Antagonism by essential divalent metals and amino acids of nickel(II)-DNA binding in vitro

In vitro binding of nickel(II) to DNA and the effects of divalent essential metals calcium, magnesium, manganese, copper, and zinc, and of amino acids histidine, cysteine, glutamine, arginine, lysine, alanine, and glycine upon that binding were investigated. Samples of 0.156 mg of calf thymus DNA (0.078 mg/ml in 5 mM ammonium acetate buffer, pH = 7.4) were incubated for 1 hr at 24 degrees C with various concentrations of nickel(II)acetate labeled with 63Ni (0.1 to 250 microM) in the absence or presence of 50 microM concentrations of the essential metal acetates, or with 100 microM concentrations of the amino acids. Free and DNA-bound nickel(II) fractions were separated by gel filtration on Sephadex G-25 and quantified by liquid scintillation counting. Scatchard analysis revealed more than two types of nickel(II)-binding sites and a positive cooperativity of binding at the bound-Ni concentrations below 0.35 microM. The high-affinity nickel(II)-binding sites at DNA were identified as phosphate groups. Their binding capacity equalled 0.043 mumol/mg DNA (approx. 1 mol Ni/70 mol of DNA bases). The apparent dissociation constant of nickel(II) from the high-affinity sites was 5.35 microM. Double reciprocal plots showed the essential divalent metals to be competitive antagonists of nickel(II)-binding to the high-affinity sites, ranking Mg(II) greater than or equal to Mn(II) greater than or equal to Ca(II) greater than or equal to Cu(II) = Zn(II). Similarly, the amino acids antagonized nickel binding to DNA with a relative strength of His greater than Gln greater than or equal to His/Cys greater than Arg greater than Cys greater than or equal to Gly = Ala greater than or equal to Lys. The strongest inhibitors of nickel(II)-DNA binding in vitro appear to be magnesium and manganese, i.e., the same metals that are capable of attenuating nickel carcinogenicity in vivo.