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Calorimetric measurements of the transition enthalpy of DNA in aqueous urea solutions
601
Biochimica et Biophysica Acta, 4 7 5 ( 1 9 7 7 ) 6 0 1 - - 6 0 4 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
BBA 98908
CALORIMETRIC MEASUREMENTS OF THE TRANSITION ENTHALPY OF DNA IN AQUEOUS U R E A SOLUTIONS
H. K L U M P * a n d W. B U R K A R T
Institut fiir Physikalische Chemie der Universitb't Freiburg, Freiburg (G.F.R.) (Received October 8th, 1976)
Summary • The helix-coil equilibrium of DNA is delicately solvent. In this investigation the helical secondary an 'increasing concentration of urea. We found transition enthalpy AH on the urea concentration as for salmon sperm DNA.
affected by the nature of the structure was destabilized b y a linear dependence of the for calf thymus DNA as well
Introduction
An extensive survey of the relative denaturing p o w e r of various organic solvents was reported by Lewin et al. [ 1 ], who measured the concentration of organic solvent necessary to bring a b o u t stated conditions of denaturation at a fixed temperature. No direct correlation was found between denaturation effectiveness and monofunctional hydrogen-bonding capacity, bifunctional hydrogen-bonding capacity, the hydrophobic character or alterations in solvent polarity for these denaturants. This paper is concerned with the direct measurement of the transition enthalpy AH on calf thymus DNA and salmon sperm DNA as a function of urea concentration in neutral aqueous solution. Material and Methods Calf thymus DNA and salmon sperm DNA were supplied by Worthington Biochem. Corporation and were used without further purification. All other c o m p o u n d s were of reagent grade. DNA solutions were prepared by first dissolving the lyophilized product in quartz-distilled water and than adding the required amounts of salt to give a neutral buffered aqueous solution, before adding the calculated a m o u n t of urea to give the final urea concentration for * To whom all correspondence should be addressed.
602 the individual calorimetric measurements. A solution with identical salt and urea concentrations was used as a blank, pH measurements were performed with the help of a Radiometer Mod. 22 pH meter. Details of the calorimeter apparatus are given elsewhere [2]. All optically measured denaturation curves we're recorded by a Pj?e-Unicam Sp 1800 spectrophotometer at 260 nm as a function of temperature. Solutions for spectrophotometric measurements were usually prepared by diluting microliter quantities of the DNA stock solutions with the appropriate salt and urea solvent. The concentration of the DNA solutions was determined via a colorimetric phosphorous analysis. Results and Discussion
When heating native calf thymus DNA at pH 7.5 in buffered aqueous solution without or with urea, the results displayed in Figs. 1 and 2 are obtained. The curves show the temperature course of the electrical compensating energy needed to maintain adiabatic conditions in the calorimeter and the heat denaturation profiles obtained by plotting the absorbance increase of the DNA solution at 260 nm against temperature. In Fig. 1 the salmon sperm DNA and calf t h y m u s DNA had been dissolved in quartz-distilled water and the final solution contained 0.01 M NaC1 plus 1 mM sodium citrate, pH 7.5, whereas in Fig. 2 the solution contained 5 M urea in addition to the salts. The temperature at which the heat absorption peak has its major maximum is the transition temperature
Tin. It is readily seen that it is the same temperature pertaining to the 50% point of hyperchromicity at 260 nm. It is evident that the temperature course of the t w o parameters signifies the transition of the DNA from the native to the randomly coiled state. As expected, the thermal stability of salmon sperm DNA and calf thymus DNA decreases with increasing urea concentration (cf. Fig. 3). In the case of calf thymus DNA, heat absorption peaks were ordinarily quite asymmetric a b o u t Tm and the curves are structured exhibiting at least two additional maxima. The secondary maximum in the compensating energytemperature curve can be associated with the well-known compositional hetero-
%~ 80
!
100
.q
,5 -6o 40 ~o
~
i
55
i
60
i
65
i
J
i
70
75 Ternperoture
,..
[°C 80
]
Fig. 1. H e a t - i n d u c e d helix-coil t r a n s i t i o n c u r v e s o f c a l f t h y m u s D N A ( ) and salmon sperm DNA (. . . . . . ) in 0.01 M NaC] plus 1 m M s o d i u m c i t r a t e b u f f e r , p H 7.5. L e f t - h a n d o r d i n a t e a b s o r b a n c e c h a n g e (o o, calf t h y m u s D N A ; o, s a l m o n s p e r m D N A ) ; r i g h t - h a n d o r d i n a t e c o m p e n s a t i n g e n e r g y , N C (t).
•,
603
~ t 100 %
:7 8°
i
/,5
50
55
60
65 68 Temper ot ure [ ° C I
Fig. 2. H e a t - i n d u c e d h e l i x - c o g t r a n s i t i o n c u r v e s o f c a l f t h y m u s D N A a n d s a l m o n s p e r m D N A in 0 . 0 1 M NaCI p l u s 1 m M s o d i u m c i t r a t e b u f f e r p H 7 . 5 / 5 M u r e a . Left-hand ordinate, absorbance change: (o o, c a l f t h y m u s D N A ; • ~, s a l m o n s p e r m D N A ) ; r i g h t - h a n d o z d i n a t e , compensating energy N C (t).
geneity of calf thymus DNA. The relative positions o f these maxima are not dependent on the urea concentration. This could indicate that the stability of G • C base pairs in equal affected by urea as the stability of A • T base pairs. Table I shows that the entropy of denaturation, calculated using the Gibbs. Helmholtz equation, also varies with urea concentration. Both A3 and AH are positive in sign as the transition proceeds from ordered to the disordered structure of the macromolecule. The slope dTm/dC urea = 2.5 ° . The interdependence of AH, AS, and Tm with urea concentration can only be described in purely phenomenological terms. The implication is that urea interacts preferentially in solution with the bases, and it is able to do this more affectively with coiled form. The question of specific binding (through use of a binding constant Ks) vs. nonspecific solvent effects implied by the use of the corrected Setschenow [3] constants ks is left open [4,5]. If the effective number of binding sites per m o n o m e r unit ~ is taken as independent of the urea concentration, the binding constant can be calculated as a function of the urea concentration. Assuming a value of 1.3 for ~ [5] the following equation yields the values listed in Table I.
7.5 m s-
7.0
,,," 6.5 -r ,,~ 6.0 5.5 0
1
2
3
4
5
6
M ureo m
Fig. 3. P l o t o f t h e t r a n s i t i o n e n t h a l p y A H vs. u r e a c o n c e n t r a t i o n m, c a l f t h y m u s D N A ; o, s a l m o n s p e r m D N A ; M B P , melted base pai~.
6O4 TABLEI Urea c o n c e n t r a t i o n (mol/1)
AH (kcal/melted base pair)
AS (cal/degree per mol)
K B - 102
Tm (o C)
1 2 3 4 5 6 --
7.000 6.750 6.500 6.125 5.750 5.450 7.500
20.86 20.34 19.60 18.62 17.64 16.82 22.06
2.97 2.88 2.97 2.65 2.50 2.39 --
63.5 61.0 58.5 56.0 53.0 51.0 66.0
KB = (ATm
"
Ah / R T m T m
o. P)/as
where KB is the association or binding constant of the denaturant with the average monomer unit, ATm is the lowering of the transition temperature of the biopolymer due to the binding of interaction of the denaturing agent with its denatured form [6], Ah is the experimental transition enthalpy per monomer unit of the polymer under study, Tm and Tm° are the midpoints of the denaturation transition in the presence and absence of denaturant and as is the activity of the denaturant, assumed to be equal to its concentration [7,8]. The binding constant decreases slightly with increasing urea concentration. The results of this or other studies provide no evidence that hydrogen bonding between urea and DNA is the main contribution to the denaturation effect. Acknowledgement W e thank Professor T. Ackermann for support and encouragement during these investigation. This work was supported by the Deutsche Forschungsgemeinschaft.
References 1 2 3 4 5 6 7 8
Levine, L. et al. ( 1 9 6 3 ) B i o c h e m i s t r y 2, 1 6 8 - - 1 7 5 G r u b e r t , M. and A c k e r m a n , T h . ( 1 9 7 4 ) Z. P h y s . C h e m . 9 1 , 2 5 5 - - 2 6 4 L o n g , F . A . a n d M c D e v i t ( 1 9 5 2 ) C h e m . Rev. 5 1 , 1 1 9 H e r s k o v i t s , T.T. and Harrington, J. ( 1 9 7 2 ) B i o c h e m i s t r y 1 1 , 4 8 0 0 H e r s k o v i t s , T.T. and B o w e n , J . J . ( 1 9 7 4 ) B i o c h e m i s t r y 1 3 , 5 4 7 4 - - 5 4 8 3 Pellet, L. ( 1 9 5 9 ) J. P h y s . C h e m . 6 3 , 1 1 9 9 S e h r i e r , E . E . , I n g w a l l , R . T . and Scheraga, H . A . ( 1 9 6 5 ) J. P h y s . C h e m . 6 9 , 2 9 8 E l b a u m , D. and Herskovits, T.T. ( 1 9 7 4 ) B i o c h e m i s t r y 1 3 , 1 2 6 8
Biochimica et Biophysica Acta, 4 7 5 ( 1 9 7 7 ) 6 0 1 - - 6 0 4 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
BBA 98908
CALORIMETRIC MEASUREMENTS OF THE TRANSITION ENTHALPY OF DNA IN AQUEOUS U R E A SOLUTIONS
H. K L U M P * a n d W. B U R K A R T
Institut fiir Physikalische Chemie der Universitb't Freiburg, Freiburg (G.F.R.) (Received October 8th, 1976)
Summary • The helix-coil equilibrium of DNA is delicately solvent. In this investigation the helical secondary an 'increasing concentration of urea. We found transition enthalpy AH on the urea concentration as for salmon sperm DNA.
affected by the nature of the structure was destabilized b y a linear dependence of the for calf thymus DNA as well
Introduction
An extensive survey of the relative denaturing p o w e r of various organic solvents was reported by Lewin et al. [ 1 ], who measured the concentration of organic solvent necessary to bring a b o u t stated conditions of denaturation at a fixed temperature. No direct correlation was found between denaturation effectiveness and monofunctional hydrogen-bonding capacity, bifunctional hydrogen-bonding capacity, the hydrophobic character or alterations in solvent polarity for these denaturants. This paper is concerned with the direct measurement of the transition enthalpy AH on calf thymus DNA and salmon sperm DNA as a function of urea concentration in neutral aqueous solution. Material and Methods Calf thymus DNA and salmon sperm DNA were supplied by Worthington Biochem. Corporation and were used without further purification. All other c o m p o u n d s were of reagent grade. DNA solutions were prepared by first dissolving the lyophilized product in quartz-distilled water and than adding the required amounts of salt to give a neutral buffered aqueous solution, before adding the calculated a m o u n t of urea to give the final urea concentration for * To whom all correspondence should be addressed.
602 the individual calorimetric measurements. A solution with identical salt and urea concentrations was used as a blank, pH measurements were performed with the help of a Radiometer Mod. 22 pH meter. Details of the calorimeter apparatus are given elsewhere [2]. All optically measured denaturation curves we're recorded by a Pj?e-Unicam Sp 1800 spectrophotometer at 260 nm as a function of temperature. Solutions for spectrophotometric measurements were usually prepared by diluting microliter quantities of the DNA stock solutions with the appropriate salt and urea solvent. The concentration of the DNA solutions was determined via a colorimetric phosphorous analysis. Results and Discussion
When heating native calf thymus DNA at pH 7.5 in buffered aqueous solution without or with urea, the results displayed in Figs. 1 and 2 are obtained. The curves show the temperature course of the electrical compensating energy needed to maintain adiabatic conditions in the calorimeter and the heat denaturation profiles obtained by plotting the absorbance increase of the DNA solution at 260 nm against temperature. In Fig. 1 the salmon sperm DNA and calf t h y m u s DNA had been dissolved in quartz-distilled water and the final solution contained 0.01 M NaC1 plus 1 mM sodium citrate, pH 7.5, whereas in Fig. 2 the solution contained 5 M urea in addition to the salts. The temperature at which the heat absorption peak has its major maximum is the transition temperature
Tin. It is readily seen that it is the same temperature pertaining to the 50% point of hyperchromicity at 260 nm. It is evident that the temperature course of the t w o parameters signifies the transition of the DNA from the native to the randomly coiled state. As expected, the thermal stability of salmon sperm DNA and calf thymus DNA decreases with increasing urea concentration (cf. Fig. 3). In the case of calf thymus DNA, heat absorption peaks were ordinarily quite asymmetric a b o u t Tm and the curves are structured exhibiting at least two additional maxima. The secondary maximum in the compensating energytemperature curve can be associated with the well-known compositional hetero-
%~ 80
!
100
.q
,5 -6o 40 ~o
~
i
55
i
60
i
65
i
J
i
70
75 Ternperoture
,..
[°C 80
]
Fig. 1. H e a t - i n d u c e d helix-coil t r a n s i t i o n c u r v e s o f c a l f t h y m u s D N A ( ) and salmon sperm DNA (. . . . . . ) in 0.01 M NaC] plus 1 m M s o d i u m c i t r a t e b u f f e r , p H 7.5. L e f t - h a n d o r d i n a t e a b s o r b a n c e c h a n g e (o o, calf t h y m u s D N A ; o, s a l m o n s p e r m D N A ) ; r i g h t - h a n d o r d i n a t e c o m p e n s a t i n g e n e r g y , N C (t).
•,
603
~ t 100 %
:7 8°
i
/,5
50
55
60
65 68 Temper ot ure [ ° C I
Fig. 2. H e a t - i n d u c e d h e l i x - c o g t r a n s i t i o n c u r v e s o f c a l f t h y m u s D N A a n d s a l m o n s p e r m D N A in 0 . 0 1 M NaCI p l u s 1 m M s o d i u m c i t r a t e b u f f e r p H 7 . 5 / 5 M u r e a . Left-hand ordinate, absorbance change: (o o, c a l f t h y m u s D N A ; • ~, s a l m o n s p e r m D N A ) ; r i g h t - h a n d o z d i n a t e , compensating energy N C (t).
geneity of calf thymus DNA. The relative positions o f these maxima are not dependent on the urea concentration. This could indicate that the stability of G • C base pairs in equal affected by urea as the stability of A • T base pairs. Table I shows that the entropy of denaturation, calculated using the Gibbs. Helmholtz equation, also varies with urea concentration. Both A3 and AH are positive in sign as the transition proceeds from ordered to the disordered structure of the macromolecule. The slope dTm/dC urea = 2.5 ° . The interdependence of AH, AS, and Tm with urea concentration can only be described in purely phenomenological terms. The implication is that urea interacts preferentially in solution with the bases, and it is able to do this more affectively with coiled form. The question of specific binding (through use of a binding constant Ks) vs. nonspecific solvent effects implied by the use of the corrected Setschenow [3] constants ks is left open [4,5]. If the effective number of binding sites per m o n o m e r unit ~ is taken as independent of the urea concentration, the binding constant can be calculated as a function of the urea concentration. Assuming a value of 1.3 for ~ [5] the following equation yields the values listed in Table I.
7.5 m s-
7.0
,,," 6.5 -r ,,~ 6.0 5.5 0
1
2
3
4
5
6
M ureo m
Fig. 3. P l o t o f t h e t r a n s i t i o n e n t h a l p y A H vs. u r e a c o n c e n t r a t i o n m, c a l f t h y m u s D N A ; o, s a l m o n s p e r m D N A ; M B P , melted base pai~.
6O4 TABLEI Urea c o n c e n t r a t i o n (mol/1)
AH (kcal/melted base pair)
AS (cal/degree per mol)
K B - 102
Tm (o C)
1 2 3 4 5 6 --
7.000 6.750 6.500 6.125 5.750 5.450 7.500
20.86 20.34 19.60 18.62 17.64 16.82 22.06
2.97 2.88 2.97 2.65 2.50 2.39 --
63.5 61.0 58.5 56.0 53.0 51.0 66.0
KB = (ATm
"
Ah / R T m T m
o. P)/as
where KB is the association or binding constant of the denaturant with the average monomer unit, ATm is the lowering of the transition temperature of the biopolymer due to the binding of interaction of the denaturing agent with its denatured form [6], Ah is the experimental transition enthalpy per monomer unit of the polymer under study, Tm and Tm° are the midpoints of the denaturation transition in the presence and absence of denaturant and as is the activity of the denaturant, assumed to be equal to its concentration [7,8]. The binding constant decreases slightly with increasing urea concentration. The results of this or other studies provide no evidence that hydrogen bonding between urea and DNA is the main contribution to the denaturation effect. Acknowledgement W e thank Professor T. Ackermann for support and encouragement during these investigation. This work was supported by the Deutsche Forschungsgemeinschaft.
References 1 2 3 4 5 6 7 8
Levine, L. et al. ( 1 9 6 3 ) B i o c h e m i s t r y 2, 1 6 8 - - 1 7 5 G r u b e r t , M. and A c k e r m a n , T h . ( 1 9 7 4 ) Z. P h y s . C h e m . 9 1 , 2 5 5 - - 2 6 4 L o n g , F . A . a n d M c D e v i t ( 1 9 5 2 ) C h e m . Rev. 5 1 , 1 1 9 H e r s k o v i t s , T.T. and Harrington, J. ( 1 9 7 2 ) B i o c h e m i s t r y 1 1 , 4 8 0 0 H e r s k o v i t s , T.T. and B o w e n , J . J . ( 1 9 7 4 ) B i o c h e m i s t r y 1 3 , 5 4 7 4 - - 5 4 8 3 Pellet, L. ( 1 9 5 9 ) J. P h y s . C h e m . 6 3 , 1 1 9 9 S e h r i e r , E . E . , I n g w a l l , R . T . and Scheraga, H . A . ( 1 9 6 5 ) J. P h y s . C h e m . 6 9 , 2 9 8 E l b a u m , D. and Herskovits, T.T. ( 1 9 7 4 ) B i o c h e m i s t r y 1 3 , 1 2 6 8