Recombinant horseradish peroxidase- and cytochrome c-based two-electrode system for detection of superoxide radicals Stepan Shipovskov a , Elena E. Ferapontova b,c, * , Irina Gazaryan d,e , Tautgirdas Ruzgas c a Department of Molecular Biophysics, Lund University, P.O. Box 124, Lund SE 22100, Sweden b Group of Bioinformatics, Web-lab, Novosibirsk IT Centre, Voskhod 26a, Novosibirsk 630102, Russia c Department of Analytical Chemistry, Lund University, P.O. Box 124, Lund SE 22100, Sweden d Department of Chemical Enzymology, Chemical Faculty, Moscow State University, Moscow 119899, Russia e Burke Medical Research Institute, Mamaroneck Ave 785, White Plains, NY 10605, USA Received 23 June 2003; received in revised form 15 September 2003; accepted 19 September 2003 Abstract The reliable detection of a superoxide anion radical O 2 S  is complicated by its spontaneous dismutation reaction to H 2 O 2 at acidic pHs. To simultaneously detect both O 2 S  and H 2 O 2 produced in the course of its spontaneous dismutation, an electrochemical two-electrode system based on cytochrome c (cyt c) and recombinant horseradish peroxidase (rHRP) was applied. Therewith, a limited applicability of the cyt c system for the reliable monitoring of O 2 S  in acidic and neutral solutions was shown. It was demonstrated that both the reaction of O 2 S  dismutation to H 2 O 2 and the reaction between the formed H 2 O 2 and O 2 S  chemically decrease the amount of the initially present O 2 S  , decreasing the sensitivity and reliability of the electrochemical detection at acidic pH. However, by appropriately varying solution pH, the concentration of O 2 S  initially injected in the system can be estimated from the analysis of calibration curves for H 2 O 2 obtained with highly sensitive rHRP-modified electrode system at pH 6.0 and 7.0. D 2004 Elsevier B.V. All rights reserved. Keywords: Superoxide radical; Hydrogen peroxide; Cytochrome c; Recombinant horseradish peroxidase; Bi-electrode system 1. Introduction Free radicals, involved in the pathogenesis of cancer and some other human severe diseases, are commonly difficult to detect directly due to their high reactivity and thus a short life time period. It is known that they produce a cascade of damaging events in the living organisms leading finally to cell death [1–4]. The superoxide anion radical (O 2 S  ), a short-lived intermediate resulting from the 1e  reduction of oxygen, is a primary component of so-called reactive oxygen species and is a potentially damaging agent, when its concentrations exceeds certain ‘‘antioxidant’’ level of the living system [1–3]. Development of the methods for the analysis of superoxide has gained considerable attention during the past decade [5–17]; reactions of superoxide with superoxide dismutase [5–8,14], superoxide reductase [9,10] and cytochrome c (cyt c) [11–17] were exploited for this purpose. Due to insufficient stability of the above-men- tioned enzymatic systems, the most popular electrodes are based on the direct in vivo detection of O 2 S  with cyt c- modified electrodes [11–16]. Therewith, the reduction of cyt c by O 2 S  can be detected by means of a subsequent oxidation of reduced cyt c at the electrode surface: O S 2 þ Cyt c ðFe 3þ Þ! Cyt c ðFe 2þ Þ þ O 2 ! 1e  Cyt c ðFe 3þ Þ ð1Þ The re-oxidation current of cyt c is then proportional to the concentration of superoxide radical. However, the reli- able determination of a real amount of O 2 S  present in the system is complicated by spontaneous dismutation of su- peroxide to H 2 O 2 : 2O S 2 þ 2H þ ! O 2 þ H 2 O 2 ð2Þ In aqueous solutions the rate constant for reaction (2) increases more then 10 12 times when changing from pH 14 to 7 [18]. Therefore it might be expected that under certain conditions the monitoring of O 2 S  can be accomplished through the analytical detection of hydrogen peroxide pro- 1567-5394/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bioelechem.2003.09.026 * Corresponding author. Tel.: +46-46-222-0103; fax: +46-46-222- 4544. E-mail addresses: elena.ferapontova@analykem.lu.se, ferapontova@nsk.fio.ru (E.E. Ferapontova). www.elsevier.com/locate/bioelechem Bioelectrochemistry 63 (2004) 277 – 280