A calorimetric study of the helix-coil transition of poly(cytidylic acid) in acid solution

Biochimica et Biophysica Acta, 383 (1975) 1--8

© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 98234

A C ALOR I METRI C STUDY OF T H E HELIX-COIL T R A N S I T I O N OF POLY(CYTIDYLIC ACID) IN ACID SOLUTION

H. KLUMP Institut fi~r Physikalische Chemie der Universitiit Freiburg, Freiburg i. Br. (G.F.R.)

(Received August 12th, 1974)

Summary In this study we show how the thermal stability of the helical structure of poly(C) is d e p e n d e n t on the degree of p r o t o n a t i o n . A H values for the thermal denaturation are measured directly by means of an adiabatic microcalorimeter in the pH range fr om pH 5.8 to 3.7. The m a x i m u m transition t e m p e r a t u r e Tm is f o u n d at pH 4.5 where poly(C) is semiprotonated. At the same pH value a m a x i m u m value of A H (AH = 5.27 Kcal/mol base pairs) was obtained. AH decreases slightly if the pH changes to lower or higher pH values. No transition was observed either at pH 6.0 or at pH 3.4. The A G ~ ( 2 5 ° C ) v a l u e s show a similar d e p e n d e n c e on the degree of p r o t o n a t i o n of poly(C).

Introduction

X-ray diffraction studies on oriented fibres of poly(C) [1] show t hat this p o l y m e r exists in a helical c o n f o r m a t i o n below pH 5.8. The t w o p o l y m e r chains are wrapped around each ot he r in parallel configuration in which t h e y are related by a t w o f o l d r ot a t i on axis. An unusual t y p e of hydrogen bonding is postulated as being responsible for holding the two chains together. In order to achieve this t y p e of hydrogen bonding one of the t w o cytosine rings must be p r o t o n a t e d . In this c o n f o r m a t i o n the base pairs are held t oget her by a set of three hydrogen bonds between the cytosine bases in the center of the molecule. Results of acid-base titration, infrared and ultraviolet spectroscopy, and thermal d en atu r atio n studies are presented, f r om which t he authors conclude that the molecule assumes a c o n f o r m a t i o n in solution which is very similar to that fo u n d for the solid state [ 3 ] . Thermal denaturation studies are r e p o r t e d by several authors [ 4] . The corresponding result is that the transition t e m p e r a t u r e (Tin) is a f u n cti on of pH and at given pH a function of t he ionic strenght a n d / o r the species of cations (see Fig. 1). There is no transition at a pH value higher than 5.7 (in 0.15 M NaC1) or lower than 3.0. In the intermediate pH

I

Trn

x~x~X ~

?O

6ok 5C 40 3E 2C ]C 0 3.5

I

~

,

,

I

i

4.0

4.5

Fig. 1. T r a n s i t i o n

temperatuxe

I

,

,

~

I

5.0

~

L

L

,

I

L

,

5.5

T m v e r s u s pI-I, p o l y m e r

L

,

I

6.0 pH concentration

N a C l (x) a n d 1 - 1 0 -2 M Mg a c e t a t e (©,/~). B u f f e r c o n c e n t r a t i o n

4 - 10 -3 M base pairs. 1 - 10 -2 M

1 • 10 -3 acetic acid/acetate.

range Tm increases sharply f r om a b o u t 30 to 80°C at pH 4.5 and decreases again to 40 °C. Increasing ionic strength or the presence of divalent cations in the solution decreases the transition t e m p e r a t u r e in general, and shifts the Tm m a x i m u m to lower pH values. It is assumed t hat the T m is proportional to the helix stability. To prove this statement, direct measurements of the transition enthalpy of helical poly(C) in the pH range 5.7 to 3.8 have been performed with the help of an adiabatic scanning calorimeter. Material and Methods Poly(C) was purchased from Miles Chemical Corporation, Elkhard/Ind., and was used w i t h o u t further purification. After each calorimetric measurement the solution was exhaustingly dialysed against deionized water and the polynucleotide was lyophilized before reuse. It was dissolved in 0.001 M acetate buffer, pH 6.5, to give a p o l y m e r c onc ent rat i on of about 2.8 mg per ml. To adjust the desired pH and ionic strength acetic acid a n d / o r NaCl was added. The ionic strength was always 0.01 M Na ÷. pH was measured by means of a R a d i o m e t e r 22 pH-meter to 0.01 pH units. Calibration of the pH-meter was p e r f o r m e d with standard buf f er solutions (Radiometer, Copenhagen). The concentration of the poly(C) solution was determined by a gravimetric phosphorus analysis [ 5 ] . Precisely weighed amounts of the poly(C) solutions used during the calorimetric measurements were digested at about 100 °C with 70% HC104. The f o r m e d inorganic phosphate was precipitated with N,N,N'~N'tetracis(2-hydroxypropyl)-ethylenediammonium~liperchlorate. The analysis yields data on weight per weight basis. The standard deviations a m o u n t e d to about 1% of the mean.

Thermal denaturation profiles Thermal d e n a t u r a t i o n profiles were r e c o rded at h = 275 nm with a Hitachi Perkin Elmer Mod. 124 double beam s p e c t r o p h o t o m e t e r fitted with a temperature controlled cell holder. The t e m p e r a t u r e was controlled by a Lauda Kryo-

mat to 0.05°C and measured by a copper-constantan t h e r m o c o u p l e . The thermovoltage was transmitted to the X-axis input of a Hewlett-Packard XY-recorder. The e x t i n c t i o n signal was transmitted to the Y-axis input of the recorder. For each sample the change of the absorbance as function of temperature was r eco r d ed as well as spectra of the solutions at t em perat ures below and above the helix coil transition were recorded. The heating rate was a b o u t 3°C per min.

Titration experiments The titration measurements were carried out on solutions which contained 3.6 mM of cytidylic acid and 0.01 M of NaC1. The titration curves were obtained by adding aliquots of 0.001 M HC1 to the solution with a microburet. The solution was stirred, and the t e m p e r a t u r e was maintained at 25°C. The pH measurements were made with a R adi om e ter 22 pH meter. The pH measurements were also p e r f o r m e d as a function of t e m p e r a t u r e to show w het her there is any d e p r o t o n a t i o n during the thermal denaturation (see Fig. 3). Calorimetric measurements Calorimetric measurements were per f orm ed with the help of an adiabatic scanning calorimeter designed by M. G r ube rt [6]. The details of this instrument are given elsewhere. T he molar ent hal py change A H was c o m p u t e d f r o m Ah/mol. wt base pair. All enthalpy measurements were p e r f o r m e d twice. The overall u n cer tain t y of the apparatus is estimated to be close to +2%. Results Fig. 2 shows the results of the titration studies. In the lower part of the

7

I 6

I 5

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pH

1 3

D

F i g . 2. U p p e r p a r t : p a i r i n g s c h e m e o f p o l y ( C ) • p o l y ( C ~ ; l o w e r part: a c i d - b a s e t i t r a t i o n c u r v e o f p o l y ( C ) i n 0 . 1 M NaC1 a t 2 5 ° C . P o l y m e r c o n c e n t r a t i o n 3 . 6 - 1 0 -3 M b a s e p a i r s .

Poty C pH 5.0 27 ? nm

>-.

0.48 O.4Z,

L

0.40 Tm =?O°C

0.36 50

610

i

70

8~0 (°C)" PoLy C

'pH 4.4 4.6 4.8 5.0

50

610

710

810 (°C)*'

F i g . 3. U p p e r p a r t : u l t r a v i o l e t a b s o r p t i o n a s a f u n c t i o n o f t e m p e r a t u r e i n 1 • 1 0 -1 M NaC1 ( n o b u f f e r ) . Polymer concentration 1 - 1 0 --4 M b a s e p a i r s . L o w e r p a r t : p H v e r s u s t e m p e r a t u r e o f t h e s a m e p o l y ( C ) solution.

figure the titration curve of poly(C) in 0.01 M NaC1 is shown. This curve corresponds to the results of Hartman and Rich [2]. It is clearly d e m o n s t r a t e d that p r o t o n a t i o n occurs in a two-step process. At pH 5.7 there is an abrupt uptake of p r o to ns by the p o l y m e r w i t hout any resulting change in the pH of the solution. Simultaneously the p o l y m e r c o n f o r m a t i o n changes from a rand o m l y coiled to a helical state. The change of the pH of the solution increases again when the degree of p r o t o n a t i o n increases from 0.4 to 0.7. This degree of p r o t o n a t i o n is reached at pH 3.0. There is a n o t h e r abrupt upt ake of protons w i t h o u t a resulting change in pH accompanied by a helix r a n d o m coil transition of the p o l y m e r until full p r o t o n a t i o n just below pH 3.0. At pH 4.5 one p r o t o n per base pair has been taken up (i.e. the degree of p r o t o n a t i o n is 0.5 per nucleotide) so that at this point the n u m b e r of hydrogen bonds f o r m e d in the helical structure should be the greatest possible. Fig. 3 (lower part) shows the change of pH as a f unct i on of t e m p e r a t u r e t h r o u g h o u t t he transition interval of a poly(C) solution. In the upper part of Fig. 3 the ultraviolet-absorbance is shown as a f u n cti on of t e m p e r a t u r e in the corresponding t e m p e r a t u r e interval. If a d e p r o t o n a t i o n occurs during the de na t u rat i on a deviation of the p H / t e m perature plot f r o m a straight line should be seen. In contrast to the proposal in [2 ], no such d e p r o t o n a t i o n was observed.

Calorimetric and spectrophotometric data on the denaturation of poly(C) in the pH interval from pH 5.78 to 3. 70. The ultraviolet absorption of poly(C) as a function of t em perat ure was measured at 275 nm. The t e m p e r a t u r e corresponding to half the final increase

TABLE I

No

Tm (o C )

1 2 3 4 5 6 7 8

40 63 75 74 72 61 54 47

pH

AH* (Kcal/MBP)

AS** (eu)

AGe(Kcai/MBP)

3.68 4.33 4.48 4.55 4.85 5.20 5.50 5.76

4.06 5.20 5.25 5.27 5.12 4.95 4.34 3.62

12.97 15.48 15.09 15.14 14.89 14.82 13.27 11.31

0.195 0.588 0.755 0.744 0.690 0.533 0.385 0.249

* Mean value of two measurements ** C a l c u l a t e d a c c o r d i n g t o A H I ( T m + 2 7 3 )

of the absorbance at 275 nm is the transition temperature Tin. In Fig. 1 the course of the Tm versus pH function is plotted for two different solutions. The triangles and dots represent a solution which contains 0.001 M acetic acid/acetate and 0.01 Mg • acetate, the crossbars represent a solution 0.001 M acetic acid/acetate and 0.01 M NaC1. The polymer concentration in all solutions was 0.001 M poly(C) base pairs. The pH value was adjusted by adding concentrated acetic acid. The pH dependence of the T m is considerably influenced by the species of the actual cations. The m a x i m u m Tm for the Na+-containing solution is 76°C and pH 4.65, the m a x i m u m Tm for the Mg2÷-containing solution is 64°C and pH 4.5. The decrease of Tm at both sides of the m a x i m u m is more pronounced for the further solution. However in both cases the m a x i m u m value of Tm is at about pH 4.5. Table I contains data concerning the variation of the transition enthalpy AH, and the transition entropy AS, with changing 12 (t)

PoLy C pH 4.&8

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F i g . 4. E x p e r i m e n t a l t r a n s i t i o n c u r v e s o f p o l y ( C ) U p p e r p a r t : concentration: 1 . 1 5 - 10 -4 M base pairs in 25 ml. L o w e r p a r t : t e m p e r a t u r e i n 1 • 1 0 -2 M NaCI 1 • 10 -3 M acetate.

calorimetric transition curve. Polymer u l t r a v i o l e t a b s o r p t i o n as a f u n c t i o n o f

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pH

Fig. 5. T r a n s i t i o n e n t h a l p y A H ( l e f t h a n d o r d i n a t e ) versus p H a n d free e n t h a l p y A G e ( r i g h t h a n d o r d i n a t e ) versus p H p o l y m e r c o n c e n t r a t i o n 4 - 1 0 - 3 M b a s e pairs in 1 • 1 0 - 2 M NaCI a n d 1 • 1 0 - 3 M a c e t a t e . ( A ) A H , (o) AG °.

pH. The maximum AH (see Fig. 5) value of 5.27 Kcal/mol base pair is measured at pH 4.5. Fig. 4 shows in the upper part an experimental transition curve from the calorimetric measurements and the corresponding spectroscopic transition curve in the lower part. Discussion Although the presented spectral and denaturation data is largely in agreement with that of Guschlbauer and of Ts'o and co-workers, a different interpretation of these results appears reasonable and will be submitted here. The course of the protonation of poly(C) can be devided into four consecutive steps: +H

Step 1: single-stranded poly(C),

+

' protonated single-stranded poly(C)

In the pH interval from pH 7.0 to 5.7 until an uptake of about 0.4 mol HC1 per mol nucleotide the native randomly coiled poly(C) becomes protonated without a major change" in the secondary structure of the polymer. Step2: poly(C)

protonated

single-stranded

poly(C)

~

protonated

double-helical

Just below pH 5.7 a coil-to-helix transition occurs when the degree of protonation exceeds 0.4 per mol nucleotide. +H

Step 3: protonated poly(C)

double-helical poly(C) ,

+

' protonated

double-helical

In the pH interval from pH 5.6 to 3.1 additional protons are bound by the

7 poly(C) helix leading to a stabilization of the secondary structure until pH 4.5 when the degree of protonation reaches 0.5, and leading to a destabilization of the helical conformation below pH 4.5. H +

Step 4: protonated double-helical poly(C) ( 0 . 7 / 1 . 0 ) ~ - p r o t o n a t e d poly(C)

coiled

Close to pH 3.0 there is another uptake of protons without a change of the pH value. The solution of poly(C) becomes turbid, suggesting that the polymer molecules become insoluble when t h e y are fully protonized. This protonation scheme is based on the results of our titration studies as well as the results of Hartman and Rich. Up till now the hydrogen-bonding scheme suggested by March et al. [1] for cytosine-5-acetic acid in the crystal form was accepted for poly(C) in solution. Akinrimisi, Sander and Ts'o [3] presented three lines of evidence suggesting that poly(C) in acidic solution asssumes the conformation of a regular, compact and perhaps helical structure. Comparable changes in physical and optical properties have been observed by Hartman and Rich [2] and by Guschlbauer [4]. All of them apply the same pairing scheme to the cytosine residues. They assume that the proton is solely bound to the N1 -position of the pyrimidine ring (see Fig. 2). Even if there are no obvious spectral data to support a partial protonation of the N~-position of the pyrimidine ring, some electrostatic and structural aspects suggest that this aspect should be taken into consideration. At pH 5.7 there is an abrupt uptake of protons onto the polymer without any change of the pH of the solution. At the same time a cooperative structural transition involving the protonated residues takes place. The polymer changes from a randomly coiled to an ordered helical state. The protons are arranged close to the helix axis while the bases form three hydrogen bonds per base pair and stack on each other. The distance between two protons is about 4 A. That would cause a strong repulsion of charges. The protonated cytosine residues will show less tendency to stack than will the unprotonated cytosine residues. The helix is formed when four out of five base pairs are protonated. It is difficult to explain how a solvated proton could get up to the N1-position of the pyrimidine ring near the twofold helix axis when the protonation goes on during the titration. But if we assume an equilibrium between the protonated imino and amino tautomers of cytosine (a), it is easy to present a pairing scheme with three hydrogen bonds where the protons are bound to the outside of the helix (b). H

H

®

H

H

~N~ ~7

il ~ HC4

~1 3 N ~ t R

~

"

7/

Ii

MC--

C

N --

C

R

R/

%

/

\\o--- . . . . .

.\\

//c" C --

H-N

/ \

a

R

C--N

/"-" .......

.c\

3 / 4 CH

NI

~O

N-H ......... 0

\N1~7

I O~ C 2

2C

H

H

CH

The repulsion between the protons is reduced if the protons are bound in the N7-position because of their enlarged distances and a further uptake of protons is eased because this position is far more accessible from the solution. The assumption of the imino-amino-tautomerism is not supported by the results of proton magnetic resonance spectroscopy or any other experimental technique so far, but perhaps not enough care has been given to this subject. Form the results of the calorimetric and titration measurements one can conclude that there is no deprotonation during the thermal denaturation at pH 5 and no protonation at pH 4, for the curve of the A H values versus pH is nearly symmetrical to pH 4.5. Additional enthalpy changes associated with a change in the degree of protonation of the r a n d o m l y coiled poly(C) will lead to a change of the s y m m e t r y of the H versus pH function, for the change of the protonation of the buffer system would also result in an enthalpy change. This result is even more clearly shown by the plot of the pH versus temperature as shown in Fig. 3. The computation of the A G ~ values from a method developed by Privalov [8] and co-workers for the computation of the change of the free enthalpy of DNA under similar conditions. AG(To) = AH(Tm)" [ ( T i n -

To)/To]

Any additional protonation or deprotonation will influence the slope of AG~ as a function of the pH. Since the curve is symmetrical around pH 4.5 it only depends on the number of hydrogen bonds involved in the helix coil transition of poly(C) in acid aqueous solution. Acknowledgements I wish to thank Professor Dr Th. Ackermann for valuable advice and the Deutsche Forschungsgemeinschaft for generous support of this research program. References 1 2 3 4 5 6 7 8

March, R.E., Bierstedt, R. and Eichhorn E.L. (1960) Acta CrystaUogt. 15, 310--314 H a x t m a n , K . A . a n d R i c h , A . ( 1 9 6 5 ) J. A m . C h e m . S o c . 8 7 , 2 0 3 3 - - 3 0 3 9 A k i n ~ i m i s i , E . O . , S a n d e r , C. a n d T s ' o , P . O . P . ( 1 9 6 3 ) B i o c h e m i s t r y 2, 3 4 0 - - 3 4 4 G u s c h l b a u e r , W. ( 1 9 6 7 ) , P r o c . N a t l . A c a d . Sci. U.S. 5 7 , 1 4 4 1 - - 1 4 4 8 A s m u s , E. a n d B a u m e r t , H.P. ( 1 9 6 8 ) Z. A n a l . C h e m . 2 5 2 - - 2 5 7 G r u b e r t , M, ( 1 9 7 4 ) , Z. P h y s i k . C h e m . , N e u e F o l g e , in p r e s s G r u e n w e d e l , D.W. ( 1 9 7 4 ) B i o p o l y m e r s , in press P r i v a l o v , P . L . P t i t s y n , O . B . a n d B i r s h t e i n , T.M. ( 1 9 6 9 ) B i o p o l y m e r s 8, 5 5 9 - - 5 7 1