J. Phys. Chem. B 1998, 102, 9181-9186
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Reexamination of Ultrasonic Relaxation Kinetics of Aqueous Solutions of Nucleotides: Evidence for Fast Syn-Anti Glycosyl Isomerization in Adenosine 5′-Monophosphate and Adenosine 5′-Diphosphate Naoki Kuramoto† and Sadakatsu Nishikawa* Department of Chemistry and Applied Chemistry, Faculty of Science and Engineering, Saga UniVersity, Saga 840-8502, Japan
Frank Jordan Department of Chemistry, Rutgers, The State UniVersity of New Jersey, Newark, New Jersey, 07102 ReceiVed: July 8, 1998; In Final Form: August 26, 1998
Ultrasonic absorption coefficients in aqueous solutions of adenosine 5′-monophosphate and adenosine 5′diphosphate were measured at 25 °C as a function of the concentration and pH in the frequency range from 0.8 to 220 MHz. At pH near 5, there existed two relaxational absorptions. One is observed at the frequency range 11, the relaxation associated with the proton transfer reaction disappeared. A second relaxation is found at around 100 MHz, and its amplitude is smaller than that due to the proton transfer reaction. This relaxation is barely observed at neutral pH, but it is clearly distinguishable at high pH because the absorption associated with the proton transfer reaction is no longer observable. The relaxation frequency of the second relaxation is independent of nucleotide concentration and the solution pH, and the maximum absorption per wavelength increases linearly with concentration. It was concluded that the source of this relaxation is an isomerization process, probably the syn-anti interconversion of nucleotides. The value of the relaxation frequency in aqueous solutions of ADP was greater than that in AMP solutions, which in turn is greater than that for adenosine. The results are discussed in relation to nucleotide molecular structures and interactions.
1. Introduction Because of the central role of the nucleic acids in biological systems, intensive studies have been carried out on the dynamic behavior of nucleic acids and their constituents in aqueous media by various theoretical and experimental approaches. Ultrasonic absorption method is one of the chemical relaxation techniques which provides useful information concerning both the fast dynamic characteristics and thermodynamic properties of reactions taking place in liquids. Rhodes and Schimmel1 carried out ultrasonic absorption measurements on aqueous solutions of adenosine 5′-monophosphate (AMP) in the vicinity of pH 5 and observed a single relaxational process with the relaxation frequency less than 10 MHz. The source of the relaxational absorption was attributed to an intermolecular proton transfer reaction between a protonated base (at N1 of adenine) of one AMP molecule and an anionic phosphate group of another molecule. However, the relaxation frequencies determined by Rhodes and Schimmel are below or at the lower limit of their experimentally accessible frequency range. Also, they predicted another relaxation process at the higher frequency range due to an isomerization reaction, although the mechanism was not * Corresponding author. E-mail:
[email protected]. † Research Fellow of the Japan Society for the Promotion of Science.
clarified. Lang et al.2 reexamined the ultrasonic absorption behavior of the same system in the 1-115 MHz frequency range and attributed the observed relaxational absorption to the intermolecular proton transfer reaction already proposed by Rhodes and Schimmel.1 It had been suggested in the prior study by Rhodes and Schimmel3 that a unimolecular relaxation process found in aqueous solutions of adenosine, 2′-deoxyadenosine, and guanosine could be associated with the syn-anti glycosyl rotational isomerization. As for nucleotides, the relaxational absorption due to the same reaction had been observed in aqueous solutions of adenosine cyclic 3′,5′-monophosphate4 and those of cytidine cyclic 2′,3′-monophosphate in the presence of ethidium bromide.5,6 However, there have been no systematic reports on the syn-anti glycosyl isomerization reaction in any noncyclic nucleotide. Kinetic and thermodynamic information on the syn-anti glycosyl isomerization in the nucleotides is important to clarify the conformational preferences of the polynucleotide chain and the interaction of nucleotides with intercalating agent. We also note that the discovery of the lefthanded Z form of DNA (in addition to the more conventional A and B forms) in which purines prefer the syn conformation has made it clear that the likelihood of observation of syn conformations in biological systems is significantly greater than had been assumed earlier.7 Thus, it is highly desirable to
10.1021/jp9829041 CCC: $15.00 © 1998 American Chemical Society Published on Web 10/21/1998
9182 J. Phys. Chem. B, Vol. 102, No. 45, 1998
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characterize the syn-anti rotational motion in noncyclic nucleosides or nucleotides. With our current instrumentation of resonators,8 it is timely to confirm the ultrasonic absorption mechanisms in aqueous solutions of nucleotides more precisely. The purpose of the present study is to examine the dynamic characteristics in aqueous solutions of adenosine 5′-monophosphate (AMP) and adenosine 5′-diphosphate (ADP), taking into account the two reactions mentioned above, the intermolecular proton-transfer reaction and the syn-anti glycosyl isomerization. 2. Experimental Section Chemicals. Adenosine 5′-monophosphate sodium salt (AMP, more than 99.7% pure) and adenosine 5′-diphosphate sodium salt (ADP, more than 93.4% pure) were purchased from Wako Pure Chemicals Co., Ltd. as the purest grade and were used without further purification. Sodium hydroxide was also from Wako Pure Chemicals Co., Ltd. and a concentrated aqueous solution was used to adjust the solution pH to the desired value. Water used in the experiment was distilled and filtered through a MilliQ SP-TOC system from Japan Millipore Ltd. Sample solutions were prepared by weighing. Measurements. The ultrasonic absorption coefficient, R, was measured by a pulse method in the frequency range of 8.5220 MHz using 0.5, 5, and 20 MHz fundamental X-cut quartz crystals.9 A resonance method was utilized to obtain the absorption coefficient in the lower frequency range. The resonator cell with 3 MHz fundamental X-cut quartz with a diameter of 3 cm was used to measure the sound absorption coefficient in the frequency range below 4.5 MHz. In the frequency range of 3-7 MHz, the cell with 5 MHz fundamental crystal with 2 cm diameter was used. Details of the instrument construction and characteristics and the procedure used for the determination of the absorption coefficient were described elsewhere.8,9 The sound velocity was measured using the resonance method at around 3 MHz. The pH was measured with a glass electrode (HM-60S Toa Denpa), while the density was measured with a vibrating density meter (DMA 60/602 Anton Paar). All the measurements were performed in dry N2 gas atmosphere to avoid as much as possible carbon dioxide contamination during the measurements. The measurements were made at 25.0 °C. 3. Results and Discussion Plots of the values of R/f 2 against the frequency near pH 5 are shown in Figure 1. A decreasing trend of R/f 2’s starts from about 1 MHz and they nearly reach plateaus at around 50 MHz, beyond which there is a slight further decrease. This suggests that there exist multiple relaxational absorptions in the solutions. However, the amplitude of the relaxation observed at AMP > ADP. However, the experimental results show the opposite trend. According to the previous report,3 the relaxation frequency for the syn-anti isomerization reaction for 2′deoxyadenosine is a factor of 2 greater than that for adenosine. It had been speculated that this difference is due to the interaction of 2′-hydroxyl group in adenosine with the N3 atom of adenine as the rotation occurs between the syn and anti isomers. This suggests that the interaction between the base and some group on the ribose plays an important role for the rotational motion around the glycosyl bond. Jordan et al.6 reported that the addition of ethidium bromide to 2′-deoxyadenosine leads to a reduction of the barrier to the syn-anti rotation. This reduction of the barrier was attributed to the participation of ethidium ion in stacking interaction with 2′deoxyadenosine molecules. This further suggests that the change in charge distribution in the base by stacking with the intercalator affects the rotational motion. Under the condition of high pH used in the present study, the phosphate groups in the nucleotides are fully ionized. The negative charges on the phosphate group may reduce the attractive interaction between the phosphates and the base, leading to the observed change in the rate of the rotational isomerization. That is, the activation energy for forming the syn form in the nucleotide may be reduced by the introduction of the ionized phosphate group, which would account for the rate constant for isomerization varying in the order adenosine < AMP < ADP. The results further confirm that in the solutions there is a mobile equilibrium in many common nucleotides and nucleosides, and, especially
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Figure 7. Ultrasonic absorption spectra in 0.135 mol dm-3 AMP solution at pH 4.919, which are analyzed by the Debye-type relaxational equation with two relaxation processes. fr1 ) (5.27 ( 0.08) MHz, A1 ) (566 ( 5) × 10-15 s2 m-1, fr2 ) (82 ( 9) MHz, A2 ) (11.1 ( 0.8) × 10-15 s2 m-1, and B ) (22.9 ( 0.3) × 10-15 s2 m-1. Curves 1 and 2 are the individual components of the two relaxational absorptions.
at high pH where the phosphates were fully ionized the energies of the two conformers cannot be too dissimilar. Had the equilibrium favored one conformer predominantly, we should not have observed the high-frequency relaxation in any of the three compounds studied. Finally, it should be confirmed that how much the ultrasonic parameters for the proton transfer reaction affect those for the syn-anti isomerization reaction in the relatively concentrated solutions of AMP at neutral pH’s because the tails of the higher relaxation are observed at 0.135 and 0.235 mol dm-3 as is seen in Figure 1. The Debye-type double relaxational equation was applied to those data in order to obtain the ultrasonic parameters.
µ ) (R/f2 - B)fc ) A1fc/[1 + (f/fr1)2] + A2fc/[1 + (f/fr2)2] (10) One of the results is shown by the modified form in Figure 7, where the ultrasonic parameters are indicated in the caption. As may be seen, the parameters in the lower frequency range are not so influenced even if the higher relaxational absorption exists. This means that the analysis of the frequency dependence of the absorption in the lower range is appropriate. However, the relaxation frequency associated with the syn-anti isomerization shifts to the lower frequency range. This may arise from a different ionization condition of phosphate moiety of AMP because the solution pH is different. To gain further insight into the relationship between the structure of a nucleotide and the syn-anti rotational motion, experiments with adenosine 5′-triphosphate are currently in progress, as are studies on the temperature dependence of the absorption. The results will be reported in due course. Acknowledgment. This work was partly supported by a Grant-in-Aid for Science Research No. 09440202 from the Ministry of Education, Science and Culture of Japan and Research Fellowship of the Japan Society for the Promotion of Science for Young Scientist to N.K. References and Notes (1) Rhodes, L. M.; Schimmel, P. R. J. Am. Chem. Soc. 1974, 96, 26092611. (2) Lang, J.; Sturm, J., Zana, R. J. Phys. Chem. 1974, 78, 80-86.
9186 J. Phys. Chem. B, Vol. 102, No. 45, 1998 (3) Rhodes, L. M.; Schimmel, P. R. Biochemistry 1971, 10, 42264433. (4) Hemmes, P.; Oppenheimer, L.; Jordan, F. J. Chem. Soc., Chem. Commun. 1976, 929-930. (5) Hemmes, P.; Oppenheimer, L.; Jordan, F.; Nishikawa, S. J. Phys. Chem. 1981, 85, 98-101. (6) Jordan, F.; Nishikawa, S.; Hemmes, P. J. Am. Chem. Soc. 1980, 102, 3913-3917. (7) Rich, A.; Nordheim, A.; Wang, A. H.-J. Annu. ReV. Biochem. 1984, 53, 791-846. (8) Kuramoto, N.; Ueda, M.; Nishikawa, S. Bull. Chem. Soc. Jpn. 1994, 67, 1560-1564.
Kuramoto et al. (9) Nishikawa, S., Kotegawa, K. J. Phys. Chem. 1985, 89, 28962900. (10) Hemmes, P.; Oppenheimer, L.; Rhinesmith, R.; Anderle, G.; Saar, D.; Jordan, F. J. Phys. Chem. 1980, 84, 911-913. (11) Robinson, R. A.; Stokes, R. H. In Electrolyte Solutions, 2nd ed.; Butterworth: London; p 232. (12) Behrends, R.; Cowman, M. K.; Eggers, F.; Eyring, E. M.; Kaatze, U.; Majewski, J.; Petrucci, S.; Richmann, K.; Riech, M. J. Am. Chem. Soc. 1997, 119, 2182-2186. (13) Aubard, J.; Dubois, J. E. J. Phys. Chem. 1980, 84, 1413-1416.
