The direct electron transfer of surface-confined horse heart cytochrome c (Cyt c) was achieved using single- or double-stranded (ss- or ds-) calf-thymus DNA immobilized on gold electrodes. The formal potential (Edegrees') for Cyt c in a 10 mM PBS (PH7.0) buffer at a scan rate of 20 mV s(-1) was -0.027 V with dsDNA and -0.016 V with ssDNA, respectively. The interaction between Cyt c and DNA makes the formal potential shift negatively when compared to that of Cyt c in solution (+0.258 V vs. SHE, i.e. +0.017 vs. SCE). Both UV-visible and reflection-absorption infrared spectroscopy indicate that the surface-confined Cyt c existed in its native form. The fractional coverage of bound Cyt c was 0.51 (with dsDNA) and 0.46 (with ssDNA) a single monolayer. The binding site sizes were determined to be 13 base pairs per Cyt c molecule with dsDNA and 26 nucleotides binding I Cyt c molecule with ssDNA. At a dsDNA/Au electrode, reduction and oxidation electron-transfer rate constant values of k(s,Red) = 21 s(-1) and k(s,Ox) = 15 s(-1) were obtained. At the ssDNA/Au electrode, the values were k(s,Red) = 24 s(-1) and k(s,Ox) = 19 s(-1), respectively. The incorporated Cyt c was strongly affected by variations in both the ionic strength and pH of the solution. The quantitative determination of Cyt c by differential pulse voltarnmetry (DPV) using DNA-modified electrodes was also explored. (C) 2003 Elsevier Science B.V. All rights reserved.