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Raman studies of nucleic acids
BIOCHIMICA ET BIOPHYSICA ACTA
I
BBA 95671
RAMAN S T U D I E S OF NUCLEIC ACIDS I I AQUEOUS P U R I N E AND P Y R I M I D I N E MIXTURES*
R
C L O R D AND G
J
THOMAS, Jn
Department o/Chemzstry and the Spectroscopy Laboratory, Massachi~setts Instztute o/ Technology, Cambrzdge, Mass (U S A ) ( R e c e i v e d M a r c h 8ttl, 1967)
SUMMARY
R a m a n spectra of aqueous solutions of complementary base pairs have been studied at neutral p H or p2H up to total solute concentrations of I.O M in the nucleoslde and nucleotide derivatives. No evidence for specific base-pamng interactions has been found Interactions between soluble nucleosides and heavy-metal ions, however, can be detected and results of a study of the cytldme-HgC12 complex in water are reported and discussed. The present investigation demonstrates the usefulness of R a m a n spectroscopy as a probe of intermolecular interactions in systems of biological Interest.
INTRODUCTION
Interactions between monomer constituents of nucleic acids m different solvent media have been the object of much recent research. Infrared spectroscopic studies have shown that in the weakly polar solvent CHC13 (see refs. 1-4), and In the non-polar solvent CC14(see ref. 5), derivatives of commonly occurring bases of RNA or DNA form hydrogen-bonded dimers with the specificity of pairing displayed in double-stranded nucleic acids, i.e. adenine and guanine associate preferentially with uracil (or thymine) and cytosine respectively. Studies by means of NMR (see ref. 6) and ultraviolet absorption 7 give independent evidence Ior specific interactions of this type in CHC13 solutions. A common purpose of these investigations is to find chemical and physical criteria for the stability and base-pairing specificity of doublestranded nucleic acids. It is therefore important to extend this kind of study to water, the universal biological solvent. Up to the present, the possibility of base pairing m aqueous solutions of the monomers has not been investigated. NMR studies6, 8 have shown that the formation of hydrogen-bonded base pairs between guanine and cytosine nucleosides is appreciable in the highly polar solvents * T h e f i r s t p a p e r in t h i s series, e n t i t l e d R a m a n S p e c t r a l S t u d i e s of N u c l e m A c i ds a n d R e l a t e d M olecu les I R l b o n u c l e i c Acid D e r i v a t i v e s , is m t h e p r e s s ( S p e c t r o c h l m m a Acta)
B*ochzm Bwphys. Acta, 142 (I967) i - t i
2
R C. LORD, G. J. THOMAS, JR.
d l m e t h y l s u l f o x l d e a n d d l m e t h y l f o r m a m i d e , even t h o u g h solvent c a r b o n y l groups can a c t as acceptors for t h e donor protons of a m i n o a n d lmino base s u b s t l t u e n t s . Therefore it seems reasonable to e x p e c t t h a t base pairs s t a b l h z e d b y i n t e r - b a s e h y d r o g e n b o n d s m a y be f o r m e d to an a p p r e c i a b l e e x t e n t in aqueous solution also. R e c e n t l y SINANO(,LU AND ABDULNUR 9,10 h a v e concluded t h a t w a t e r should be more f a v o r a b l e t h a n o t h e r p o l a r solvents, including f o r m a m l d e , in stabilizing the association of solute species, even those w i t h sizable dipoles, as in the case of the nucleotlde bases of D N A This conclusion has been s u b s t a n t i a t e d b y the evidence for parallel s t a c k i n g of p u r m e a n d p y n m l d i n e bases in aqueous solution from the N M R studies of T s ' o a n d others H is. However, d e t a i l e d studies have n o t been e x t e n d e d to m i x t u r e s of base d e r i v a t i v e s c a p a b l e of forming strong h y d r o g e n b o n d s w i t h one another. The N M R results H-la m o r e o v e r do not distinguish b e t w e e n s t a c k e d m o n o m e r s a n d s t a c k e d dimers, so t h a t the existence of base p a i r i n g in a d d i t i o n to s t a c k i n g c a n n o t be excluded on the basis of N M R proton-shift d a t a alone, as has been p o i n t e d out b y JARDETZKY 14. On the o t h e r hand, h y d r o g e n - b o n d e d base pairs should be r e a d i l y d e t e c t e d b y m e t h o d s of v i b r a t i o n a l s p e c t o s c o p y The a d v a n t a g e s of the R a m a n effect for studies of this t y p e in aqueous solutmn have been cited earherT, is a n d d e t a i l e d R a m a n studies of the c o m m o n nucleic acid bases a n d r e l a t e d molecules have been reportedT, 16. I n the first p a r t of the present work t h e results of a R a m a n i n v e s t i g a t i o n of aqueous m i x t u r e s of p u r m e n u c l e o h d e s a n d p y r l m i d m e nucleosldes are r e p o r t e d I n a d d l t m n to p u r m e - p y n m l d i n e associations, the b i n d i n g of m e t a l ions to nucleic acids a n d their m o n o m e r c o n s t i t u e n t s has received m u c h attentlon~7-ss, 3s. T w o different t y p e s of ionic i n t e r a c t i o n s m a y be d i s t i n g u i s h e d for present purposes: (~) i o n - r a n interaction, z.e. the b i n d i n g of cationic species to the mono- a n d p o l y nucleotlde p h o s p h a t e groups, - - P O a2- a n d ~ P O 2- respectively, whmh m a y include b o t h h g h t - a n d h e a v y - m e t a l runs, for e x a m p l e Mg 2+ a n d Co 2+, (2) 1on dipole interaction, ~ e. the b i n d i n g of t r a n s i t i o n - m e t a l ions to polar sites of the base rings, for example Hg(II) ....
N Q . I n t e r a c t i o n s of t y p e (i) h a v e been e x t e n s i v e l y i n v e s t i g a t e d
b y m a g n e t i c resonance methodsiS, 3s. These are n o t well s u i t e d to s t u d y b y R a m a n spectroscopy, since only small p o l a r i z a b i l i t y changes are e x p e c t e d to a c c o m p a n y s t r e t c h i n g v l b r a t m n s of such electrostatic bonds. Resulting p e r t u r b a t i o n s of t h e p h o s p h a t e group v i b r a t i o n s are also e x p e c t e d to be small*. I n t e r a c t i o n s of t y p e (2), however, are well s u i t e d to R a m a n i n v e s t i g a t i o n since these are e x p e c t e d to cause significant changes in the ~-electron d i s t r i b u t i o n of the p u r m e or p y r l m l d i n e rings. Such changes in t u r n will have considerable effect on the p o l a r l z a b i h t y changes acc o m p a n y m g t h e v i b r a t i o n s of the r m g a t o m s a n d the a t o m s of the 1on-&pole bond. I n the second p a r t of this work a R a m a n s t u d y of the complex formed between cytldine a n d HgC12 in aqueous solution l~ r e p o r t e d
* ~Ve have obtained Raman spectra of aqueous sodmm monomethylphosphate, a mononucleotlde analog, and fred only small displacements of tile phosphate group frequencies upon a(hhtlon of excess Mg2+ Notably the symmetrm PO]- stretching frequency is displaced from 985 to 99o cm ~ at most, a shift too small to allow meaningful quantitative investigation of such interactions by the Raman techmque i3~och~'~ H~ophw Arta, i42 (I907) I - I I
RAMAN S T U D I E S OF P U R I N E S AND P Y R I M I D I N E S EXPERIMENTAL
3
METHODS
The general methods of Raman instrumentation have been describe&, 16. For the present work quantitative comparison of Raman line mtensities was also important. Therefore, in the spectra of purane and pyrimidlne mixtures, transmission of the filter solution for isolation of the 4358-• mercury line was carefully checked on a Beckman Model DK-2 spectrophotometer after each Raman spectrum to assure that a nearly constant exciting-line intensity was admitted. In addition, in the spectra of cytidlne and HgCI~ solutions a "standard" scan of the 459-cm -1 line of CC14 was obtained after each solution spectrum. The relative hght-scattermg volumes of all sample cells were also computed from measurements of this standard line intensity. Raman scattermg intensities m the solutions were then normahzed to the integrated intensity of the standard with correction also for any cell volume differences. This method eliminates sources of error from filter solution deterioration, variations of arc intensity during the time lapse between successive spectra, differences in cell volumes, and the hke. No corrections were found necessary for such "internal" factors as refractive index changes from one solution to another accompanying changes in solution composition or concentration. It was verified that the intensity of Raman scattering an HgClz solutions and in cytidine solutions is linearly related to solute concentration. The same was found for solutions containing HgC12 and 1,3-dimethyluracil or Hg (CN)2 and cytidlne, in which no complexes are formed (below). It is concluded therefore that to good approximation the intensity of a Raman line in solutions examined here is directly proportional to the molarity of the species which gives rise to the line. Integrated lane intensities were measured with a planimeter from spectra recorded with an expanded abscissa (20 cm-1/inch). Base lines were drawn to fit the background contours of the pure solvent scattering. Base line determinations were not a problem in the cytldme-HgC12 spectra since reliable area measurements were needed only for the 322-cm -1 line of HgC12 (v1, symmetric stretching), which occurs An a frequency region nearly free from Raman scattering of the solvent or other solute species. The error in line area determinations was less than =L2 %. The reproducibility of mdividual measurements is within ± 5 ~o for the HgC12 hne areas and within ::~IO O/ ,o in other cases. Nucleosldes (Sigma Chemical Co.) and nucleotldes (Calbiochem Co.) were the highest grades commercially available. The purest guanine nucleotide, guanosine 5'-monophosphate tetrahydrate, disodmm salt (Calblochem lot 3o288), still contained a trace amount of impurity which caused partial absorption of the blue excltmg radiation. This did not seriously affect the qualitative conclusions obtained from the spectra of its solution mixtures. The background scattering due to H20 (or 2H20 ) has been subtracted from all Raman spectra shown below. All measurements in cytldine-HgCl~ solutions were made at 35 °. The pH or p~H values of solution mixtures were unadjusted, ~ e. no additional H + or O H ions were added. The resulting pH values of solutions were always in the range 5 ~ pH =< 6. Since the pK for protonatlon of cytldine is 4.2, the nucleosade was essentially in the non-protonated form. The interaction between HgC12 and Irnethylcytoslne, an analog of cytldme, produces an insoluble complex which could not be studied. B,ochzm B,ophys. ~tcta, 142 (1967) i - i ~
4
R.C.
LORD, G. J. THOMAS,
JR.
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F i g i R a m a n s p e c t r a of A d o - 5 ' - P , u r l d l n e a n d A d o - 5 ' - P + U r d s o l u t m n s (a) o 25 M A d o - 5 ' - P i n 2 H 2 0 , (b) o z5 M u n d m e i n ~ H 2 0 , (c) A d o - 5 ' - P + U r d e q u l m o l a r m i x t u r e , o 5 ° M t o t a l in ~H~O; (d) s u m m a t i o n of (a) a n d (b) S p e c t r a r e c o r d e d a t p 2 H = 7 5== ° 3 a n d 3 5 ~ I ° S p e c t r u m (c) u n c h a n g e d b y a d d ~ t t o n of e x c e s s NaC1 S p e c t r a l s h t - w i d t h i o c m ~ S c a n n i n g s p e e d o 25 c m ~/ see P e r i o d 2 sec
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F i g 2 R a m a n s p e c t r a of A d o - 5 ' - P + U r d i n t h e I 7 5 O - I 4 5 o - c m - 1 r e g i o n (a) S p e c t r u m o b s e r v e d for e q m m o l a r A d o - 5 ' - P + U r d m i x t u r e , o 5o M t o t a l i n ~H20, p~H = 7 5, 35 ° (See t e x t ) (b) S p e c t r u m o b t a i n e d b y a d d i n g s p e c t r a of o 25 M A d o - 5 ' - P a n d o 25 M u r l d l n e an 2H2(), pZH = 7 5, 35 ° S p e c t r a l s h t - w l d t h i o c m 1 S c a n n i n g s p e e d o i o c m 1/sec P e r i o d 15 sec S i g n a l ( m t e n m t y for s t r o n g e s t l i n e ) S = 3 ° N o s e (at p e a k h e i g h t ) N
B*ophys B*ophys Acta, 142 (19(77) I i i
I~4,MAN STUDIES OF PURINES AND PYRIMIDINES
5
Raman study o/ Ado-5'-P+Urd and Cuo-5'-P+Cyd mixtures ~n aqueous soluhon Fig. I shows the spectra of Ado-5'-P, uridlne and an equxmolar mixture of Ado-5'-P and urldine in 2H20 solution. It is seen that the observed spectrum of the mixture agrees closely in R a m a n line frequencies and intensities with that obtained from summing the spectra of the constituents. This is clearly evident in the I75o-I45o-cm -1 region (Fig. 2) when the spectra are corrected for changes in filter transmission. { c m 15 1800
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i! F i g 3 R a m a n s p e c t r a of Guo-5"-P, c y t l d m e a n d G u o - 5 ' - P + C y d s o l u t i o n s (a) o 25 M G u o - 5 ' - P m ~H20, (b) o 25 M c y t l d m e in 2H20, (c) G u o - 5 ' - P + C y d e q m m o l a r m i x t u r e , o 5 ° M t o t a l in ~HzO, (d) s u m m a t m n of (a) a n d (b) S p e c t r a r e c o r d e d a t p2H = 7 5:t: ° 3 a n d 3 5 ± 1 ° S p e c t r u m (c) u n c h a n g e d b y a d d m o n of e x c e s s NaC1 S p e c t r a l s h t - w l d t h i o c m 1 S c a n n i n g s pe e d o 25 c m l/ sec P e r m d 2 see
Fig. 3 shows the spectra of Guo-5'-P, cytidme and an equlmolar mixture of Guo-5'-P and cytidlne in 2H20 solution. The mixture spectrum gives no evidence of frequency changes but large intensity losses (approx. 5o %) are apparent in the lines of the cytldlne component. This can be a t m b u t e d entirely to the fact that the extinction coefficients in the blue for solutions of Guo-5'-P are not zero. Therefore transferring cytidlne from its own solutions (optically transparent near 4358 ~) to the absorbing solutions reduces the relative intensity of its R a m a n scattering significantly*. When a correction is made for this effect, the spectrum of the mixture is found to correspond closely to the summation spectrum, as indicated in Fig. 4 for the I75O-I45o-cm -1 region. Sunllar results are obtained for A d o - 5 ' - P + U r d and G u o - 5 ' - P + C y d systems under varying conditions of observation in both H~O and 2H20 solutions (Table I). If significant spectral changes in the I75O-I45o-cm -1 region occurred in the mixed solutions, these could be attributed to the formation of hydrogen-bonded * T h e i n t e n s i t y of R a m a n s c a t t e r i n g as a f u n c t i o n of s o l u t e c o n c e n t r a t i o n m a n a b s o r b i n g m e d i u m h as b e e n t r e a t e d q u a n t x t a t l v e l y , c~ ref 29.
B~och,m. B,ophys. Acta, 142 (1967) I - i i
0
R. C. L O R D ,
IOO
G. J. T H O M A S , J R
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Fig 4 Raman spectra of G u o - 5 ' - P + C y d m the i75o-i45o-cm 1 r e g i o n (a) Spectrum observed for eqmmolar G u o - 5 ' - P + C y d mixture o 2 o M t o t a l m 2 H 2 0 , p 2 H - - 7 5; 35 ° (See t e x t ) (b) Spectrum obtained by adding spectra o f o I o M G u o - 5 ' - P a n d o 1o M cytldme in 2 H 2 0 , p 2 H ~ 7 5, 35 °. Spectral sht-wldth i o c m ~ Scanning speed o o 5 cm-I/sec, P e r i o d 3 ° s e c S / N = i o TABLE
1
PURINE--PYRIMIDINtg
Solutes
SOLUTION
MIXTURES*
Solvent
pH or p~H
Concentration ga~4ge
(total molar) Ad°-5'-P+Urd Ado-5'-P+ Urd Ado-5'-P+ Urd-5"-P Ado + Urd Gu°-5'-P+ Cyd Gu°-5'-P+ Cyd
Guo-5'-P+Cyd-5"-P Guo+Cyd
H20 2H20 HzO 2H20 H2(-) 2H20 2H20 2HzO
7 7 7 2 7 7 7 II
5 5 5 o 5 5 5 o
0 i0--I
0
O IO--O 50
o 5° o 5° o2o o5o 0 IO--I 0
o 5° o 5°
* N o evidence for base-base interactions in all solutions
complexes 1,a°. The absence of any spectral changes here, together with the results of NMR studies u-14 which indicate that at these concentrations the base rings are stacked parallel and one above another ("vertical stacking"), ]mplies that Inter-base hydrogen bonding is absent Aggregates of stacked base pairs would largely exclude water molecules from the immediate environment of the polar ring substituents but aggregates of stacked monomers would not The resulting difference in hydrogen-bonding strength of base-base and base-solvent interactions should then be evident in the I75O-i45o-cm -1 region where frequencies of the coupled C = O stretching and N H deformation vibrations occur ~6. Since no frequency changes are observed it is concluded that no appreciable base pairing accompanies such vemcal stacking as exists in these systems at the concentration levels examined. Bwchzm
Bwphys .dcta, 142 ( 1 9 6 7 ) I - - I I
7
RAMAN STUDIES OF PURINES AND PYRIMIDINES
This is consistent with the w~despread belief that the main source ot stabilization energy of double-hehcal D N A m aqueous solution is provided by forces other than those resulting from the formation of hydrogen-bonded base pairsamm,a",4°. Apparently a necessary requirement for base pairing in aqueous solution is the geometric restriction imposed on the bases by their attachment to the sugar-phosphate backbone of the polymer. i) IOOC (cm
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Fig 5 R a m a n s p e c t r a of H , O s o l u t i o n s of c y t l d m e and c y t l d m e + H g C 1 2 (a) o 25 M c y t l d m e ; 5 < p H < i i , (b) o 2 5 M c y t l d m e and o 2 5 M HgC12, p H ~ 5 3 ~ o 2 S p e c t r a recorded at 35 ° S p e c t r a l s h t - w l d t h IO crn -1 S c a n n i n g speed o i o cm-1/sec Period i o sec S / N = 3 °
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Fig 6 R a m a n s p e c t r a of iH~O s o l u t i o n s of cyt~dlne and c y t l d l n e + H g C l 2 (a) o 20 M c y t l d m e , 5 < p*H < i i , (b) o 2 o M c y t l d l n e a n d o e o M HgC12, p~H ~ 5 2 ! o 2 S p e c t r a recorded at 35 ° S p e c t r a l s l l t - w l d t h i o c m 1 S c a n n i n g speed o 25 cln-~/sec Period Io sec S / N = 15 t?~ochzm
Bzophys _dcta,
142 (1967) i - I i
R
C. LORD, G. J. THOMAS, JR.
Rarnan study o/the cyt,dme-HgCl 2 complex I n Fig. 5, R a m a n s p e c t r a of H~O solutions of c y t l d m e a n d of an e q m m o l a r m i x t u r e of c y t l d m e a n d HgC12 are c o m p a r e d over t h e f r e q u e n c y region 18oo-2oo cm 1 C o r r e s p o n d m g s p e c t r a of ~H20 solutions are shown in Fig. 6. The n u m e r o u s changes in line frequencies a n d m t e n s l t i e s m the c y t i d m e s p e c t r u m caused b y HgCI 2 i n d i c a t e t h e f o r m a t i o n of a c o m p l e x The line at 322 cm -1 m the m i x t u r e s is due to undlssocAated HgC12 (ref. 33) b u t Ats i n t e n s i t y in the m i x t u r e s is g r e a t l y d l m i m s h e d from t h a t o b s e r v e d in pure HgC12 solutions at the same concentratAon T h u s a large a n d m e a s u r able a m o u n t of the m o l e c u l a r species HgClo is d i s s o c i a t e d b y the n u e l e o , l d e T h e i n t e n s i t y decrease at 322 cm -A a n d the absence of new lAnes in this f r e q u e n c y region a t t e n d i n g c o m p l e x f o r m a t i o n are consistent w i t h the o b s e r v a t i o n s of others 21,e2 t h a t the specle~ b o u n d to t h e nucleoside AS n o t HgC12 or HgC1 + b u t H g 2+. This is in accord w i t h the fact t h a t no change in the R a m a n s p e c t r u m of c y t l d i n e occurs on the a d d i t i o n of Hg(CN)2, which AS far less d i s s o c i a t e d t h a n HgC12 (ref. 34) u n d e r c o m p a r a b l e e x p e r i m e n t a l conditions. As in the case of the D N A - H g ( I I ) c o m p l e x 2~, the Anteractlon o b s e r v e d here is c o m p l e t e l y r e v e r s e d b y the a d d i t i o n of a sufficient excess of C1- to the solution m i x t u r e s . Thus, for e x a m p l e , w i t h the a d d i t i o n of a 4-fold excess of Cl- An the solution which gives the s p e c t r u m of FAg 5, all of the c o m p l e x is dAssoclated, as e v i d e n c e d b y t h e fact t h a t t h e s p e c t r u m of u n b o u n d cytAdme is recovered. I n additAon the line at 322 cm 1 becomes v a n l s h l n g l y weak a n d new lines a p p e a r a t 285 a n d 270 cm 1 a t t r i b u t a b l e r e s p e c t i v e l y to the m o l e c u l a r species H g C l f a n d HgCI~(ref 35-37) l:Ig~. 5 a n d 6 show t h a t t h e largest p e r t u r b a t i o n s in t h e s p e c t r u m of c y t l d m e occur in the I~OO-I55O- a n d 13oo 12oo-cm -1 regAons where r e s p e c t i v e l y rAng doubleb o n d a n d single-bond s t r e t c h m g v i b r a t i o n s h a v e been assigned 16. Moreover, for b o t h H~O a n d 2H20 solutions, the b m d m g of H g ( I I ) t o c y t l d i n e causes q u a h t a t l v e l y slmAlar s p e c t r a l shAfts T h u s a r e m a r k a b l e m t e n s i t y reversal occurs in the pair of lines n e a r 13oo a n d 125o cm -1 wAth shifts of a b o u t lO cm ~ in the lAne centers, a n d t h e r e is a large i n t e n s i t y m c r e a s e n e a r 165o cm ~ wAth an i n t e n s i t y decrease n e a r 162o cm -~ An b o t h solutions. The characteristAc c y t o s m e rAng frequencies near IOOO c m ~ are also shifted some 15-25 cm -~ to higher f r e q u e n c y in b o t h solutAonb These effects are best e x p l a i n e d b y assuming t h a t bAnding of H g ( I I ) occurs at t h e a v a d a b l e N-3 r a g posltAon. If i n s t e a d H g ( I I ) r e p l a c e d a p r o t o n a t the e x t e r n a l a m i n o group 24 it w o u l d be dAfficult to e x p l a i n the s p e c t r a l changes An t h e I3OO-I2OO-Cm -1 region of H20 solutAons a n d in the I8OO-i5oo-cm -~ region of 2 H 2 0 solutions On the o t h e r hand, b i n d i n g at the N- 3 position would be e x p e c t e d to have a p r o n o u n c e d effect on the x - e l e c t r o n d i s t r i b u t i o n of the h e t e r o c y c h c ring, t h e r e b y alterAng b o t h the C - - N a n d C - N s t r e t c h i n g frequencAes a n d a c c o u n t i n g for the slgnlfAcant a n d closely almilar s p e c t r a l change~ n o t e d in b o t h H 2 0 a n d ~H20 solution s p e c t r a A t t a c h m e n t of t h e m e r c u r y catAon to t h e nucleoslde i n d e e d produces m u c h the s a m e s p e c t r a l change as t h a t of protonatAon (~ c a t t a c h m e n t of H + or 2H+), as one can see b y c o m p a r i n g t h e s p e c t r a of FAgs 5 a n d 6 wAth the s p e c t r a of p r o t o n a t e d c y t l d l n e ~. T h u s for b o t h m e r c u r a t e d a n d p r o t o n a t e d c y t i d i n e there is a shift to hAgher f r e q u e n c y of t h e t o t a l i n t e n s i t y in the d o u b l e - b o n d region (18oo-15oo cm -~) a n d a shift to lower f r e q u e n c y of t h e t o t a l i n t e n s i t y in the single-bond region (13oo12oo cm 1). This IS Anterpreted as a "fAXAng" of the double a n d sAngle b o n d s of the ring. Bzochzm
B~ophy~ Acta, 142 (1007) i I i
RAMAN
STUDIES
OF
PURINES
AND
9
PYRIMIDINES
Since the present data indicate that all the perturbations in the cytidine spect r u m increase monotonically with increasing molar ratio of HgCI 2 to cytidine, it is concluded further that binding occurs only at the N-3 ring position under the present experimental conditions. The binding of H g ( I I ) to cytldine m a y be further treated by considering the following equilibria*. HgC12 + Cyd = HgCyd e+ + 2 C1-
K1
(I)
HgCyd 2+ + Cyd = Hg(Cyd)22÷
K2
(2)
Binding of more than two cytidine molecules to H g ( I I ) is neither sterically feasible nor consistent with the results of previous investigatlonsel, 22,24. In the following discussion equlhbria involving hydrolysis of Hg 2+ are neglected. This is a good approximation since the present data indicate that the equilibrium of Eqn. I lies well to the right and therefore at the p H values of the present measurements (5 G p H G 6) appreciable hydrolysis of Hg z+ IS prohibited aS,a~. The integrated intensity of the line at 322 cm -~ gives a reliable value of the concentration of HgC12 remaining undissociated, and therefore unbound to cytidine. [C1-] is approximated b y [HgCI~] 0 2 ~HgC12]. One can see 111 Figs. 5 and 6 that the stronger R a m a n hnes of cytldme and "complex" overlap, so that independent measurements of their respective line intensities (and therefore concentrations) cannot be obtained with high accuracy. Therefore in solutions with [HgC12] 0 << [Cyd]0, where the 322-cm -1 line is vanishmgly weak, the relative concentrations of cytldme and complex can be obtained from the total R a m a n Intensity at some frequency v where the R a m a n intensities of bound and unbound cytldine differ greatly. The scattering (I~) due only to cytldine can be P can be approxobtained from solution spectra of cytidlne, that due to complex (ox) iinated b y the intensity in the solution containing [HgC12]0 = 5[Cyd]0 = 0.25 M TABLE
II
E Q U I L I B R I U M DATA OF C y d - M g C 1 2 SOLUTIONS (r)
(2)
(3)
(4)
(5)
(6)
(7)
[HgClz]o a
ECyd]o
IHgC12]b
[Cyd]e
[Cyd] a
Kle
Ka r
(average)
o 25 o 25 o25 o 05 o 125 o 05 o o5 oo 5 o 05
o 05 o 125 o25 o 05 o 25 o io o 15 o2o o 25
o 19o o 126 oo625 o 0237 o o179 o o13o o o4 ooo2 o
(o) o o18 o lO 7
(o) o 035 o 12o o 022 o 14o o 068 o ioo o 153 o 203
-o 31 o61 o 44 o 59 o 5° o 28 o46 --
-o 73 o75 o 37 o 5° o 57 o 22 o35 --
-o 52 o68 o 41 o 55 o 54 o 25 o4 I --
a b e a e r
o 020 o o o o o
13o 065 092 143 2oo
S o l u b i l i t y h m l t = o 25 M a t 3 5 ° A l l m e a s u r e m e n t s F r o m 3 2 2 - c m -1 h n e i n t e n s i t y Eqn 5, v = 1 2 5 3 c m -1 Eqn 5, v = 1 2 7 o c m - t . Data of Columns ( I ) , (2), (3), (4). D a t a o f C o l u m n s ( i ) , (2), (3), (5)
* F o r t h e m o l e c u l a r s p e c i e s z, ~z]0 a n d [z] d e n o t e t r a t i o n s , w h e r e z m a y b e H g C 1 z, C y d , C1 o r c o m p l e x
were
made
a t 35 °.
the initial and eqmhbrlum
t3,och*m
B~ophys
K1
molar concen-
./lcta, 142 ( 1 9 6 7 ) i - I I
io
R.
c
LORD,
G.
J.
THOMAS,
JR.
where nearly all the cytidme is bound (Table II, above). The total intensity at v is then 2+
v
/total-- [CydlI~ + [UgCyd2+]I;x + 2[Hg(Cyd)2 llcx
(3)
where it is assumed that both complexes have the same scattering intensity per molecule of bound cytldlne. From Eqns. 1 and 2 [Cyd]0 = [ C y d l + [HgCyd 2+] + 2[Hg(Cyd)22+]
(4)
and from Eqns. 3 and 4 [Cydl --
/~otal - -
I~
-
-
[Cyd]0 I~x
(5)
I~x
The quantity [Cyd] was calculated from Eqn. 5 at v = 1253 and 127o cm -1. The results are presented in Table II. It is seen that in the solutions containing excess HgC12, the amount of HgC12 "removed" by binding to cytidine, as determined from the 322-cm -~ hne area, is very nearly equal to the amount of cytldine initially present, z.e. only a I : I binding ratio is suggested. In solutions containing excess cytldme, the amount of cytldlne removed by binding is also equal to the initial concentration of HgC12, within experimental uncertainty. Therefore, over the entire concentration range studied A [HgC12] - [ngCl~] 0 -- [HgC121 A [Cydl
[Cyd]0 -- [Cydl
and it is concluded that only a 1:1 complex, HgCyd 2+, is formed z.e K 1 >> K 2. The apparent equilibrium constant*,/£1, is defined as ]k.1 __ [HgCyd 2+] [C1 ~ 2 [HgC121 [Cydl
([Cyd]0_[Cyd])([HgCl~]0_z[HgC12])2 ~
[HgC12] [Cyd]
(EHgCl~10--[HgC12]) ([HgC12]0-- 2 IHgCl2~)~ [HgC12~ [Cyd] and is calculated as indicated in Table II. A mean value of K~ =: (5o:£2o]" IO-2 M is obtained, the devmtion being largely due to the difficulty m obtaining accurate Raman intensities an dilute solutions (o 05 M in HgC12 or cytidme) The system cytidme HgCI2 was chosen because of its suitabdlty to a Raman study. Although complexes are readily formed between HgC12 and derivatives of adenine, guanine and lnoslne, under comparable experimental conditions 22,24, these are not soluble at the concentration levels reqmred to obtain Raman spectra of satisfactory quahty Derivatives of uracil, including uradme, I-methyluracfl and 1,3-dlmethyluracil, gave no interaction with HgC12 up to the addition of a 5-fold excess of the salt. This ~s consistent with the conclusion reached earlier that strong binding occurs preferentially at unsaturated nitrogen sites EICHHORN AND C L A R K 21 have observed the urldme-HgC12 interaction under considerably different experimental condmons, with a 32-fold excess of the mercury * No attempt
was made
B~ochzm B~ophys. Acla,
142
to correct (1967)
i-iI
for actlvltms
RAMAN STUDIES OF PURINES AND PYRIMIDINES
II
salt and at pH 9. Under such conditions, replacement of the imino N-3 proton of uracil by Hg z+ is greatly favored. It is concluded therefore that the interaction observed here between Hg 2+ and cytldine is very different from that expected at high pH. Salts which cause no change in the aqueous Raman spectra of either cytidme or uridine are ZnC12, ZnSO a, CdC12, CuSO, and MgCI 2. AgN03 gives spectral evidence of interaction only with cytidine. These results are inconclusive, however, since detailed analysis of solutions of Ag + is prohibited with mercury-arc sources, which cause rapid photodecomposltion. In view of the recent interest in the polynucleotldeAg + interaction26, 2r, it would appear worthwhile to examine such systems using a laser-source Raman instrument.
REFERENCES I R M HAMLIN, J R , R C LORD AND A RICH, Science, 148 (1965)1734 2 Y. KYOGOKU, R C LORD AND i RICH, J A m Soc, 89 (1967) 496 3 Y KYOGOKO, R C LORD AND i RICH, Sc,ence, 154 (1966) 518 4 J PITHA, R N JONES AND P PITHOVA, Can. J Chem, 44 (1966) lO44. 5 E KUCHLER AND J DERKOSCH, Z. Natur[orseh, 2113 (1966) 209 6 L, KATZ AND S PENMAN, J Mol Bzol , 15 (1966) 220 7 G. J THOMAS, JR , P h D Thesis, M I T , Cambridge, M a s s , 1967 8 R. R SHOUP, T H MILES AND E D BECKER, B*ochem B*ophys Res Commun, 23 (1966)194 9 0 . SINANO(~LU AND S ABDULNUR, Federat,on Proc, 24 (1965) S-I2 IO O SINANOGLU AND S ABDULNUR, Photochem. Photob~ol, 3 (1964) 333 I I S. [ CHAN, M P SCHWEIZER, P O P TS'O AND G K HELMKAMP, J A m Chem. Soc, 86 (1964) 4182 I2 M P SCHWEIZER, S I CHAN AND P. O P Ts'o, J A m Chem Soc, 87 (1965)5241. I3 S I. CHAN, B W 13ANGERTER AND H H PETER, B*ophys Soc Ioth Ann Meeting, Boston, Mass., 1966, A b s t r a c t s , Biophysical Society, p 17 14 O JARDETZKY, Bzopolymers Syrup , I (1964) 5Ol 15 R. A MALT, B*ochzm Bzophys Acla, 12o (1966) 461 16 n . C. LORD AND G J THOMAS, JR , Spectrochzm Acta, in the press 17 R G SHULMAN, H STERNLICHT AND B J WYLUDA, J Chem. Phys , 43 (1965) 3116 18 H. STERNLICHT, R G SItULMAN AND E W* ANDERSON, J Chem P h y s , 43 (1965) 3123 . 19 S KATZ, J A m Chem Soc, 74 (1952) 2238 20 C A THOMAS, J A m Chem Soc , 76 (1954) 6052 21 T YAMANE AND N I)AVIDSON, J A m Chem Soc, 83 (1961) 2599 22 R B. SIMPSON, J Am. Chem Soc, 86 (1964) 2059 23 M N LIPSETT, J B,ol Chem, 239 (1964)125o 24 G. L EICHrlORN AND P CLARK, J A m Chem Soc , 85 (1963) 4020 25 Y [5[A'~VADA,Bzochem Bzophys Res Commun, io (1963) 204 26 R H JENSEN AND N. I)AVIDSON, Bzopolymers, 4 (1966) 17 27 M DUANE, C A DEKKER AND N I( SCHACHMAN, B*opolymers, 4 (1966) 51. 28 K A. }{ARTMAN, J R , Bzochzm Bzophys Acta, 138 (1967) 192 29 E R LIPPINCOTT, J P SIBILIA AND R D FISHER, J Opt Soc A m , 49 (1959) 85 3 ° G. C PIMENTEL AND A L McCELLAN, The Hydrogen Bond, F r e e m e n , S a n Francisco, 196o 31 H D E V O E A N D I . TINOCO, J R , J Mol B z o l , 4 (1962) 500 32 M. FALK, t( A HARTMAN AND R C LORD, dr A m Chem Soc, 84 (1962)3843 33 H. BRAUNE AND G ENGELBRECHT, Z Physzk Chem , S e r B, 21 (1933) 297 34 J KLEINBERG, W J ARGERSlNGER, JR AND E GRISWOLD, Inorgamc Chem,stry, H e a t h and Co., 13oston, 196o 35 L G SILL~N, Acla Chem Scand, 3 (1949) 539 36 J BJERRUM, G SCHWARZENBACHAND L G SILLI~N, Stabzl*ty Constants o/MetalIon Complexes, Part II [norganzc Lzgands, The Chemical Society, London, 1958 37 Z. KECKt, Spectrochzm Acla, 18 (1962) 1165 38 H. STERNLICHT, R G SHULMAN AND E W ANDERSON, J Chem. P h y s , 43 (1965) 3133 39 M. FALK, K A t-IARTMAN AND R C. LORD, J A m Chem Soc , 85 (1963) 387 4 ° M. FALK, I( A HARTMAN AND R C LORD, J A m Chem Soc, 85 (1963) 391
B~och~rn B,ophys
Acta, 142 (1967) I - I I
I
BBA 95671
RAMAN S T U D I E S OF NUCLEIC ACIDS I I AQUEOUS P U R I N E AND P Y R I M I D I N E MIXTURES*
R
C L O R D AND G
J
THOMAS, Jn
Department o/Chemzstry and the Spectroscopy Laboratory, Massachi~setts Instztute o/ Technology, Cambrzdge, Mass (U S A ) ( R e c e i v e d M a r c h 8ttl, 1967)
SUMMARY
R a m a n spectra of aqueous solutions of complementary base pairs have been studied at neutral p H or p2H up to total solute concentrations of I.O M in the nucleoslde and nucleotide derivatives. No evidence for specific base-pamng interactions has been found Interactions between soluble nucleosides and heavy-metal ions, however, can be detected and results of a study of the cytldme-HgC12 complex in water are reported and discussed. The present investigation demonstrates the usefulness of R a m a n spectroscopy as a probe of intermolecular interactions in systems of biological Interest.
INTRODUCTION
Interactions between monomer constituents of nucleic acids m different solvent media have been the object of much recent research. Infrared spectroscopic studies have shown that in the weakly polar solvent CHC13 (see refs. 1-4), and In the non-polar solvent CC14(see ref. 5), derivatives of commonly occurring bases of RNA or DNA form hydrogen-bonded dimers with the specificity of pairing displayed in double-stranded nucleic acids, i.e. adenine and guanine associate preferentially with uracil (or thymine) and cytosine respectively. Studies by means of NMR (see ref. 6) and ultraviolet absorption 7 give independent evidence Ior specific interactions of this type in CHC13 solutions. A common purpose of these investigations is to find chemical and physical criteria for the stability and base-pairing specificity of doublestranded nucleic acids. It is therefore important to extend this kind of study to water, the universal biological solvent. Up to the present, the possibility of base pairing m aqueous solutions of the monomers has not been investigated. NMR studies6, 8 have shown that the formation of hydrogen-bonded base pairs between guanine and cytosine nucleosides is appreciable in the highly polar solvents * T h e f i r s t p a p e r in t h i s series, e n t i t l e d R a m a n S p e c t r a l S t u d i e s of N u c l e m A c i ds a n d R e l a t e d M olecu les I R l b o n u c l e i c Acid D e r i v a t i v e s , is m t h e p r e s s ( S p e c t r o c h l m m a Acta)
B*ochzm Bwphys. Acta, 142 (I967) i - t i
2
R C. LORD, G. J. THOMAS, JR.
d l m e t h y l s u l f o x l d e a n d d l m e t h y l f o r m a m i d e , even t h o u g h solvent c a r b o n y l groups can a c t as acceptors for t h e donor protons of a m i n o a n d lmino base s u b s t l t u e n t s . Therefore it seems reasonable to e x p e c t t h a t base pairs s t a b l h z e d b y i n t e r - b a s e h y d r o g e n b o n d s m a y be f o r m e d to an a p p r e c i a b l e e x t e n t in aqueous solution also. R e c e n t l y SINANO(,LU AND ABDULNUR 9,10 h a v e concluded t h a t w a t e r should be more f a v o r a b l e t h a n o t h e r p o l a r solvents, including f o r m a m l d e , in stabilizing the association of solute species, even those w i t h sizable dipoles, as in the case of the nucleotlde bases of D N A This conclusion has been s u b s t a n t i a t e d b y the evidence for parallel s t a c k i n g of p u r m e a n d p y n m l d i n e bases in aqueous solution from the N M R studies of T s ' o a n d others H is. However, d e t a i l e d studies have n o t been e x t e n d e d to m i x t u r e s of base d e r i v a t i v e s c a p a b l e of forming strong h y d r o g e n b o n d s w i t h one another. The N M R results H-la m o r e o v e r do not distinguish b e t w e e n s t a c k e d m o n o m e r s a n d s t a c k e d dimers, so t h a t the existence of base p a i r i n g in a d d i t i o n to s t a c k i n g c a n n o t be excluded on the basis of N M R proton-shift d a t a alone, as has been p o i n t e d out b y JARDETZKY 14. On the o t h e r hand, h y d r o g e n - b o n d e d base pairs should be r e a d i l y d e t e c t e d b y m e t h o d s of v i b r a t i o n a l s p e c t o s c o p y The a d v a n t a g e s of the R a m a n effect for studies of this t y p e in aqueous solutmn have been cited earherT, is a n d d e t a i l e d R a m a n studies of the c o m m o n nucleic acid bases a n d r e l a t e d molecules have been reportedT, 16. I n the first p a r t of the present work t h e results of a R a m a n i n v e s t i g a t i o n of aqueous m i x t u r e s of p u r m e n u c l e o h d e s a n d p y r l m i d m e nucleosldes are r e p o r t e d I n a d d l t m n to p u r m e - p y n m l d i n e associations, the b i n d i n g of m e t a l ions to nucleic acids a n d their m o n o m e r c o n s t i t u e n t s has received m u c h attentlon~7-ss, 3s. T w o different t y p e s of ionic i n t e r a c t i o n s m a y be d i s t i n g u i s h e d for present purposes: (~) i o n - r a n interaction, z.e. the b i n d i n g of cationic species to the mono- a n d p o l y nucleotlde p h o s p h a t e groups, - - P O a2- a n d ~ P O 2- respectively, whmh m a y include b o t h h g h t - a n d h e a v y - m e t a l runs, for e x a m p l e Mg 2+ a n d Co 2+, (2) 1on dipole interaction, ~ e. the b i n d i n g of t r a n s i t i o n - m e t a l ions to polar sites of the base rings, for example Hg(II) ....
N Q . I n t e r a c t i o n s of t y p e (i) h a v e been e x t e n s i v e l y i n v e s t i g a t e d
b y m a g n e t i c resonance methodsiS, 3s. These are n o t well s u i t e d to s t u d y b y R a m a n spectroscopy, since only small p o l a r i z a b i l i t y changes are e x p e c t e d to a c c o m p a n y s t r e t c h i n g v l b r a t m n s of such electrostatic bonds. Resulting p e r t u r b a t i o n s of t h e p h o s p h a t e group v i b r a t i o n s are also e x p e c t e d to be small*. I n t e r a c t i o n s of t y p e (2), however, are well s u i t e d to R a m a n i n v e s t i g a t i o n since these are e x p e c t e d to cause significant changes in the ~-electron d i s t r i b u t i o n of the p u r m e or p y r l m l d i n e rings. Such changes in t u r n will have considerable effect on the p o l a r l z a b i h t y changes acc o m p a n y m g t h e v i b r a t i o n s of the r m g a t o m s a n d the a t o m s of the 1on-&pole bond. I n the second p a r t of this work a R a m a n s t u d y of the complex formed between cytldine a n d HgC12 in aqueous solution l~ r e p o r t e d
* ~Ve have obtained Raman spectra of aqueous sodmm monomethylphosphate, a mononucleotlde analog, and fred only small displacements of tile phosphate group frequencies upon a(hhtlon of excess Mg2+ Notably the symmetrm PO]- stretching frequency is displaced from 985 to 99o cm ~ at most, a shift too small to allow meaningful quantitative investigation of such interactions by the Raman techmque i3~och~'~ H~ophw Arta, i42 (I907) I - I I
RAMAN S T U D I E S OF P U R I N E S AND P Y R I M I D I N E S EXPERIMENTAL
3
METHODS
The general methods of Raman instrumentation have been describe&, 16. For the present work quantitative comparison of Raman line mtensities was also important. Therefore, in the spectra of purane and pyrimidlne mixtures, transmission of the filter solution for isolation of the 4358-• mercury line was carefully checked on a Beckman Model DK-2 spectrophotometer after each Raman spectrum to assure that a nearly constant exciting-line intensity was admitted. In addition, in the spectra of cytidlne and HgCI~ solutions a "standard" scan of the 459-cm -1 line of CC14 was obtained after each solution spectrum. The relative hght-scattermg volumes of all sample cells were also computed from measurements of this standard line intensity. Raman scattermg intensities m the solutions were then normahzed to the integrated intensity of the standard with correction also for any cell volume differences. This method eliminates sources of error from filter solution deterioration, variations of arc intensity during the time lapse between successive spectra, differences in cell volumes, and the hke. No corrections were found necessary for such "internal" factors as refractive index changes from one solution to another accompanying changes in solution composition or concentration. It was verified that the intensity of Raman scattering an HgClz solutions and in cytidine solutions is linearly related to solute concentration. The same was found for solutions containing HgC12 and 1,3-dimethyluracil or Hg (CN)2 and cytidlne, in which no complexes are formed (below). It is concluded therefore that to good approximation the intensity of a Raman line in solutions examined here is directly proportional to the molarity of the species which gives rise to the line. Integrated lane intensities were measured with a planimeter from spectra recorded with an expanded abscissa (20 cm-1/inch). Base lines were drawn to fit the background contours of the pure solvent scattering. Base line determinations were not a problem in the cytldme-HgC12 spectra since reliable area measurements were needed only for the 322-cm -1 line of HgC12 (v1, symmetric stretching), which occurs An a frequency region nearly free from Raman scattering of the solvent or other solute species. The error in line area determinations was less than =L2 %. The reproducibility of mdividual measurements is within ± 5 ~o for the HgC12 hne areas and within ::~IO O/ ,o in other cases. Nucleosldes (Sigma Chemical Co.) and nucleotldes (Calbiochem Co.) were the highest grades commercially available. The purest guanine nucleotide, guanosine 5'-monophosphate tetrahydrate, disodmm salt (Calblochem lot 3o288), still contained a trace amount of impurity which caused partial absorption of the blue excltmg radiation. This did not seriously affect the qualitative conclusions obtained from the spectra of its solution mixtures. The background scattering due to H20 (or 2H20 ) has been subtracted from all Raman spectra shown below. All measurements in cytldine-HgCl~ solutions were made at 35 °. The pH or p~H values of solution mixtures were unadjusted, ~ e. no additional H + or O H ions were added. The resulting pH values of solutions were always in the range 5 ~ pH =< 6. Since the pK for protonatlon of cytldine is 4.2, the nucleosade was essentially in the non-protonated form. The interaction between HgC12 and Irnethylcytoslne, an analog of cytldme, produces an insoluble complex which could not be studied. B,ochzm B,ophys. ~tcta, 142 (1967) i - i ~
4
R.C.
LORD, G. J. THOMAS,
JR.
SPt~CTROSCOPIC R E S U L T S AND DISCUSSION (era I) 1800
1600
1400
1200
1000
800
600
400
--
¢
{d)
-
-
200
-
it)
q
I
,
,
-
-
-
(b)
-
{u)
F i g i R a m a n s p e c t r a of A d o - 5 ' - P , u r l d l n e a n d A d o - 5 ' - P + U r d s o l u t m n s (a) o 25 M A d o - 5 ' - P i n 2 H 2 0 , (b) o z5 M u n d m e i n ~ H 2 0 , (c) A d o - 5 ' - P + U r d e q u l m o l a r m i x t u r e , o 5 ° M t o t a l in ~H~O; (d) s u m m a t i o n of (a) a n d (b) S p e c t r a r e c o r d e d a t p 2 H = 7 5== ° 3 a n d 3 5 ~ I ° S p e c t r u m (c) u n c h a n g e d b y a d d ~ t t o n of e x c e s s NaC1 S p e c t r a l s h t - w i d t h i o c m ~ S c a n n i n g s p e e d o 25 c m ~/ see P e r i o d 2 sec
100
I
I
-
---
;b) -
(a)
< v
z z
i/
0c
\Ix I 1750
IYOO
1650
1600 [ c m -I )
1550
///
, 1500
\\'~ 1480
F i g 2 R a m a n s p e c t r a of A d o - 5 ' - P + U r d i n t h e I 7 5 O - I 4 5 o - c m - 1 r e g i o n (a) S p e c t r u m o b s e r v e d for e q m m o l a r A d o - 5 ' - P + U r d m i x t u r e , o 5o M t o t a l i n ~H20, p~H = 7 5, 35 ° (See t e x t ) (b) S p e c t r u m o b t a i n e d b y a d d i n g s p e c t r a of o 25 M A d o - 5 ' - P a n d o 25 M u r l d l n e an 2H2(), pZH = 7 5, 35 ° S p e c t r a l s h t - w l d t h i o c m 1 S c a n n i n g s p e e d o i o c m 1/sec P e r i o d 15 sec S i g n a l ( m t e n m t y for s t r o n g e s t l i n e ) S = 3 ° N o s e (at p e a k h e i g h t ) N
B*ophys B*ophys Acta, 142 (19(77) I i i
I~4,MAN STUDIES OF PURINES AND PYRIMIDINES
5
Raman study o/ Ado-5'-P+Urd and Cuo-5'-P+Cyd mixtures ~n aqueous soluhon Fig. I shows the spectra of Ado-5'-P, uridlne and an equxmolar mixture of Ado-5'-P and urldine in 2H20 solution. It is seen that the observed spectrum of the mixture agrees closely in R a m a n line frequencies and intensities with that obtained from summing the spectra of the constituents. This is clearly evident in the I75o-I45o-cm -1 region (Fig. 2) when the spectra are corrected for changes in filter transmission. { c m 15 1800
1600
1400
1200
IO00
800
600 I
400 - -
200
i
.......
(d)
-
-
(¢)
- - - -
(b)
-
(e)
? t~q /~s
/
iii
,
/"
,i ] ,
-
i! F i g 3 R a m a n s p e c t r a of Guo-5"-P, c y t l d m e a n d G u o - 5 ' - P + C y d s o l u t i o n s (a) o 25 M G u o - 5 ' - P m ~H20, (b) o 25 M c y t l d m e in 2H20, (c) G u o - 5 ' - P + C y d e q m m o l a r m i x t u r e , o 5 ° M t o t a l in ~HzO, (d) s u m m a t m n of (a) a n d (b) S p e c t r a r e c o r d e d a t p2H = 7 5:t: ° 3 a n d 3 5 ± 1 ° S p e c t r u m (c) u n c h a n g e d b y a d d m o n of e x c e s s NaC1 S p e c t r a l s h t - w l d t h i o c m 1 S c a n n i n g s pe e d o 25 c m l/ sec P e r m d 2 see
Fig. 3 shows the spectra of Guo-5'-P, cytidme and an equlmolar mixture of Guo-5'-P and cytidlne in 2H20 solution. The mixture spectrum gives no evidence of frequency changes but large intensity losses (approx. 5o %) are apparent in the lines of the cytldlne component. This can be a t m b u t e d entirely to the fact that the extinction coefficients in the blue for solutions of Guo-5'-P are not zero. Therefore transferring cytidlne from its own solutions (optically transparent near 4358 ~) to the absorbing solutions reduces the relative intensity of its R a m a n scattering significantly*. When a correction is made for this effect, the spectrum of the mixture is found to correspond closely to the summation spectrum, as indicated in Fig. 4 for the I75O-I45o-cm -1 region. Sunllar results are obtained for A d o - 5 ' - P + U r d and G u o - 5 ' - P + C y d systems under varying conditions of observation in both H~O and 2H20 solutions (Table I). If significant spectral changes in the I75O-I45o-cm -1 region occurred in the mixed solutions, these could be attributed to the formation of hydrogen-bonded * T h e i n t e n s i t y of R a m a n s c a t t e r i n g as a f u n c t i o n of s o l u t e c o n c e n t r a t i o n m a n a b s o r b i n g m e d i u m h as b e e n t r e a t e d q u a n t x t a t l v e l y , c~ ref 29.
B~och,m. B,ophys. Acta, 142 (1967) I - i i
0
R. C. L O R D ,
IOO
G. J. T H O M A S , J R
7 -
(b)
~-
-
-
(a}
I i-
t
0
.
.
.
.
1750
WOO
[650
1600 (Crll II
1550
1500
1450
Fig 4 Raman spectra of G u o - 5 ' - P + C y d m the i75o-i45o-cm 1 r e g i o n (a) Spectrum observed for eqmmolar G u o - 5 ' - P + C y d mixture o 2 o M t o t a l m 2 H 2 0 , p 2 H - - 7 5; 35 ° (See t e x t ) (b) Spectrum obtained by adding spectra o f o I o M G u o - 5 ' - P a n d o 1o M cytldme in 2 H 2 0 , p 2 H ~ 7 5, 35 °. Spectral sht-wldth i o c m ~ Scanning speed o o 5 cm-I/sec, P e r i o d 3 ° s e c S / N = i o TABLE
1
PURINE--PYRIMIDINtg
Solutes
SOLUTION
MIXTURES*
Solvent
pH or p~H
Concentration ga~4ge
(total molar) Ad°-5'-P+Urd Ado-5'-P+ Urd Ado-5'-P+ Urd-5"-P Ado + Urd Gu°-5'-P+ Cyd Gu°-5'-P+ Cyd
Guo-5'-P+Cyd-5"-P Guo+Cyd
H20 2H20 HzO 2H20 H2(-) 2H20 2H20 2HzO
7 7 7 2 7 7 7 II
5 5 5 o 5 5 5 o
0 i0--I
0
O IO--O 50
o 5° o 5° o2o o5o 0 IO--I 0
o 5° o 5°
* N o evidence for base-base interactions in all solutions
complexes 1,a°. The absence of any spectral changes here, together with the results of NMR studies u-14 which indicate that at these concentrations the base rings are stacked parallel and one above another ("vertical stacking"), ]mplies that Inter-base hydrogen bonding is absent Aggregates of stacked base pairs would largely exclude water molecules from the immediate environment of the polar ring substituents but aggregates of stacked monomers would not The resulting difference in hydrogen-bonding strength of base-base and base-solvent interactions should then be evident in the I75O-i45o-cm -1 region where frequencies of the coupled C = O stretching and N H deformation vibrations occur ~6. Since no frequency changes are observed it is concluded that no appreciable base pairing accompanies such vemcal stacking as exists in these systems at the concentration levels examined. Bwchzm
Bwphys .dcta, 142 ( 1 9 6 7 ) I - - I I
7
RAMAN STUDIES OF PURINES AND PYRIMIDINES
This is consistent with the w~despread belief that the main source ot stabilization energy of double-hehcal D N A m aqueous solution is provided by forces other than those resulting from the formation of hydrogen-bonded base pairsamm,a",4°. Apparently a necessary requirement for base pairing in aqueous solution is the geometric restriction imposed on the bases by their attachment to the sugar-phosphate backbone of the polymer. i) IOOC (cm
1800
1600
1400
1200
800
600
400
206
I00 1 -
-
(b)
.....
Io)
u~ z 2"
Fig 5 R a m a n s p e c t r a of H , O s o l u t i o n s of c y t l d m e and c y t l d m e + H g C 1 2 (a) o 25 M c y t l d m e ; 5 < p H < i i , (b) o 2 5 M c y t l d m e and o 2 5 M HgC12, p H ~ 5 3 ~ o 2 S p e c t r a recorded at 35 ° S p e c t r a l s h t - w l d t h IO crn -1 S c a n n i n g speed o i o cm-1/sec Period i o sec S / N = 3 °
(cm
1800 I00
t6OO
1400
1200 --
-
•
~1
I000 ]
~
800 T
T
600
4 O0
200
i- -'2,' '--
,,
0
I
I
-
--
Fig 6 R a m a n s p e c t r a of iH~O s o l u t i o n s of cyt~dlne and c y t l d l n e + H g C l 2 (a) o 20 M c y t l d m e , 5 < p*H < i i , (b) o 2 o M c y t l d l n e a n d o e o M HgC12, p~H ~ 5 2 ! o 2 S p e c t r a recorded at 35 ° S p e c t r a l s l l t - w l d t h i o c m 1 S c a n n i n g speed o 25 cln-~/sec Period Io sec S / N = 15 t?~ochzm
Bzophys _dcta,
142 (1967) i - I i
R
C. LORD, G. J. THOMAS, JR.
Rarnan study o/the cyt,dme-HgCl 2 complex I n Fig. 5, R a m a n s p e c t r a of H~O solutions of c y t l d m e a n d of an e q m m o l a r m i x t u r e of c y t l d m e a n d HgC12 are c o m p a r e d over t h e f r e q u e n c y region 18oo-2oo cm 1 C o r r e s p o n d m g s p e c t r a of ~H20 solutions are shown in Fig. 6. The n u m e r o u s changes in line frequencies a n d m t e n s l t i e s m the c y t i d m e s p e c t r u m caused b y HgCI 2 i n d i c a t e t h e f o r m a t i o n of a c o m p l e x The line at 322 cm -1 m the m i x t u r e s is due to undlssocAated HgC12 (ref. 33) b u t Ats i n t e n s i t y in the m i x t u r e s is g r e a t l y d l m i m s h e d from t h a t o b s e r v e d in pure HgC12 solutions at the same concentratAon T h u s a large a n d m e a s u r able a m o u n t of the m o l e c u l a r species HgClo is d i s s o c i a t e d b y the n u e l e o , l d e T h e i n t e n s i t y decrease at 322 cm -A a n d the absence of new lAnes in this f r e q u e n c y region a t t e n d i n g c o m p l e x f o r m a t i o n are consistent w i t h the o b s e r v a t i o n s of others 21,e2 t h a t the specle~ b o u n d to t h e nucleoside AS n o t HgC12 or HgC1 + b u t H g 2+. This is in accord w i t h the fact t h a t no change in the R a m a n s p e c t r u m of c y t l d i n e occurs on the a d d i t i o n of Hg(CN)2, which AS far less d i s s o c i a t e d t h a n HgC12 (ref. 34) u n d e r c o m p a r a b l e e x p e r i m e n t a l conditions. As in the case of the D N A - H g ( I I ) c o m p l e x 2~, the Anteractlon o b s e r v e d here is c o m p l e t e l y r e v e r s e d b y the a d d i t i o n of a sufficient excess of C1- to the solution m i x t u r e s . Thus, for e x a m p l e , w i t h the a d d i t i o n of a 4-fold excess of Cl- An the solution which gives the s p e c t r u m of FAg 5, all of the c o m p l e x is dAssoclated, as e v i d e n c e d b y t h e fact t h a t t h e s p e c t r u m of u n b o u n d cytAdme is recovered. I n additAon the line at 322 cm 1 becomes v a n l s h l n g l y weak a n d new lines a p p e a r a t 285 a n d 270 cm 1 a t t r i b u t a b l e r e s p e c t i v e l y to the m o l e c u l a r species H g C l f a n d HgCI~(ref 35-37) l:Ig~. 5 a n d 6 show t h a t t h e largest p e r t u r b a t i o n s in t h e s p e c t r u m of c y t l d m e occur in the I~OO-I55O- a n d 13oo 12oo-cm -1 regAons where r e s p e c t i v e l y rAng doubleb o n d a n d single-bond s t r e t c h m g v i b r a t i o n s h a v e been assigned 16. Moreover, for b o t h H~O a n d 2H20 solutions, the b m d m g of H g ( I I ) t o c y t l d i n e causes q u a h t a t l v e l y slmAlar s p e c t r a l shAfts T h u s a r e m a r k a b l e m t e n s i t y reversal occurs in the pair of lines n e a r 13oo a n d 125o cm -1 wAth shifts of a b o u t lO cm ~ in the lAne centers, a n d t h e r e is a large i n t e n s i t y m c r e a s e n e a r 165o cm ~ wAth an i n t e n s i t y decrease n e a r 162o cm -~ An b o t h solutions. The characteristAc c y t o s m e rAng frequencies near IOOO c m ~ are also shifted some 15-25 cm -~ to higher f r e q u e n c y in b o t h solutAonb These effects are best e x p l a i n e d b y assuming t h a t bAnding of H g ( I I ) occurs at t h e a v a d a b l e N-3 r a g posltAon. If i n s t e a d H g ( I I ) r e p l a c e d a p r o t o n a t the e x t e r n a l a m i n o group 24 it w o u l d be dAfficult to e x p l a i n the s p e c t r a l changes An t h e I3OO-I2OO-Cm -1 region of H20 solutAons a n d in the I8OO-i5oo-cm -~ region of 2 H 2 0 solutions On the o t h e r hand, b i n d i n g at the N- 3 position would be e x p e c t e d to have a p r o n o u n c e d effect on the x - e l e c t r o n d i s t r i b u t i o n of the h e t e r o c y c h c ring, t h e r e b y alterAng b o t h the C - - N a n d C - N s t r e t c h i n g frequencAes a n d a c c o u n t i n g for the slgnlfAcant a n d closely almilar s p e c t r a l change~ n o t e d in b o t h H 2 0 a n d ~H20 solution s p e c t r a A t t a c h m e n t of t h e m e r c u r y catAon to t h e nucleoslde i n d e e d produces m u c h the s a m e s p e c t r a l change as t h a t of protonatAon (~ c a t t a c h m e n t of H + or 2H+), as one can see b y c o m p a r i n g t h e s p e c t r a of FAgs 5 a n d 6 wAth the s p e c t r a of p r o t o n a t e d c y t l d l n e ~. T h u s for b o t h m e r c u r a t e d a n d p r o t o n a t e d c y t i d i n e there is a shift to hAgher f r e q u e n c y of t h e t o t a l i n t e n s i t y in the d o u b l e - b o n d region (18oo-15oo cm -~) a n d a shift to lower f r e q u e n c y of t h e t o t a l i n t e n s i t y in the single-bond region (13oo12oo cm 1). This IS Anterpreted as a "fAXAng" of the double a n d sAngle b o n d s of the ring. Bzochzm
B~ophy~ Acta, 142 (1007) i I i
RAMAN
STUDIES
OF
PURINES
AND
9
PYRIMIDINES
Since the present data indicate that all the perturbations in the cytidine spect r u m increase monotonically with increasing molar ratio of HgCI 2 to cytidine, it is concluded further that binding occurs only at the N-3 ring position under the present experimental conditions. The binding of H g ( I I ) to cytldine m a y be further treated by considering the following equilibria*. HgC12 + Cyd = HgCyd e+ + 2 C1-
K1
(I)
HgCyd 2+ + Cyd = Hg(Cyd)22÷
K2
(2)
Binding of more than two cytidine molecules to H g ( I I ) is neither sterically feasible nor consistent with the results of previous investigatlonsel, 22,24. In the following discussion equlhbria involving hydrolysis of Hg 2+ are neglected. This is a good approximation since the present data indicate that the equilibrium of Eqn. I lies well to the right and therefore at the p H values of the present measurements (5 G p H G 6) appreciable hydrolysis of Hg z+ IS prohibited aS,a~. The integrated intensity of the line at 322 cm -~ gives a reliable value of the concentration of HgC12 remaining undissociated, and therefore unbound to cytidine. [C1-] is approximated b y [HgCI~] 0 2 ~HgC12]. One can see 111 Figs. 5 and 6 that the stronger R a m a n hnes of cytldme and "complex" overlap, so that independent measurements of their respective line intensities (and therefore concentrations) cannot be obtained with high accuracy. Therefore in solutions with [HgC12] 0 << [Cyd]0, where the 322-cm -1 line is vanishmgly weak, the relative concentrations of cytldme and complex can be obtained from the total R a m a n Intensity at some frequency v where the R a m a n intensities of bound and unbound cytldine differ greatly. The scattering (I~) due only to cytldine can be P can be approxobtained from solution spectra of cytidlne, that due to complex (ox) iinated b y the intensity in the solution containing [HgC12]0 = 5[Cyd]0 = 0.25 M TABLE
II
E Q U I L I B R I U M DATA OF C y d - M g C 1 2 SOLUTIONS (r)
(2)
(3)
(4)
(5)
(6)
(7)
[HgClz]o a
ECyd]o
IHgC12]b
[Cyd]e
[Cyd] a
Kle
Ka r
(average)
o 25 o 25 o25 o 05 o 125 o 05 o o5 oo 5 o 05
o 05 o 125 o25 o 05 o 25 o io o 15 o2o o 25
o 19o o 126 oo625 o 0237 o o179 o o13o o o4 ooo2 o
(o) o o18 o lO 7
(o) o 035 o 12o o 022 o 14o o 068 o ioo o 153 o 203
-o 31 o61 o 44 o 59 o 5° o 28 o46 --
-o 73 o75 o 37 o 5° o 57 o 22 o35 --
-o 52 o68 o 41 o 55 o 54 o 25 o4 I --
a b e a e r
o 020 o o o o o
13o 065 092 143 2oo
S o l u b i l i t y h m l t = o 25 M a t 3 5 ° A l l m e a s u r e m e n t s F r o m 3 2 2 - c m -1 h n e i n t e n s i t y Eqn 5, v = 1 2 5 3 c m -1 Eqn 5, v = 1 2 7 o c m - t . Data of Columns ( I ) , (2), (3), (4). D a t a o f C o l u m n s ( i ) , (2), (3), (5)
* F o r t h e m o l e c u l a r s p e c i e s z, ~z]0 a n d [z] d e n o t e t r a t i o n s , w h e r e z m a y b e H g C 1 z, C y d , C1 o r c o m p l e x
were
made
a t 35 °.
the initial and eqmhbrlum
t3,och*m
B~ophys
K1
molar concen-
./lcta, 142 ( 1 9 6 7 ) i - I I
io
R.
c
LORD,
G.
J.
THOMAS,
JR.
where nearly all the cytidme is bound (Table II, above). The total intensity at v is then 2+
v
/total-- [CydlI~ + [UgCyd2+]I;x + 2[Hg(Cyd)2 llcx
(3)
where it is assumed that both complexes have the same scattering intensity per molecule of bound cytldlne. From Eqns. 1 and 2 [Cyd]0 = [ C y d l + [HgCyd 2+] + 2[Hg(Cyd)22+]
(4)
and from Eqns. 3 and 4 [Cydl --
/~otal - -
I~
-
-
[Cyd]0 I~x
(5)
I~x
The quantity [Cyd] was calculated from Eqn. 5 at v = 1253 and 127o cm -1. The results are presented in Table II. It is seen that in the solutions containing excess HgC12, the amount of HgC12 "removed" by binding to cytidine, as determined from the 322-cm -~ hne area, is very nearly equal to the amount of cytldine initially present, z.e. only a I : I binding ratio is suggested. In solutions containing excess cytldme, the amount of cytldlne removed by binding is also equal to the initial concentration of HgC12, within experimental uncertainty. Therefore, over the entire concentration range studied A [HgC12] - [ngCl~] 0 -- [HgC121 A [Cydl
[Cyd]0 -- [Cydl
and it is concluded that only a 1:1 complex, HgCyd 2+, is formed z.e K 1 >> K 2. The apparent equilibrium constant*,/£1, is defined as ]k.1 __ [HgCyd 2+] [C1 ~ 2 [HgC121 [Cydl
([Cyd]0_[Cyd])([HgCl~]0_z[HgC12])2 ~
[HgC12] [Cyd]
(EHgCl~10--[HgC12]) ([HgC12]0-- 2 IHgCl2~)~ [HgC12~ [Cyd] and is calculated as indicated in Table II. A mean value of K~ =: (5o:£2o]" IO-2 M is obtained, the devmtion being largely due to the difficulty m obtaining accurate Raman intensities an dilute solutions (o 05 M in HgC12 or cytidme) The system cytidme HgCI2 was chosen because of its suitabdlty to a Raman study. Although complexes are readily formed between HgC12 and derivatives of adenine, guanine and lnoslne, under comparable experimental conditions 22,24, these are not soluble at the concentration levels reqmred to obtain Raman spectra of satisfactory quahty Derivatives of uracil, including uradme, I-methyluracfl and 1,3-dlmethyluracil, gave no interaction with HgC12 up to the addition of a 5-fold excess of the salt. This ~s consistent with the conclusion reached earlier that strong binding occurs preferentially at unsaturated nitrogen sites EICHHORN AND C L A R K 21 have observed the urldme-HgC12 interaction under considerably different experimental condmons, with a 32-fold excess of the mercury * No attempt
was made
B~ochzm B~ophys. Acla,
142
to correct (1967)
i-iI
for actlvltms
RAMAN STUDIES OF PURINES AND PYRIMIDINES
II
salt and at pH 9. Under such conditions, replacement of the imino N-3 proton of uracil by Hg z+ is greatly favored. It is concluded therefore that the interaction observed here between Hg 2+ and cytldine is very different from that expected at high pH. Salts which cause no change in the aqueous Raman spectra of either cytidme or uridine are ZnC12, ZnSO a, CdC12, CuSO, and MgCI 2. AgN03 gives spectral evidence of interaction only with cytidine. These results are inconclusive, however, since detailed analysis of solutions of Ag + is prohibited with mercury-arc sources, which cause rapid photodecomposltion. In view of the recent interest in the polynucleotldeAg + interaction26, 2r, it would appear worthwhile to examine such systems using a laser-source Raman instrument.
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