1762
Acknowledgments. We thank Mr. David Lee for experimental assistance, Professors Hubbell and Sauer for the use of their equipment, and Dr. Eileen N. Duesler
for helpful discussions. This research was supported by the National Institutes of Health (Grant AI 1 1744) and, in part, by the National Science Foundation.
Heavy Metal-Nucleotide Interactions. Binding of Methylmercury( 11) to Pyrimidine Nucleosides and Nucleotides. Studies by Raman Difference Spectroscopy’ Samir Mansy, Thomas E. Wood, Jolyon C. Sprowles, and R. Stuart Tobias*
Contribution from the Department of Chemistry, Purdue University, West Lafayette, Indiana 47907. Received October 12, 1973 Abstract: Using data on the equilibrium constants for hydrolysis of CH3Hgf and for its binding to uridine and cytidine, solutions in the 10-100 mM range have been prepared for which a particular complex should predominate.
Raman spectra and particularly Raman difference spectra have been used to determine the perturbations of the cation and of the nucleoside ligand vibrations upon metal binding. The differencetechnique, applied here for the first time, is particularly effectivefor observing small spectral changes. Spectra have been obtained for the complex with polyuridilic acid to show that the perturbations are very similar with polynucleotides. A procedure for determining heavy metal binding sites on polynucleotides with two or more base moieties in aqueous solution is outlined. The methylmercury(I1)ion binds to uridine (Urd) with displacement of a proton and coordination to N(3). Binding to cytidine also occurs at N(31, although, at pH 7, coordination to Urd is favored. The behavior of CH,Hg+ and Hg2+are compared.
T
he binding of metals by nucleosides and nucleotides has been investigated for a number of years. Because of recent observations that CH3Hg’ causes chromosome damage and consequently is and that certain platinum(I1) compounds inhibit mitosis by selective inhibition of DNA ~ y n t h e s i s , there ~ ’ ~ is renewed interest in the binding of heavy metals to polynucleotides. In this work we have examined the interaction between CH3Hg” and pyrimidine nucleosides and nucleotides. Equilibria of CH3Hg+ are relatively simple, since it is primarily a unifunctional electrophile. Consequently, the methylmercury cation should serve as a model for the binding of heavy metals to nucleosides, nucleotides, and polynucleotides. The mutagenic effect of the unifunctional CH3Hg+ electrophile compared to the antimitotic effect in tumor tissue of the (presumed) bifunctional (NH3)2Pt11parallels the activity of uni- and bifunctional organic alkylating agents.6,’ In 1961, Ferreira, et aZ.,8 noted that CH3Hg+and CzH5Hg+ formed complexes with dThd,g and the competition reaction between H+ and CH3Hg+was studied. (1) Work supported by Public Health Service Grant AM-16101 from the National Institute for Arthritis, Metabolism, and Digestive Diseases and by the National Science Foundation Grant GP-23208. ( 2 ) G. Lofroth, “Methylmercury,” Bulletin No. 4, Swedish National Science Research Council, Stockholm, 1969. (3) J. J. Mulvihill, Science, 176,132 (1972). (4) H . C. Harder and B. Rosenberg, Int. J . Cancer, 6,207 (1970). (5) G . R . Gale J. A. Howle, and E. M. Walker, Jr., Cancer Res., 31, 950 (1971). ( 6 ) W. C. J. Ross, “Biological Alkylating Agents,” Butterworth, London, 1962. (7) A. Haddow, Adcan. Cancer Res., 16,181 (1972). (8) R . Ferreira, E. Ben-Zvi, T. Yamane, J. Vasilevskis, and N. Davidson, “Advances in the Chemistry of the Coordination Compounds,” S . Kirschner, Ed., Macmillan, New York, 1961, p 457. (9) The IUPAC-IUB abbreviations for nucleosides, etc., are employed throughout; see Biochemisfry, 9,4022 (1970).
Journal of the American Chemical Society
1 96:6
Because of the proton dependence, binding was assumed to occur at N(3). In 1966, Gruenwedel and Davidson’O obtained a value for the equilibrium constant for dThd reacting with CH3HgOH by measuring the effect of dThd on the distribution between an aqueous and a toluene phase. Simpson” in 1964 used uv spectrophotometric measurements to obtain equilibrium constants for binding to Urd and to Cyd. Binding was assumed to occur at N(3)in both cases. With Cyd at high pH, a second reaction was observed, and this was assumed to be C H 3 H g binding ~ t s the C(4)NH2group. Carrabine and Sundaralingham12 recently determined the structure of the crystalline adduct HgClz.2Ura (Ura = uracil). This consists of a linear HgClz molecule coordinated to one oxygen (CC4,=O)from each of two Ura m9lecules with rather short Hg-0 interactions of 2.71 (2) A . In addition there are two chlorides from adjacent HgC12 molecules coordinated about mercury giving distorted octahedral coordination. On the basis of the HgClz.2Ura structure, Carrabine and Sundaralingham suggested that reaction of mercury(11) with Ura, Urd, and dThd in aqueous solution occurs by coordination only with the oxygen of the C(q=O group. The pH dependence of the binding was suggested to be caused by proton transfer from a water molecule in the first coordination sphere of the bound Hg2+. This, however, does not account for the similar proton dependence observed for CH3Hg+ and C2Hr Hg+ 8 where the second principal coordination site of mercury is blocked by the inert carbanion ligands. Recently the usefulness of Raman spectroscopy in (10) D. W. Gruenwedel and N. Davidson, J. Mol. Biol., 21, 129 (1966). (1 1) R. B. Simpson, J . Amer. Chem. Soc., 86,2059 (1964). (12) J. A. Carrabine and M. Sundaralingham, Biochemisfry, 10, 292(1971).
March 20, 1974
1763 Ligand Solutions. Cyd was obtained from Clyco Chemical, polynucleotide chemistry has been demonstrated amply.13 The binding of H+, Na+, Mg2+, and Ca2+ LOS Angeles, Calif., and International Chemical and Nuclear, Irvine, Calif., Ura from Sigma Chemical, St. Louis, Mo., Urd from to the phosphate of ATP has been studied,l4Il5 and Aldrich Chemical, Milwaukee, Wis., and Poly U from Miles Labconsiderable progress has been made in using Raman oratories, Kankakee, Ill. Weighed amounts were dissolved in deionized, doubly distilled H 2 0 or 993Z D20 to provide stock spectroscopy to follow conformational changes in polysolutions. The pH’s (pD’s) of solutions containing nucleosides nucleotides. l6-l8 or nucleotides and CHaHgC were adjusted with HC10, (DClO4) The present work was begun with several aims in or NaOH (NaOD) solutions using a Radiometer PHM-4 pH meter. mind. Firstly, we wished to examine the reactions of For the deuterium oxide solutions, a standard glass electrode was CH3Hg+ because of its known physiological effects. used, and the meter reading was corrected by the procedure of Glascoe and Long. 2 3 Secondly, it appears that this cation can serve as a Raman Spectra. Spectra were excited with the 5145-A line of a model for heavy metal binding to nucleosides. ConCoherent Radiation Model 52 Ar+ laser at ca. 800 mW. The sequently, if information on the perturbation of the Raman scattering was dispersed with a Spex 1400 double mononucleoside or nucleotide vibrations caused by metal cochromator and detected with an RCA 31034A photomultiplier tube. For the difference spectra, a programmed sample carrier ordination at specific sites can be cataloged, Raman for alternate positioning of reference and sample cells in the laser spectroscopy can be used in a straightforward fashion beam was employed. Single photon counting equipment was used to ascertain the sites at which other heavy metals bind. to count for a preset period of time, usually 10 sec, at each waveIn this context, we were interested in resolving the queslength value. This number and a digital signal from a photo diode tion concerning the site of the binding of Hg(I1) to laser power monitor used to normalize the spectra were punched on paper tape. Spectra were plotted and band maxima located rouUrd and dThd. Finally, it has been shown that the tinely off-line with a Hewlett Packard 2116A computer (8K X principal spectral features of polynucleotides can be 16 bit memory) system using PROGRAM RSPC written by R. W. obtained by summing the spectra of the constituent Chrisman. 2 4 More complex spectral analyses were performed offnucleotides in the proper ratio. l 9 Similarly, it should line with a CDC 6500 computer using PROGRAM RAMAN written by be possible to reproduce the spectra of polynucleotides J. W. L ~ n d e e n . ’ ~A detailed description of the difference spectrophotometer has been given elsewhere.26 which have a heavy metal bound to some of the base In order to compare intensities in a series of spectra, c.g., in a moieties by summation of the spectra of metallated and continuous variation experiment, the intensities were scaled so YI unmetallated nucleotides in the proper ratios. On this of the internal Clod- reference was the same. This is an option basis, laser Raman spectroscopy would appear to be a in RAMAN, and the procedure involves the construction of a baseline and a numerical integration over the P I envelope. very useful tool for determining the bases to which bindIn general, solutions were clarified by filtration through 100-nm ing occurs preferentially and in establishing conformapore size ultrafilters or, with small samples, through fine frits as tional changes brought about by metal binding. used for Rayleigh scattering studies on biological molecules. * 6 Mansy, et U Z . , ~ O recorded Raman spectra of several The samples were contained in 1-ml cells with optically flat winsynthetic polynucleotides as well as calf thymus DNA dows. The cell was maintained at 25 f 1 “ by mounting it in a ] ~ +block through which water was circulated from a constant in the presence of cis- and t r u n ~ - [ P t ( N H ~ ) ~ ( H ~ 0 ) ~brass temperature bath. In the difference measurements, the sample and suggested binding sites and conformational changes temperature was ca. 22 ’. on the basis of changes in the spectra from those of the pure polynucleotides. Lord and Thomas2 investigated Data and Results the interaction of HgClz with the nucleosides Cyd and Species Distribution. Since approximate equilibrium Urd using Raman spectroscopy. Changes in the constantsll for binding of CH3Hg-t to Urd and Cyd spectrum of Cyd were observed upon addition of Hgare known, we have attempted to obtain Raman spectra Clz which were similar to those occurring upon protonaunder conditions such that one methylmercury(I1)tion, and N(3)was assigned as the binding site. N o innucleoside complex predominates in the solution. The teraction could be detected with Ura, Urd, 1-MeUra, or methylmercury(I1) cation is rather acidic, and, un1,3-Me2Ura. fortunately, upon hydrolysis it forms polynuclear comIn this work, we also give the first demonstration of plexes. In the determination of the hydrolysis conthe usefulness of Raman difference spectroscopy (RDS) s t a n t ~ , *CH3Hg+ ~ concentrations of only up to 22 m M in the study of changes in biological molecules. were employed; even at these concentrations the binuclear complex (CH3Hg),0H+ is an important species. Experimental Section In more concentrated solutions, it is likely that a triMethylmercury(I1) Perchlorate. Methylmercuric iodide obnuclear species also is produced, since [(CH3Hg)30+]tained from Alfa Inorganics was recrystallized from ethanol, mp Clod- precipitates when (CH3Hg)20is treated with per144” (lit. 143”). This was treated with a standard aqueous solution (HzO or DzO) of AgC104 (G. F. Smith) while stirring conchloric acid in aqueous solution.28 Because of the tinuously to give a stock solution of CH3HgC10a. A test was run uncertainty about the methylmercury(I1) species present for unreacted Ag+ by titrating the solution with base, log K , , A , ~ E in solution at higher concentrations, the total stoi= -7.4.” chiometric concentration was no larger than 50 m M in any of the solutions studied (13) Raman spectra of biological molecules have been reviewed recently: J. L. Koenig, J . Polym. Sci., Part D,6,59 (1972). (14) L. Rimai and M. E. Heyde, Biochem. Biophys. Res. Commun., 41,313 (1970). (15) M. E. Heyde and L. Rimai, Biochemistry, 10,1121 (1971). (16) E. W. Small and W. Peticolas, Biopolymers, 10,69 (1971). (17) E. W. Small and W. Peticolas, Biopolymers, 10, 1377 (1971). (18) K . G . Brown, E. J. Kiser, and W. L. Peticolas, Biopolymers, 11, 1855 (1972). (19) M . Tsuboi, S . Takahashi, S. Muraishi, T. Kajiura, and S. Nishimura, Science, 174, 1142 (1971). (20) S . Mansy and B. Rosenberg, unpublished data. (21) R. C. Lord and G. J. Thomas, Biochim. Biophys. Acta, 142, 1 (1967).
(22) L. G. Sillen and A. E. Martell, Chem. Soc., Spec. Publ., NO.17, 61 (1964). (23) P. I
