ related to an increase in order within the water structure surrounding the polymer; (b) that rupture of similar bonds by a variety of agents leads to the denaturation (i.e., helix -~ coil transition) of double-stranded DNA2S,24; (c) the explicit suggestion by HAMAGUCHIAND GEIDUSCHEKlz that the denaturing effect on DNA of anions at high concentration is due to their action as hydrophobic bond breaking agents, i.e., their ability to disrupt ordered water structures responsible for helix stability and (d) our demonstration by means of nuclear magnetic resonance techni* This presents a distinct possibility since for each individual base pair we must take into account three different types of hydrogen bonds broken and/or made: those between bases, those between bases and water and those of water itself. Some of these combinations may well be such as to lead to no net change in dF and/lt-I (c[. footnote 3 of ref. 19 and ref. 3). It is also of interest that exchange of deuterium for protons on films of DNA salts is exceedingly rapid it.
Biochim. Biophys. Acta, 55 (1962) 789-792
792
PRELIMINARY NOTES
ques that a considerable proportion of the solvent molecules around native DNA is immobilized in a highly ordered form and that the effect disappears with denaturation ~5, 2e. We are indebted to Professor W. J. MOORE and Professor V. J. SHINER, Jr., for many helpful and illuminating discussions, and to the National Science Foundation (Grant G 8959) and the U. S. Public Health Service (Grant E 1854) for supporting this research.
Chemical Laboratories, Indiana University, Bloomington, Ind. (U.S.A.)
H. R. MAHLER B. D. MEHROTRA
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Received February 9th, I962 Biochim. Biophys. Acta, 55 (1962) 789-792