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Pressure effect on deoxyribonucleic acid transition
SHORT COMMUNICATIONS
179
sc 43 064
Pressure effect on deoxyribonucleic acid transition The thermodynamic description of the strand separation process of nucleic acids requires information on: (a) the effect of temperature on the transition process and the heat of transition and (b) the effect of pressure on the process and the volume of transition. A number of studies have been made on the effect of temperature on the denaturation of DNA (see for example refs. I and 2), and estimates of heat of transition have been obtained from either the effect of acid denaturation studies 3 or the heats of reaction of synthetic polynucleotides 4. Direct heats of transition measurements have been reported by PRIVALOV and co-workers5. The pressure effect on the transition process has not been as amenable to investigation. However, if the stability of the double-chain nucleic acid molecule were sensitive to pressure change, interesting biological implications could be imagined. For example, strand separation in the DNA synthesis within a cell might be promoted by a build-up of osmotic pressure. In principle it is possible to calculate the pressure sensitivity on the process using information on specific volume changes and helix-coil theory. Measurements of the density of native and denatured DNA by DoT¥ and co-workers6,7 in 7.8 M CsC1 at room temperature have led to the conclusion that the density increases in strand separation by o.o15 to O.Ol7 g/cc and the density of the material is about 1. 7 g/cc. This corresponds to a change in specific volume of about 0.005 cc/g. Because the specific volume of the material is less in the denatured than in its natural states in CsC1 an effect of pressure should then promote denaturation. Unfortunately direct measurements of the volume change of the transition process in low salt concentration and at temperatures, comparable to those where thermal transition occurs, are not available. Thus a direct attempt at measurement of the pressure effect appeared desirable. In the course of this investigation the results of HEDEN and co-workers8 using a different technique for evaluating a pressure effect on the transition process have become available and are compatible with our findings. The calf-thymus DNA was either prepared in our laboratory under the direction of Professor M. DOWNINGusing the method of KAY and co-workers9, or acquired from CalBiochem. Solutions were made with 0.03 M NaC1 and o.ooi M EDTA at concentrations of approx. 0.5 %. The transition process was studied by following the effect of pressure upon the optical rotation of the solutions by means of apparatus described by GILL AND GLOGOVSKY10. It was quickly established that the effect of pressure was small and could best be studied by effecting thermal transitions at different fixed pressures, a method which had been used for other polymer transition processes n. The measurements were complicated by the necessity of using as high a concentration of DNA as possible to obtain reasonable changes in the optical rotation and by the need for going to relatively high (90 °) temperatures to promote the complete transition process. The high temperature causes a deterioration of the Polaroids used in the apparatus, with the result that absolute optical-rotation measurements could not be obtained since the calibration factor did not remain constant. Relative changes could be observed and were based upon the fraction of change observed between the minimum and maximum readings. Reproducibility of optical readings was -~ 5 %. The temperature was known to -~ i o. Biochirn. Biophys. Acta, 112 (1966) 179-181
I80
SHORT
COMMUNICATIONS
The fractional change in optical rotation as a function of temperature for three different pressures is illustrated for a typical set of runs in Fig. I. The decrease in optical rotation with the initial increase in temperature is consistent with observations by DOTY and co-workers 1. FR~SCO12 has suggested that this initial dip might be ascribed to configurational changes of the pentose portion of the chain. Evidence of an intermediate structure, which might explain this observation, has been given recently by LuzzATI and co-workers a3. Further increase in temperature promotes the most significant change in optical rotation which is associated with the separation of base-pair links. The effect of pressure upon this region of the transition process is not particularly large, but is such that an increase in pressure raises the transition temperature.
I. 000
0.800
.~ 0.600
o
C)
"6 0.400
3 0.200
o ,.~
o I
20
o
4O
60 80 Ternperatur'e
I
100
Fig. I. R e l a t i o n s h i p b e t w e e n c h a n g e in optical r o t a t i o n a n d t e m p e r a t u r e a t different pressures. O--O, atmospheric pressure; ~--~, i o o o o l b . i n c h 3; A - - ~ , 2 o o o o l b ' i n c h -2.
This result is opposite to what one would predict from density changes as measured in density gradients of CsC1 solution at room temperature. It is, however, consistent with results of HEDEN and co-workers s, who found that pressures to 27oo arm promoted stabilization of the double-helix structure. Since the pressure effect is observed to be quite small, it does not appear to be of any mechanistic importance in biological synthesis of nucleic acids. An estimate of the volume change for the transition under the conditions of this experiment can be found by applying the theory of ZI]VIM14 to displacement of the transition temperature Tm from the effect of pressure upon the equilibrium-constant parameter s for the process of rupturing a given base-pair interaction. CROZIERS AXD ZIMM15 have shown how the observed breadth of the thermal transition can be explained by sample heterogeneity. Thus is a distribution of parameters sl which depend upon base-pair composition. However, for a particular chain with a given base-pair composition a fixed fraction of denaturation will be maintained provided: dp dT
AHI T~Vt
Biochim. Biophys. Acla, i12 (1966) I 7 9 - x 8 I
(i)
181
SHORT COMMUNICATION S
There is substantial evidence that the heat of base-pair disruption AHI, depends upon base-pair composition 15, and one might anticipate a similar effect for the volume change per mole of base-pair disruption AVI. However an estimate of an average value of AV can be obtained from Eqn. (I) by using the value of dp/dT determined from Fig. I at 0 --~ 1/2 along with an average value of AH = 9 kcal/mole base-pair. Using a value of dp/dT ~ 2.2. lOs atm.degree -1 at 35o°K for T m w e find AV = 4.5 cc/mole base-pairs or with an average molecular weight of 650 for a base-pair the specific volume change is 0.007 cc/g which corresponds to a density decrease of approx. o.o21 g/cc. We are now left with the situation that the pressure measurements indicate a decrease in the density of denatured state whereas the measurements in CsC1 density gradients indicate just the opposite. The pressure measurements are not completely free of experimental difficulty, as noted with Polaroid deterioration, but we feel this problem should not affect the semi-quantitative aspects of our observations. Three complete sets of measurements gave a similar pressure temperature effect. One is therefore left with at least two possible explanations: (I) native and denatured DNA in a solution of approx. 8 M CsC1 have a different density than it would have in a solution of low salt concentration or (2) as KAUTZMANNTM has suggested and HEDEN et al. s choose to explain their results which are similar to ours, a reasonably different temperature coefficient might exist for the partial molar volumes of native and denatured DNA so that an inversion in sign could occur at higher temperature. From a biological point of view a resolution of these explanations does not seem particularly important since it appears the volume change is far too small to be of interest in discussion of the mechanism of strand separation in cell division. This work was supported in part by a National Science Foundation Grant GP734. It is a pleasure to acknowledge many discussions on matters pertinent to this note with Prof. M. DOWNING.
Department of Chemistry, University of Colorado, Boulder, Colo. (U.S.A.)
BETTY W E I D A S. J . GILL
I P. DOTY, H. BOEDTKER, J. R. FRESCO, R. HASELKORN AND M. LITT, Proc. Natl. Acad. Sci. U.S., 45 (196o) 482. 2 D. M. CROTHERS, N. R. KALLENBACH AND ]3. H. ZIMM, J. Mol. Biol., i i (1965) 802. 3 J. M. STURTEVANT, S. A. RICE AND E. P. GEIDUSCHEK, Discussions Faraday Soc., 25 (1958) 138. 4 M. A. RAWITZER, P. D. ROSS AND J. M. STURTEVANT, J. Am. Chem. Soc., 85 (1963) 1915. 5 P. L. PRIVALOV, K. A. KAFIANI AND D. R. MONASELIDZE, Dokl. Ahad. Nauk SSSR, 156 (1964) 951 . 6 P. DOTY, J. MARMUR, J. EIGNER AND C. SHILDKRAUT, Proc. Natl. Acad. Sci, U.S., 46 (196o) 461. 7 C. SHILDKRAUT, J. MARMUR AND P. DOTY, jr. Mol. Biol., 3 (1961) 595. 8 C. G. HEDEN, T. LINDAHL AND I. TOPLIN, Acta Chem. Scand., 18 (1964) 115o. 9 E. R. M. KAY, ~XT.S. SIMMONS AND A. L. DOUNCE, jr. Am. Chem. Soc., 74 (1952) 1724. Io S. J. GILL AND R. GLOGOVSKY, Rev. Sci. Instr., 35 (1964) 1281. I I S. J. GILL AND R. GLOGOVSKY,J . Phys. Chem., 69 (1965) 1515. 12 J. R. FRESCO, Tetrahedron, 13 (1961) 185. 13 V. LUZZATI, A. MATHIS, F. MASSON AND J. WITZ, J. Mol. Biol., io (1964) 28. 14 ]3. H. ZIMM, J. Chem. Phys., 33 (196o) 1349. 15 D. M. CROTHERS AND B. H. ZIMM, jr. Mol. Biol., 9 (1964) I. 16 W. I~AUTZMANN,Advan. Protein Chem., 14 (1959) 16.
Received August 9th, I965 Biochim. Biophys. Acta, i i 2 (1966) 179-181
179
sc 43 064
Pressure effect on deoxyribonucleic acid transition The thermodynamic description of the strand separation process of nucleic acids requires information on: (a) the effect of temperature on the transition process and the heat of transition and (b) the effect of pressure on the process and the volume of transition. A number of studies have been made on the effect of temperature on the denaturation of DNA (see for example refs. I and 2), and estimates of heat of transition have been obtained from either the effect of acid denaturation studies 3 or the heats of reaction of synthetic polynucleotides 4. Direct heats of transition measurements have been reported by PRIVALOV and co-workers5. The pressure effect on the transition process has not been as amenable to investigation. However, if the stability of the double-chain nucleic acid molecule were sensitive to pressure change, interesting biological implications could be imagined. For example, strand separation in the DNA synthesis within a cell might be promoted by a build-up of osmotic pressure. In principle it is possible to calculate the pressure sensitivity on the process using information on specific volume changes and helix-coil theory. Measurements of the density of native and denatured DNA by DoT¥ and co-workers6,7 in 7.8 M CsC1 at room temperature have led to the conclusion that the density increases in strand separation by o.o15 to O.Ol7 g/cc and the density of the material is about 1. 7 g/cc. This corresponds to a change in specific volume of about 0.005 cc/g. Because the specific volume of the material is less in the denatured than in its natural states in CsC1 an effect of pressure should then promote denaturation. Unfortunately direct measurements of the volume change of the transition process in low salt concentration and at temperatures, comparable to those where thermal transition occurs, are not available. Thus a direct attempt at measurement of the pressure effect appeared desirable. In the course of this investigation the results of HEDEN and co-workers8 using a different technique for evaluating a pressure effect on the transition process have become available and are compatible with our findings. The calf-thymus DNA was either prepared in our laboratory under the direction of Professor M. DOWNINGusing the method of KAY and co-workers9, or acquired from CalBiochem. Solutions were made with 0.03 M NaC1 and o.ooi M EDTA at concentrations of approx. 0.5 %. The transition process was studied by following the effect of pressure upon the optical rotation of the solutions by means of apparatus described by GILL AND GLOGOVSKY10. It was quickly established that the effect of pressure was small and could best be studied by effecting thermal transitions at different fixed pressures, a method which had been used for other polymer transition processes n. The measurements were complicated by the necessity of using as high a concentration of DNA as possible to obtain reasonable changes in the optical rotation and by the need for going to relatively high (90 °) temperatures to promote the complete transition process. The high temperature causes a deterioration of the Polaroids used in the apparatus, with the result that absolute optical-rotation measurements could not be obtained since the calibration factor did not remain constant. Relative changes could be observed and were based upon the fraction of change observed between the minimum and maximum readings. Reproducibility of optical readings was -~ 5 %. The temperature was known to -~ i o. Biochirn. Biophys. Acta, 112 (1966) 179-181
I80
SHORT
COMMUNICATIONS
The fractional change in optical rotation as a function of temperature for three different pressures is illustrated for a typical set of runs in Fig. I. The decrease in optical rotation with the initial increase in temperature is consistent with observations by DOTY and co-workers 1. FR~SCO12 has suggested that this initial dip might be ascribed to configurational changes of the pentose portion of the chain. Evidence of an intermediate structure, which might explain this observation, has been given recently by LuzzATI and co-workers a3. Further increase in temperature promotes the most significant change in optical rotation which is associated with the separation of base-pair links. The effect of pressure upon this region of the transition process is not particularly large, but is such that an increase in pressure raises the transition temperature.
I. 000
0.800
.~ 0.600
o
C)
"6 0.400
3 0.200
o ,.~
o I
20
o
4O
60 80 Ternperatur'e
I
100
Fig. I. R e l a t i o n s h i p b e t w e e n c h a n g e in optical r o t a t i o n a n d t e m p e r a t u r e a t different pressures. O--O, atmospheric pressure; ~--~, i o o o o l b . i n c h 3; A - - ~ , 2 o o o o l b ' i n c h -2.
This result is opposite to what one would predict from density changes as measured in density gradients of CsC1 solution at room temperature. It is, however, consistent with results of HEDEN and co-workers s, who found that pressures to 27oo arm promoted stabilization of the double-helix structure. Since the pressure effect is observed to be quite small, it does not appear to be of any mechanistic importance in biological synthesis of nucleic acids. An estimate of the volume change for the transition under the conditions of this experiment can be found by applying the theory of ZI]VIM14 to displacement of the transition temperature Tm from the effect of pressure upon the equilibrium-constant parameter s for the process of rupturing a given base-pair interaction. CROZIERS AXD ZIMM15 have shown how the observed breadth of the thermal transition can be explained by sample heterogeneity. Thus is a distribution of parameters sl which depend upon base-pair composition. However, for a particular chain with a given base-pair composition a fixed fraction of denaturation will be maintained provided: dp dT
AHI T~Vt
Biochim. Biophys. Acla, i12 (1966) I 7 9 - x 8 I
(i)
181
SHORT COMMUNICATION S
There is substantial evidence that the heat of base-pair disruption AHI, depends upon base-pair composition 15, and one might anticipate a similar effect for the volume change per mole of base-pair disruption AVI. However an estimate of an average value of AV can be obtained from Eqn. (I) by using the value of dp/dT determined from Fig. I at 0 --~ 1/2 along with an average value of AH = 9 kcal/mole base-pair. Using a value of dp/dT ~ 2.2. lOs atm.degree -1 at 35o°K for T m w e find AV = 4.5 cc/mole base-pairs or with an average molecular weight of 650 for a base-pair the specific volume change is 0.007 cc/g which corresponds to a density decrease of approx. o.o21 g/cc. We are now left with the situation that the pressure measurements indicate a decrease in the density of denatured state whereas the measurements in CsC1 density gradients indicate just the opposite. The pressure measurements are not completely free of experimental difficulty, as noted with Polaroid deterioration, but we feel this problem should not affect the semi-quantitative aspects of our observations. Three complete sets of measurements gave a similar pressure temperature effect. One is therefore left with at least two possible explanations: (I) native and denatured DNA in a solution of approx. 8 M CsC1 have a different density than it would have in a solution of low salt concentration or (2) as KAUTZMANNTM has suggested and HEDEN et al. s choose to explain their results which are similar to ours, a reasonably different temperature coefficient might exist for the partial molar volumes of native and denatured DNA so that an inversion in sign could occur at higher temperature. From a biological point of view a resolution of these explanations does not seem particularly important since it appears the volume change is far too small to be of interest in discussion of the mechanism of strand separation in cell division. This work was supported in part by a National Science Foundation Grant GP734. It is a pleasure to acknowledge many discussions on matters pertinent to this note with Prof. M. DOWNING.
Department of Chemistry, University of Colorado, Boulder, Colo. (U.S.A.)
BETTY W E I D A S. J . GILL
I P. DOTY, H. BOEDTKER, J. R. FRESCO, R. HASELKORN AND M. LITT, Proc. Natl. Acad. Sci. U.S., 45 (196o) 482. 2 D. M. CROTHERS, N. R. KALLENBACH AND ]3. H. ZIMM, J. Mol. Biol., i i (1965) 802. 3 J. M. STURTEVANT, S. A. RICE AND E. P. GEIDUSCHEK, Discussions Faraday Soc., 25 (1958) 138. 4 M. A. RAWITZER, P. D. ROSS AND J. M. STURTEVANT, J. Am. Chem. Soc., 85 (1963) 1915. 5 P. L. PRIVALOV, K. A. KAFIANI AND D. R. MONASELIDZE, Dokl. Ahad. Nauk SSSR, 156 (1964) 951 . 6 P. DOTY, J. MARMUR, J. EIGNER AND C. SHILDKRAUT, Proc. Natl. Acad. Sci, U.S., 46 (196o) 461. 7 C. SHILDKRAUT, J. MARMUR AND P. DOTY, jr. Mol. Biol., 3 (1961) 595. 8 C. G. HEDEN, T. LINDAHL AND I. TOPLIN, Acta Chem. Scand., 18 (1964) 115o. 9 E. R. M. KAY, ~XT.S. SIMMONS AND A. L. DOUNCE, jr. Am. Chem. Soc., 74 (1952) 1724. Io S. J. GILL AND R. GLOGOVSKY, Rev. Sci. Instr., 35 (1964) 1281. I I S. J. GILL AND R. GLOGOVSKY,J . Phys. Chem., 69 (1965) 1515. 12 J. R. FRESCO, Tetrahedron, 13 (1961) 185. 13 V. LUZZATI, A. MATHIS, F. MASSON AND J. WITZ, J. Mol. Biol., io (1964) 28. 14 ]3. H. ZIMM, J. Chem. Phys., 33 (196o) 1349. 15 D. M. CROTHERS AND B. H. ZIMM, jr. Mol. Biol., 9 (1964) I. 16 W. I~AUTZMANN,Advan. Protein Chem., 14 (1959) 16.
Received August 9th, I965 Biochim. Biophys. Acta, i i 2 (1966) 179-181