The electrocatalytic oxidation of guanine in DNA and oligonucleotides by RU(bpy)(3)(3+/2+) was investigated using cyclic voltammetry (CV) and chronoamperometry (CA) (bpy = 2,2'-bipyridine), Oxidation of RU(bpy)(3)(2+) to the Ru(III) form at tin-doped indium oxide (ITO) electrodes in the presence of DNA produces catalytic current due to the oxidation of guanine by Ru(III). CA traces of RU(bpy)(3)(2+) With calf thymus DNA at high salt concentration (50 mM sodium phosphate + 700 mM NaCl) give a single kinetic process with a rate constant of 3500 +/-300 M-1 s(-1) that is independent of DNA concentration and similar to values determined previously by fitting CV data under the same conditions. Under low salt conditions (50 mM sodium phosphate + 0 mM NaCl), the CA data show two linear regions that give rate constants of 2.7 x 10(4) and 6 x 10(5) M-1 s(-1). Digital simulation of CV data at low salt requires a careful accounting for the binding of the metal complex to the DNA polyanion, which can be accomplished using binding constants that are independently determined. This analysis gives rate constants that are independent of DNA concentration and range from 2.3 x 10(5) to 1.4 x 10(6) M-1 s(-1) as the scan rate is increased from 25 to 250 mV/s. The variation in rate constant with scan rate can be attributed to the two kinetic processes observed in the CA results. Resolution of Ru(bpy)(3)(2+) into the Delta and Lambda stereoisomers showed that the two kinetic processes were not due to the stereoisomerism. Satisfactory fitting of the CV data requires addition of a second electron-transfer step from the oxidized guanine to Ru(III); this rate constant was always less than 1% of the rate constant for the first homogeneous electron transfer. In addition, the fitting at low salt requires accounting for the density of guanines in the DNA sequence. Calf thymus DNA is 20% guanine; however, the fitting shows that binding of the mediator by at least 60% of the nucleotides produces catalytic turnover. Oligonucleotides containing a single guanine gave similar rate constants to those observed by CV and CA on calf thymus DNA, and the fitting suggested that binding of the mediator by 5-10 of the 30 nucleotides (defined as "active binding sites") in the oligomer produced catalyst cycling. Thus, the electron is able to transfer to a mediator that is bound in a region that spans 2.5-5 base pairs and contains the oxidized guanine. The number of "active binding sites" increased predictably with the number of guanines in the sequence, ranging from 15% to 33% of the total nucleotides for a 15-mer duplex with one guanine to 75-100% for a 15-mer duplex with six guanines. Decreasing the salt concentration enhances the catalytic current both by increasing the number of active binding sites by a factor of 5-10 and by increasing the intrinsic oxidation rate by an order of magnitude.