F. Gudratova, A. Aliyeva, S. Mahmudova, K. Gasimov, T. Yusifov*

Institute of Biophysics, Ministry of Science and Education
of the Republic of Azerbaijan, Baku;
*e-mail: tjussifo@ucla.edu

Received: 23 May 2025; Revised: 24 July 2025;
Accepted: 12 September 2025; Available on-line: 17 September2025

Cytochrome c, as an electron carrier within the mitochondria, plays a crucial role in the electron transport chain. To meet the demand for rapid methods that assess the electron transport properties of cytochrome c, we used the electron donor 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonate) (ABTS) as a substrate and suitable spectrophotometric reporter of cytochrome c peroxidase-like activity. ABTS and cytochrome c from bovine were purchased from Sigma-Aldrich Inc. The time course of the cytochrome c-driven ABTS oxidation reaction was studied using H₂O₂ as a second substrate. It was demonstrated that CytC addition is a prerequisite for the transfer of electrons from ABTS to H₂O₂. The reaction kinetic analysis with determination of V_max, K_m, k_cat, and k_cat/K_m values for both substrates was performed. Our results demonstrate that the cytochrome c-catalyzed ABTS oxidation reaction can be effectively employed as a model for studying the functional role of cytochrome c in various conditions.