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Heat of the reaction between polyribocytidylic acid and polyriboinosinic acid
J. Mol. Biol. (1969) 45, 567-569
LETTER
TO THE EDITOR
Heat of the Reaction between Polyribocytidylic Polyriboinosinic Acid
Acid and
The synthetic polynucleotides, polyribocytidylic acid and polyriboinosinic acid react in aqueous salt solutions to form a two-stranded helical complex, poly (rC:rI)t (Davies & Rich, 1958). In order to gain a fuller understanding of the thermodynamics of polynucleotide helix formation and as part of a comprehensive study of the effect of chemical structure upon the energetics of base pairing in nucleic acid complexes we have measured the enthalpy change accompanying this reaction. Calorimetric measurements of the heat of reaction upon mixing were made in an LKB model 10700 batch microcalorimeter (Wadso, 1968) by the techniques described elsewhere (Scruggs t Ross, 1969). The experiments were carried out at pH 8-O and at low concentrations of salt (NaCl) and of polymer (typically 7 x 10e4 M-phosphorus in each polymer) in order to minimize the formation of partially ordered single stranded poly rC (Ts’o, Helmkamp & Sander, 1962; Fasman, Lindblow & Grossman, 1964) and any formation of the multi-stranded ordered form of poly r1 (Rich, 1958). The calculated heats of reaction are based upon a value for the molar extinction coefficient of poly rC in O-1 M-Nacl at 268 rnp of c(P) = 6200 l./mole-cm. Since it was felt that the molar extinction coefficient of poly rC was known with greater certainty than that of poly r1, the experiments were carried out with the former component in limiting amount. The composition of the solution in the calorimeter TABLE
1
The heat of interaction of poly rC and poly rI at pH 8.0
Molarity
of NaCl 0.02 0.05 0.10 0.20 Average
20°C -AH cal./mole
of base pairs
Molarity
5649 5638 5598 5525 6589 5476 5600 5609
of NaCl
37°C -AH
cal./mole
of base pairs
0.06
(5082H
0.10
5582 6609 5594 5586
0.40
5585&20 (S.E.) Average Average of all work (12 experiments) 5588&13
569356
(S.E.)
(S.E.)
t This reaction w&s not complete. The measured heat was 91 oh of that found in the other cases shown in Table 1 for complete reaction. The reaction was judged to be 92% complete from spectrophotometric investigation and when corrected gave a value of AH = - 5530 cal./mole base pairs. This experiment was excluded from the averaging. t Abbreviations used: rC, polyribocytidylic acid; rU, polyribouridylic acid.
acid; r1, polyriboinosinic 667
acid;
rA, polyriboadenylic
568
P. D.
ROSS
AND
R.
L.
SCRUGGS
cell after mixing was approximately 45 mole per cent poly rC. Variation of the mole fraction produced no change in the measured values of AH, a result which is fully consistent with the absence of any reported three-stranded complexes in the poly rC-poly r1 system (Chamberlin & Patterson, 1965). The value obtained for the heat of reaction to form the poly (rC : r1) complex is close to AH = -5600 cal./mole of base pairs and is independent of the conditions of salt concentration and temperature employed. These results, obtained at 20 and 37”C, are presented in Table 1. The precision obtained of better than 0.3% for these experiments in which approximately 25 meal. of heat was evolved is also noteworthy. The invariance of these heat measurements to solvent conditions (Table 1) for the poly rC-poly r1 system stands in sharp contrast to the results obtained for the formation of poly (rA:rU), where the measured value of AH was found to depend upon both the salt concentration and the temperature (Steiner $ Kitzinger, 1962 ; Rawitscher, Ross & Sturtevant, 1963; Ross $ Scruggs, 1965; Neumann $ Ackermann, 1967 ; Krakauer & Sturtevant, 1968). It may be seen in Table 1 that the heat ofthe interaction between poly rC and poly r1 does not change with temperature. Tentatively accepting the result as general, that the apparent change in heat capacity, AC,, is zero for this reaction it is possible to extrapolate the measured heats to the melting temperatures, T,, of the poly (rC:rI) complexes to obtain AH;, at T,. Upon extrapolation and reversing the sign of AH, one then obtains a value of AH;, of 5.6 kcal./ mole of base pairs for the melting of poly (rC :rI) at the transition temperature. This is a considerably lower value of AH;, than was found for the poly (rA : rU) system where the value of AH;, varied from 7.5 to 8.5 kcal./mole of base pairs depending upon T, (Rawitscher, Ross & Sturtevant, 1963 ; Neumann & Ackermann, 1967 ; Krakauer & Sturtevant, 1968). If the extrapolated value at T, of the measured heat corresponds to the true AH;, of transition, then it is possible to calculate the standard entropy change upon melting from the relationship AS” = AHGJT,. The values of AX” so obtained over the range of T,‘s corresponding to the salt concentrations employed in this work are approximately 17.5 cal./deg-mole of base pairs for poly (rC:rI), which is considerably lower than the values in the neighborhood of 23 cal./deg-mole of base pairs found for dissociation of the poly (rS:rU) complex (Rawitscher, Ross & Sturtevant, 1963; Ross & Scruggs, 1965; Neumann & Ackermann 1967; Krakauer & Sturtevant, 1968). Thus, both the standard heats, AH”, and the standard entropies, AS”, at the transition temperature, may be different for different nucleic acid base pairs. The most immediate interpretation of the insensitivity of the measured heats to environmental conditions in the poly rC-poly r1 system is that the single strand forms of poly rC and poly r1 contain litt,le, if any, self-structure which affects the heat of the complex formation reaction, This result is obtained for a system in which there is spectrophotometric evidence of short-range interaction in the single strand form of poly rC with an estimated AH of 9.6 kcal./mole of base pairs (Leng & Michelson, 1968). The measurements reported in this paper conclusively show for the first time in a polymer-polymer reaction that the heats and the entropies of the interaction between different types of nucleic acid base pairs can be different. This conclusion follows from the contrasting results that have been obtained for the poly (rA:rU) and poly
LETTER
TO THE
(rC:rI) systems. A more detailed interpretation elsewhere. Laboratory of Molecular Biology National Institute of Arthritis and Metabolic National Institutes of Health Bethesda, Maryland 20014, U.S.A.
EDITOR
569
of these findings will be reported
Diseases
PHILIP D. Ross ROBERT L. SCRUCGS
Received 3 July 1969 REFERENCES Chamberlin, M. J. & Patterson, D. L. (1965). J. Mol. Biol. 12, 410. Davies, D. R. & Rich, A. (1958). J. Amer. Chem. Sot. 80, 1003. Fasman, G. D., Lindblow, C. & Grossman, L. (1964). Biochemistry, 3, 1015. Krakauer, H. & Sturtevant, J. M. (1968). Biopolymers, 6, 491. Leng, M. & Michelson, A. M. (1968). Biochim. biophys. Acta, 155, 91. Neumann, E. & Ackerman, T. (1967). J. Phys. Chem. 71, 2377. Rawitscher, M. A., Ross, P. D. & Sturtevant, J. M. (1963). J. Amer. Chem. Sot. 85, 1915. Rich, A. (1958). Biochim. biophys. Actu, 29, 502. Ross, P. D. & Scruggs, R. L. (1965). Biopolymers, 3, 491. Scruggs, R. L. & Ross, P. D. (1969). J. Mol. Biol. in the press. Steiner, R. F. & Kitzinger, C. (1962). Nature, 194, 1172. Ts’o, P. 0. P., Helmkamp, G. K. & Sander, C. (1962). Biochim. biophys. Actu, 55, 584. Wadso, I. (1968). Actu them. Scud. 22, 927.
LETTER
TO THE EDITOR
Heat of the Reaction between Polyribocytidylic Polyriboinosinic Acid
Acid and
The synthetic polynucleotides, polyribocytidylic acid and polyriboinosinic acid react in aqueous salt solutions to form a two-stranded helical complex, poly (rC:rI)t (Davies & Rich, 1958). In order to gain a fuller understanding of the thermodynamics of polynucleotide helix formation and as part of a comprehensive study of the effect of chemical structure upon the energetics of base pairing in nucleic acid complexes we have measured the enthalpy change accompanying this reaction. Calorimetric measurements of the heat of reaction upon mixing were made in an LKB model 10700 batch microcalorimeter (Wadso, 1968) by the techniques described elsewhere (Scruggs t Ross, 1969). The experiments were carried out at pH 8-O and at low concentrations of salt (NaCl) and of polymer (typically 7 x 10e4 M-phosphorus in each polymer) in order to minimize the formation of partially ordered single stranded poly rC (Ts’o, Helmkamp & Sander, 1962; Fasman, Lindblow & Grossman, 1964) and any formation of the multi-stranded ordered form of poly r1 (Rich, 1958). The calculated heats of reaction are based upon a value for the molar extinction coefficient of poly rC in O-1 M-Nacl at 268 rnp of c(P) = 6200 l./mole-cm. Since it was felt that the molar extinction coefficient of poly rC was known with greater certainty than that of poly r1, the experiments were carried out with the former component in limiting amount. The composition of the solution in the calorimeter TABLE
1
The heat of interaction of poly rC and poly rI at pH 8.0
Molarity
of NaCl 0.02 0.05 0.10 0.20 Average
20°C -AH cal./mole
of base pairs
Molarity
5649 5638 5598 5525 6589 5476 5600 5609
of NaCl
37°C -AH
cal./mole
of base pairs
0.06
(5082H
0.10
5582 6609 5594 5586
0.40
5585&20 (S.E.) Average Average of all work (12 experiments) 5588&13
569356
(S.E.)
(S.E.)
t This reaction w&s not complete. The measured heat was 91 oh of that found in the other cases shown in Table 1 for complete reaction. The reaction was judged to be 92% complete from spectrophotometric investigation and when corrected gave a value of AH = - 5530 cal./mole base pairs. This experiment was excluded from the averaging. t Abbreviations used: rC, polyribocytidylic acid; rU, polyribouridylic acid.
acid; r1, polyriboinosinic 667
acid;
rA, polyriboadenylic
568
P. D.
ROSS
AND
R.
L.
SCRUGGS
cell after mixing was approximately 45 mole per cent poly rC. Variation of the mole fraction produced no change in the measured values of AH, a result which is fully consistent with the absence of any reported three-stranded complexes in the poly rC-poly r1 system (Chamberlin & Patterson, 1965). The value obtained for the heat of reaction to form the poly (rC : r1) complex is close to AH = -5600 cal./mole of base pairs and is independent of the conditions of salt concentration and temperature employed. These results, obtained at 20 and 37”C, are presented in Table 1. The precision obtained of better than 0.3% for these experiments in which approximately 25 meal. of heat was evolved is also noteworthy. The invariance of these heat measurements to solvent conditions (Table 1) for the poly rC-poly r1 system stands in sharp contrast to the results obtained for the formation of poly (rA:rU), where the measured value of AH was found to depend upon both the salt concentration and the temperature (Steiner $ Kitzinger, 1962 ; Rawitscher, Ross & Sturtevant, 1963; Ross $ Scruggs, 1965; Neumann $ Ackermann, 1967 ; Krakauer & Sturtevant, 1968). It may be seen in Table 1 that the heat ofthe interaction between poly rC and poly r1 does not change with temperature. Tentatively accepting the result as general, that the apparent change in heat capacity, AC,, is zero for this reaction it is possible to extrapolate the measured heats to the melting temperatures, T,, of the poly (rC:rI) complexes to obtain AH;, at T,. Upon extrapolation and reversing the sign of AH, one then obtains a value of AH;, of 5.6 kcal./ mole of base pairs for the melting of poly (rC :rI) at the transition temperature. This is a considerably lower value of AH;, than was found for the poly (rA : rU) system where the value of AH;, varied from 7.5 to 8.5 kcal./mole of base pairs depending upon T, (Rawitscher, Ross & Sturtevant, 1963 ; Neumann & Ackermann, 1967 ; Krakauer & Sturtevant, 1968). If the extrapolated value at T, of the measured heat corresponds to the true AH;, of transition, then it is possible to calculate the standard entropy change upon melting from the relationship AS” = AHGJT,. The values of AX” so obtained over the range of T,‘s corresponding to the salt concentrations employed in this work are approximately 17.5 cal./deg-mole of base pairs for poly (rC:rI), which is considerably lower than the values in the neighborhood of 23 cal./deg-mole of base pairs found for dissociation of the poly (rS:rU) complex (Rawitscher, Ross & Sturtevant, 1963; Ross & Scruggs, 1965; Neumann & Ackermann 1967; Krakauer & Sturtevant, 1968). Thus, both the standard heats, AH”, and the standard entropies, AS”, at the transition temperature, may be different for different nucleic acid base pairs. The most immediate interpretation of the insensitivity of the measured heats to environmental conditions in the poly rC-poly r1 system is that the single strand forms of poly rC and poly r1 contain litt,le, if any, self-structure which affects the heat of the complex formation reaction, This result is obtained for a system in which there is spectrophotometric evidence of short-range interaction in the single strand form of poly rC with an estimated AH of 9.6 kcal./mole of base pairs (Leng & Michelson, 1968). The measurements reported in this paper conclusively show for the first time in a polymer-polymer reaction that the heats and the entropies of the interaction between different types of nucleic acid base pairs can be different. This conclusion follows from the contrasting results that have been obtained for the poly (rA:rU) and poly
LETTER
TO THE
(rC:rI) systems. A more detailed interpretation elsewhere. Laboratory of Molecular Biology National Institute of Arthritis and Metabolic National Institutes of Health Bethesda, Maryland 20014, U.S.A.
EDITOR
569
of these findings will be reported
Diseases
PHILIP D. Ross ROBERT L. SCRUCGS
Received 3 July 1969 REFERENCES Chamberlin, M. J. & Patterson, D. L. (1965). J. Mol. Biol. 12, 410. Davies, D. R. & Rich, A. (1958). J. Amer. Chem. Sot. 80, 1003. Fasman, G. D., Lindblow, C. & Grossman, L. (1964). Biochemistry, 3, 1015. Krakauer, H. & Sturtevant, J. M. (1968). Biopolymers, 6, 491. Leng, M. & Michelson, A. M. (1968). Biochim. biophys. Acta, 155, 91. Neumann, E. & Ackerman, T. (1967). J. Phys. Chem. 71, 2377. Rawitscher, M. A., Ross, P. D. & Sturtevant, J. M. (1963). J. Amer. Chem. Sot. 85, 1915. Rich, A. (1958). Biochim. biophys. Actu, 29, 502. Ross, P. D. & Scruggs, R. L. (1965). Biopolymers, 3, 491. Scruggs, R. L. & Ross, P. D. (1969). J. Mol. Biol. in the press. Steiner, R. F. & Kitzinger, C. (1962). Nature, 194, 1172. Ts’o, P. 0. P., Helmkamp, G. K. & Sander, C. (1962). Biochim. biophys. Actu, 55, 584. Wadso, I. (1968). Actu them. Scud. 22, 927.