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A potentiometric and spectroscopic (UV/vis and CD) study of Cu(II) and Ni(II) binding to the N-terminal pentadecapeptide of human protamine HP2 (HP2(1-15)) was performed. The results indicate that the N-terminal tripeptide motif Arg-Thr-His is the exclusive binding site for both metal ions at a metal to HP2(1-15) molar ratio not higher than 1. The very high value of protonation-corrected stability constant (log *K) for Ni(II)-HP2(1-15) complex, -19.29, indicates that HP2 has the potential to sequester Ni(II) from other peptide and protein carriers, including albumin. The same is likely for Cu(II) (log *K = -13.13). The CD spectra of Cu(II) and Ni(II) complexes of HP2(1-15) indicate that the N-terminal metal binding affects the overall conformation of the peptide that, in turn, may alter interaction of HP2 with DNA. These results imply HP2 as a likely target for the toxic metals Ni(II) and Cu(II).

The extent to which the replacement of a ribose unit by a 2'-deoxyribose unit influences the acid-base properties of nucleotides has not hitherto been determined in detail. In this study, by potentiometric pH titrations in aqueous solution, we have measured the acidity constants of the 5'-di- and 5'-triphosphates of 2'-deoxyguanosine [i.e., of H(2)(dGDP)(-) and H(2)(dGTP)(2-)] as well as of the 5'-mono-, 5'-di-, and 5'-triphosphates of 2'-deoxyadenosine [i.e., of H(2)(dAMP)(+/-), H(2)(dADP)(-), and H(2)(dATP)(2-)]. These 12 acidity constants (of the 56 that are listed) are compared with those of the corresponding ribose derivatives (published data) measured under the same experimental conditions. The results show that all protonation sites in the 2'-deoxynucleotides are more basic than those in their ribose counterparts. The influence of the 2'-OH group is dependent on the number of 5'-phosphate groups as well as on the nature of the purine nucleobase. The basicity of N7 in guanine nucleotides is most significantly enhanced (by about 0.2 pK units), while the effect on the phosphate groups and the N1H or N1H(+) sites is less pronounced but clearly present. In addition, (1)H NMR chemical shift change studies in dependence on pD in D(2)O have been carried out for the dAMP, dADP, and dATP systems, which confirmed the results from the potentiometric pH titrations and showed the nucleotides to be in their anti conformations. Overall, our results are not only of relevance for metal ion binding to nucleotides or nucleic acids, but also constitute an exact basis for the calculation, determination, and understanding of perturbed pK(a) values in DNAzymes and ribozymes, as needed for the delineation of acid-base mechanisms in catalysis.

Nickel(II) compounds are established human carcinogens, but the molecular mechanisms underlying their activity are only partially known. One mechanism may include mediation by nickel of promutagenic oxidative DNA damage that depends on Ni(II) binding to chromatin. To characterize such binding at the histone moiety of chromatin, we synthesized the peptide CH3CO-Cys-Ala-Ile-His-NH2 (L), a model of the evolutionarily conserved motif in histone H3 with expected affinity for transition metals, and evaluated its reactivity toward Ni(II). Combined spectroscopic (UV/vis, CD, NMR) and potentiometric measurements showed that, at physiological pH, mixtures of Ni(II) and L yielded unusual macrochelate complexes, NiL and NiL2, in which the metal cation was bound through Cys and His side chains in a square-planar arrangement. Above pH 9, a NiH-3L complex was formed, structurally analogous to typical square-planar nickel complexes. These complexes are expected to catalyze oxidation reactions, and therefore, coordination of Ni(II) by the L motif in core histone H3 may be a key event in oxidative DNA base damage observed in the process of Ni(II)-induced carcinogenesis.

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2-Amino-2-hydroxymethyl-propane-1,3-diol, or tris(hydroxymethyl)aminomethane (Tris), is probably the most common biochemical buffer used alone or in combination with other buffers because it is stable, unreactive, and compatible with most proteins and other biomolecules. Being nontoxic, it has even found applications in medicine. Tris is known, however, to coordinate transition metal ions, Cu(II) among them. Although often ignored, this feature affects interactions of Cu(II) ions with biomolecules, as Tris is usually used in high molar excess. Therefore, it is important to have precise knowledge on the stoichiometry, stability, and reactivity of cupric Tris complexes. The literature data are incoherent in this respect. We reinvestigated the complex formation in the Tris-Cu(II) system by potentiometry, UV-vis, ESI-MS, and EPR at a broad range of concentrations and ratios. We found, contrary to several previous papers, that the maximum stoichiometry of Tris to Cu(II) is 2 and at neutral pH, dimeric complexes are formed. The apparent affinity of Tris buffer for Cu(II), determined by the competitivity index (CI) approach [Krężel, A.; Wójcik, J.; Maciejczyk, M.; Bal, W. Chem. Commun. 2003, 6, 704-705] at pH 7.4 varies between 2 × 10(6) and 4 × 10(4) M(-1), depending on the Tris and Cu(II) concentrations and molar ratio.

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