Interactions of DNA with divalent metal ions. I. 31P-NMR studies.

Abstract

Abstract31P‐nmr has been used to investigate the specific interaction of three divalent metal ions, Mg2+, Mn2+, and Co+2, with the phosphate groups of DNA. Mg2+ is found to have no significant effect on any of the 31P‐nmr parameters (chemical shift, line‐width, T1, T2, and NOE) over a concentration range extending from 20 to 160 mM. The two paramagnetic ions, Mn2+ and Co2+, on the other hand, significantly change the 31P relaxation rates even at very low levels. From an analysis of the paramagnetic contributions to the spin–lattice and spin–spin relaxation rates, the effective internuclear metal–phosphorus distances are found to be 4.5 ± 0.5 and 4.1 ± 0.5 Å for Mn2+ and Co2+, respectively, corresponding to only 15 ± 5% of the total bound Mn2+ and Co2+ being directly coordinated to the phosphate groups (inner‐sphere complexes). This result is independent of any assumptions regarding the location of the remaining metal ions which may be bound either as outer‐sphere complexes relative to the phosphate groups or elsewhere on the DNA, possibly to the bases. Studies of the temperature effects on the 31P relaxation rates of DNA in the absence and presence of Mn2+ and Co2+ yielded kinetic and thermodynamic parameters which characterize the association and dissociation of the metal ions from the phosphate groups. A two‐step model was used in the analysis of the kinetic data. The lifetimes of the inner‐sphere complexes are 3 × 10−7 and 1.4 × 10−5 s for Mn2+ and Co2+, respectively. The rates of formation of the inner‐sphere complexes with the phosphate are found to be about two orders of magnitude slower than the rate of the exchange of the water of hydration of the metal ions, suggesting that expulsion of water is not the rate‐determining step in the formation of the inner‐sphere complexes. Competition experiments demonstrate that the binding of Mg2+ ions is 3–4 times weaker than the binding of either Mn2+ or Co2+. Since the contribution from direct phosphate coordination to the total binding strength of these metal ion complexes is small (∼15%), the higher binding strength of Mn2+ and Co2+ may be attributed either to base binding or to formation of stronger outer‐sphere metal–phosphate complexes. At high levels of divalent metal ions, and when the metal ion concentration exceeds the DNA–phosphate concentration, the fraction of inner‐sphere phosphate binding increases. In the presence of very high levels of Mg2+ (e.g., 3.1M), the inner‐sphere ⇄ outer‐sphere equilibrium is shifted toward ∼100% inner‐sphere binding. A comparison of our DNA results and previous results obtained with tRNA indicates that tRNA and DNA have very similar divalent metal ion binding properties. A comparison of the present results with the predictions of polyelectrolyte theories is presented.