Interaction of nucleic acids: VIII. Binding of magnesium ions by nucleic acids

Abstract

The lowering of the activity of magnesium ions in aqueous solution due to the presence of nucleic acids (5 to 20 m m) was measured by a divalent cation specific electrode which shows a large decrease in potential. The electrode response over a wide range of MgCl 2 concentration (10 −1 to 10 −4 m) in water is linear as described by the Nernst equation. The binding studies were carried out in ~ 5 m m-sodium phosphate buffer, at neutral pH, with a variety of nucleic acids. The results are treated as site-binding to the phosphate in a multiple equilibrium using the equation of Scatchard (1949). The binding isotherms in the Scatchard plot (v̄/A 0 versus v̄, where v̄ is the degree of saturation of binding sites and A 0 is the concentration of free Mg 2+) show a limited linear portion from approximately 30 to 70% saturation of binding sites and then the slopes decrease markedly. As a first-order approximation to account for the interference factor (electrostatic, conformational, etc.) in the binding process of magnesium ions by the polymer with only one type of binding site (the phosphate ligand), two additional treatments were added to the Scatchard equation: (1) the binding constant, k, is replaced by ke −φv where φ represents the interference factor; (2) the derivative of the equation was taken to construct a [(δ v ̄ /A 0)/δ v ̄ ] n versus v̄ plot. In these plots, there is a region of zero slope (horizontal region) and a region of negative slope(s). The value from the horizontal region is taken as the apparent intrinsic constant for magnesium ion binding by the nucleic acids in the neutral sodium phosphate buffer at room temperature: transfer RNA (≈1.7 × 10 4) > poly A ≈ poly A· poly U ≈ poly I ≈ poly I · poly C ≈ native DNA ≈ stabilized stored denatured DNA (≈6 × 10 3) > freshly denatured DNA ≈ poly U ≈ poly C ≈ (≈3 × 10 3) ⪢ monomers (≈2 × 10 2). The slope in the derivative plot is related to the quantity −φ, varying from −φ to −2φ. The slopes for all the nucleic acids are similar except for transfer RNA, which has a much larger slope. These results indicate the importance of the conformation of nucleic acid in the binding of magnesium ions as reflected in both the apparent binding constant and the interference factor. Since the binding constant and the interference factor of transfer RNA are larger than those of other nucleic acids, these observations indicate that transfer RNA has a more folded conformation.