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(3) (a) W. Y. Wen, "Water and Aqueous Solutlons, Structure, Thermodynamics and Transport Processes," R . A. Horne, Ed., Wiley-lnterscience, New York, N. Y., 1971,Chapter XV. (b) H. L. Anderson and R. H. Wood, "Water Comprehensive Treatise," Vol. 3, F. Franks, Ed., Plenum Press, New York, N. Y.. 1973,Chapter XIV. (4) H. S . Frank and M. 9. Evans, J. Chem. Phys., 13,507(1945). (5) H. S. Frank and W. Y. Wen, Discuss. Faraday SOC., 124, 133 11957\ I . - - ' , .
(6) H. S.Frank, 2. Phys. Chem., 228,367 (1965). (7) W. Y. Wen and S.Saito, J. Phys. Chem., 68,2639 (1964). (8) W. Y. Wen, K. Miyajuma, and A. Otsuka, J. Phys. Chem., 75, 2148 (19711. (9) R. H. Wood and H. L. Anderson, J. Phys. Chem., 71,1871 (1967). (IO) S.Lindenbaum, J. Phys. Chem., 70,814 (1966). (11) R. H. Wood, H. L. Anderson, J. D. Beck, J. R. France, W. E. deVry, and L. J. Soltzberg, J. Phys. Chem., 71,2149(1967). (12) R. L. Kay, T. Vituccio, C. Zawoyoki, and D. F. Evans, J. Phys. Chem., 70,2336 (1966).
(13) R. L. Kay and D. F. Evans, J. Phys. Chem., 70,2325 (1966). (14) S. Lindenbaum, L. Leifer, G. E. Boyd, and J. W. Chase, J. Phys. Chem., 74,761 (1970). (15) H. L. Anderson and L. A. Petree, J. Phys. Chem., 74,1455 (1970). (16) H. L. Anderson, R. D. Wilson, and 0.E. Smith, J. Phys. Chem., 75, 1125 (1971). (17) H. L. Friedman, J. Phys. Chem., 32,1134 (1960). (18) See paragraph at end of text regarding supplementary material. (19) Y. C. Wu, M. 9. Smith, and T. F. Young, J. Phys. Chem., 69, 1868 (1965). (20) R. H.Wood and R. W. Smith, J. Phys. Chem., 69,2974 (1965). (21) H. S. Jongenburger and R. H. Wood, J. Phys. Chem., 69, 4231 (1965). (22) D. D. Ensor and H. L. Anderson, J. Chem. Eng. Data, 18, 205 (1973). (23) P. J. Reilly and R. H. Wood, J . Phys. Chem., 73,4292(1969). (24) D. D. Eiey, Trans. FaradaySoc., 35,128 (1939). (25) R. H. Wood, private communication.
Ultrasonic Absorption in Aqueous Solutions of Nucleotides and Nucleosides. 11. Kinetics of Proton Exchange in Adenosine 5'-Monophosphate Jacques Lang, Jean Sturm, and Raoul Zana" C. N.R.S., Centre de Recherches sur les Macromolecules, 67083 Strasbourg-Cedex, France
(Received July 6, 1973)
Publication costs assisted by the Centre National de la Recherche Scientitique, France
The excess ultrasonic absorption of aqueous solutions of 5'-adenosine mononucleotide (5'AMP) solutions has been measured in the frequency range 1-115 MHz and a t pH 5.05 where the absorption is maximum. The relaxation spectra can be fitted by a relaxation equation with a single relaxation frequency. The excess absorption is attributed to a proton exchange between two molecules of 5'AMP in differently ionized forms coupled with the stacking of the nucleotide molecules. (In this work stacking is assumed to be limited to dimerization.) Several reaction mechanisms are proposed and discussed. The results permit us to obtain the following new information: (1) the rate constants for the proton exchange between 5'AMP molecules and for the proton transfer involving the phosphate moiety of the nucleotide molecule and (2) the dimerization constant of 5'AMP and the volume change upon dimerization. This last quantity is found to be positive, i.e., of the same sign as for hydrophobic bonding.
Introduction In the first part of this work1 we reported ultrasonic absorption measurements on aqueous solutions of nucleosides and nucleotides showing maxima on the plots of alp ( a = ultrasonic absorption coefficient, f = frequency) us. pH, in the pH range 1-13. These maxima were attributed to the following two types of proton transfer equilibria: (a) hydrolysis and protolysis equilibria which appear to be responsible for the absorption maxima of small amplitude observed in the acid and/or in the alkaline range (Such equilibria have been extensively studied by means of ultrasonic methods for amino acid^,^,^ polypeptides,4 and protein^.^.^) and (b) proton exchange equilibria between three differently ionized forms of the same nucleotide. These processes occur only with nucleotides which contain a nitrogen atom protonable in the acid range. Thus far ultrasonic methods have been used only for the study of proton exchange between two different molecules.6 The purpose of this paper is to report new experimental results which allow a quantitative study of proton exchange for adenosine 5'-monophosphate (5'AMP) around The Journal ot Physical Chemistry, Voi. 78,No. 7, 1974
pH 5. In addition to the values of the rate constants and volume change for the proton exchange our results provide new evidence for the association of VAMP through base stacking. Materials a n d Methods 5'AMP has been purchased from Sigma and used without further purification. All solutions were prepared with freshly deionized distilled water. The pH values were adjusted by addition of small amounts of HC1 and NaOH solutions and measured using a Tacussel pH meter with a glass electrode and a standard calomel electrode. The pH meter was calibrated using Merck buffers. A two crystal interferometer' was used for the ultrasonic absorption measurements at frequencies between 1 and 10 MHz, and the standard pulse techniqueE in the range 6-150 MHz. Experimental Results We have previously shown (in ref 1 see ref 7 , Figure 5) that for aqueous solutions of 5'AMP the absorption maxi-
ai
Kinetics of Proton Exchange in Adenosine 5'-Monophosphate mum occurs at pH = pHa = 5.05 k 0.1, independent of concentration (in the range 0.02-0.15 M ) and ionic strength (in the range 0-0.15 M KC1). As part of the present work we have also found that pH.4 is independent of frequency. Within experimental error, pHA = (pKp2 + pKN)/2 where p K p ~= 6.45 and pKN = 3.8 are the pK,'s of the secondary phosphoric acid function and of the protonable N1 nitrogen atom of 5'AMP, respectively. In this work ultrasonic absorption measurements have been performed in the range 1-115 MHz on four solutions of 5'AMP with concentrations 0.016, 0.05, 0.099, and 0.148 M a t pH = ~ H and A at pH >9 where the proton exchange contribution to the excess absorption of the solution is neg1igible.l The difference Aa/f2 between the values of a/f2 at a given pH and at pH >9 can be taken as the contribution of the proton exchange reaction. In Figure 1 the value ( A a / f 2 )of~ Aa/f2 at pH = pHA, has been plotted as a function o f f for four solutions of S'AMP. (See paragraph at end of paper regarding supplementary material.) These results obey eq 1, where fR is the relaxation frequency and A R the relaxation amplitude at f
