10492 Langmuir 2005, 21, 10492-10496 Changes in Hydration of Lanthanide Ions on Binding to DNA in Aqueous Solution Diana Costa,* Hugh D. Burrows, and M. da Grac¸a Miguel Departamento de Quı´mica, Universidade de Coimbra, 3004-535 Coimbra, Portugal Received June 6, 2005. In Final Form: August 11, 2005 The interaction of the trivalent lanthanides Ce(III), Eu(III), and Tb(III) with sodium deoxyribonucleic acid (DNA) in aqueous solution has been studied using their luminescence spectra and decays. Complexation with DNA is indicated by changes in luminescence intensity. In the system terbium(III)-DNA, changes in luminescence with pH are suggested to be due to the protonation of phosphate groups. The degree of hydration of Tb(III) on binding to DNA is followed by luminescence lifetime measurements in water and deuterium oxide solutions, and it is found that the lanthanide ion loses at least one hydration water on binding to long double stranded DNA at pH 4.7 and pH 7. Rather different behavior is observed on binding to long or short single stranded DNA, where six water molecules are lost, independent of pH. It is suggested that in this case the lanthanide probably binds to the bases of the DNA backbone. The DNA conformation seems to be an important factor in the binding. In addition, the isotopic effect on terbium luminescence lifetime may provide a useful method to distinguish between single and double stranded DNA. DSC results are consistent with cleavage of the double helix of DNA at pH 9 in the presence of terbium. 1. Introduction of the interactions of metal ions with DNA. Nucleic acids may be considered to be ambidentate ligands, with various DNA is a negatively charged polyelectrolyte, and the potential binding sites, including nitrogen and oxygen binding of metal cations to it is of importance since this donors on the bases, hydroxyl groups on the ribose sugar, plays a crucial role in the biological activity of nucleotides and negatively charged oxygen atoms in the phosphate and nucleic acids, changing their properties in ways that groups. As a consequence of the abundance of negatively depend on the nature of the metal ion.1 Knowledge of the charged oxygen donor groups, the DNA molecule readily mechanisms responsible for the activity of these systems interacts with Ln3+ ions and may be expected to occupy requires the study of metal ion-nucleic acid interactions. These can lead to DNA denaturation, compaction, ag- at least some of the inner-sphere coordination sites of the 2,3 cations, contributing to the coordination process by gregation, or precipitation. It has been shown that 14 cations with a valence g3 are generally required to induce completing chelate bridges. Interactions are expected to have pronounced effects on the spectral properties of these the condensation of DNA in aqueous solution. Among the 3+ 3+ 3+ multivalent cations, spermidine(IV),4 spermine(III),5 and ions. The luminescence properties of Tb ,Eu , and Ce the hexaamminecobalt complex, [Co(NH ) ]3+ 6 have been make them versatile in their applications to examination 3 6 15 shown to induce DNA condensation through studies of biomolecular structures. The first two of these cations involving light-scattering, sedimentation, electron mi- are of special interest due to the fact that their resonance croscopy, and various spectroscopic methods.7,8 Although energy levels overlap the triplet energy states of protein electrostatic interactions are likely to be of major impor- and nucleic acid aromatic residues leading to enhancement tance in the binding, other factors may also be involved,9-12 of their natural fluorescence by energy transfer when and many questions still remain unanswered on the irradiated with ultraviolet light.16 Similar behavior has Downloaded by PORTUGAL CONSORTIA MASTER on June 29, 2009 driving force for this process. been observed on binding by other polyelectrolyte sys- tems.17 Binding stoichiometries remain a problem in Published on September 21, 2005 http://pubs.acs.org | doi: 10.1021/la051493u Trivalent lanthanide ions have attractive, spectroscopic, 3+ and magnetic properties13 and have been used as probes carrying out quantitative staining studies, but when Tb or Eu3+ luminescence exhibits significant sensitivity to + binding these may be estimated from luminescence * To whom correspondence should be addressed. Fax: 351-239 18-21 82 7703. E-mail: [email protected]. titration measurements. These cations possess many (1) Stryer, L. Biochemistry; W. H. Freeman and Company: San similarities in their properties to calcium(II) and the Francisco, CA, 1994. isomorphous replacement of this cation by Ln3+ ions holds (2) Bloomfield, V. A. Biopolymers 1997, 44, 269-282. out the promise of exploiting the rich and variety (3) Duguid, J. A.; Bloomfield, V. A. Biophys. J. 1995, 69, 2623-2641. (4) Gosule, L. C.; Schellman, J. A. Nature 1976, 259, 333-335. spectroscopic properties of the lanthanide ions for obtain- (5) Gosule, L. C.; Schellman, J. A. J. Mol. Biol. 1978, 121, 311-333. ing information on the structure of Ca(II) in biological (6) Widom, J.; Baldwin, R. L. J. Mol. Biol. 1980, 144, 431-453. (7) Bloomfield, V. A. Curr. Opin. Struct. Biol. 1996, 6, 334-341. (8) Bloomfield, V. A.; Ma, C.; Arscott, P. G. In Macro-Ion Charac- (14) Marzilli, L. G. Prog. Inorg. Chem. 1977, 23, 255-378. terization: From Dilute Solutions to Complex Fluids; Schmitz, K. S., (15) Richardson, F. S. Chem. Rev. 1982, 82, 541-552. Ed.; American Chemical Society: Washington, DC, 1994; pp 195. (16) Yonushot, G.; Mushrush, G. W. Biochemistry 1975, 14, 1677- (9) Manning, G. S. Acc. Chem. Res. 1979, 12, 443-449. 1681. (10) Lau, A. W. C.; Pincus, P. Phys. Rev. E 2002, 66, 041501. (17) Tapia, M. J.; Burrows, H. D. Langmuir 2002, 18, 1872-1876. (11) Schwinefus, J. J.; Bloomfield, V. A. Biopolymers 2000, 54, 572- (18) Barala, T. D.; Burchett, J.; Kizer, D. E. Biochemistry 1975, 14, 577. 4887-4892. (12) Wennerstrom, H. Curr. Opin. Colloid Interface Sci. 2004, 9, 163- (19) Furie, B. C.; Furie, B. J. Biol. Chem. 1975, 250, 601-608. 164. (20) Martin, R. B.; Richardson, F. S. Q. Rev. Biophys. 1979, 12, 181- (13) Bu¨ nzli, J.-C. G.; Choppin, G. R. Lanthanide Probes in Life, 209. Chemical and Earth Sciences: Theory and Practice; Elsevier: Am- (21) Snyder, A. P.; Sudnick, D. R.; Aekle, V. K.; Horrocks, W. DeW., sterdam, 1989. Jr. Biochemistry 1981, 20, 3334-3339. 10.1021/la051493u CCC: $30.25 © 2005 American Chemical Society Published on Web 09/21/2005 Changes in Hydration of Lanthanide Ions Langmuir, Vol. 21, No. 23, 2005 10493 systems.22,23 Also, their ability to substitute Zn(II)24,25 and involving lanthanide ions.50,51 However, in the latter case, Mg(II) at their binding sites makes lanthanide lumines- this requires relatively close contact and is favored by cence a useful tool to study interaction between DNA and complexation52 or binding of complexes of the ions to these ions.26-29 In addition, polyelectrolyte/lanthanide ion charged species, such as micelles,53 phospholipid or various systems are amenable to modeling by Monte Carlo and vesicles,54 or other biologically relevant systems.55 other techniques, which can help provide a more detailed Luminescence decay lifetimes of lanthanides provide a understanding of the binding behavior.30 direct measure of the number of metal-coordinated water The luminescence of the lanthanide ions arises from f molecules. Replacement of OH oscillators by the OD ones f f electron transitions and this can give information on causes the vibronic deexcitation pathway to become both the coordination environment31,32 and degree of exceedingly inefficient, and the resultant isotope effect hydration of these ions.23,33 There is increasing interest on luminescence lifetimes permits the determination of - in long-lived luminescent probes34 36 and lanthanide ions the number of water molecules in the first coordination would appear to be good candidates for this, particularly sphere of metal ion,56-62 in addition to the changes in as their emission is not quenched by oxygen. The hydration on lanthanide ion binding. luminescence of Ln(III) ions is extremely weak when We have carried out a detailed study of the association compared to organic fluorophores, principally because of of trivalent lanthanide ions Ce3+,Eu3+, and Tb3+ with ∼ -6 the low oscillator strength ( 10 ) of their absorption double stranded DNA (2000 base pairs) using their 37,38 f bands. This is because lanthanide f f transitions are luminescence. We are aware that lanthanide ions catalyze generally forbidden by both spin and Laporte selection the hydrolysis of nucleic acids.63 However, this effect is 39,40 rules. In contrast, with cerium(III), the lowest energy not important under our experimental conditions. To study electronic band in absorption corresponds to the allowed the mechanisms of binding, the following well-defined f 4f 5d transition. Although this results in a much broader DNA samples have been used: (1) long ds-DNA (2000 band than with the other trivalent lanthanides, it does base pairs), long ss-DNA (heat-denatured 2000 bases), mean that the transition has a reasonable molar absorp- and short ss-DNA (580 and 831 base pair fragments). 41,42 tion coefficient. In certain cases, the inherent weakness Particular emphasis is placed on the effect of binding on of Ln(III) ion luminescence may be overcome by an energy the cation hydration sphere. transfer process, and interest in energy transfer in these systems has increased dramatically in the past few 2. Materials and Methods years43-47 due to applications in luminescent assays in physics, chemistry, and biochemistry. For this phenomena, Cerium (III), europium (III), and terbium (III) perchlorates both dipole-dipole (Fo¨rster)48 and exchange (Dexter)49 from Aldrich were of the purest grade available and were used mechanisms have been suggested for energy transfer as received.