Analytical and Bioanalytical Chemistry

, Volume 408, Issue 18, pp 4935–4941

Redox-dependent interactions between reduced/oxidized cytochrome c and cytochrome c oxidase evaluated by in-situ electrochemical surface plasmon resonance

Research Paper

DOI: 10.1007/s00216-016-9586-9

Cite this article as:
Hou, Y., An, J., Deng, C. et al. Anal Bioanal Chem (2016) 408: 4935. doi:10.1007/s00216-016-9586-9

Abstract

The interactions between the redox couple of cytochrome c (Cyt c) and cytochrome c oxidase (COX) were investigated at a mimic redox-modulated interface by using an electrochemical surface plasmon resonance (EC-SPR) system. Although early studies of the binding between COX and Cyt c have been conducted using several techniques in homogeneous solutions, a problem still inherent is that ferro-cytochrome c (Cyt cred), the reduced form of Cyt c, can be easily oxidized into ferri-cytochrome c (Cyt cox) and adversely impact the accuracy and reproducibility of the binding measurements. In order to realize reliable redox-dependent binding tests, here the Cyt cred is quantitatively electro-generated from Cyt cox by in situ cathodic polarization in a flow cell. Then the kinetic and dissociation constants of the bindings between COX and Cyt cred/Cyt cox can be evaluated accurately. In this study, the values of association/dissociation rate constants (ka, kd) for both COX/Cyt cred and COX/Cyt cox were obtained. The dissociation constants, KD, were finally calculated as 3.33 × 10–8 mol · L–1 for COX/Cyt cred and 4.25 × 10–5 mol · L–1 for COX/Cyt cox, respectively. In-situ EC-SPR is promising for better mimicking the in vivo condition that COX is embedded in the inner mitochondrial membrane and Cyt c acts as an electron shuttle in the mobile phase. It is an effective method for the investigation of redox-dependent biomolecular interactions.

Graphical Abstract

Schematic representation of the experimental designs using EC-SPR system. (a) the Au-Cys-COX SPR chip with SAM layers. (b) redox-modulated Cyt c and its binding onto pre-immobilized COX

Keywords

Electrochemical surface plasmon resonance (EC-SPR) Cytochrome c (Cyt cCytochrome c oxidase (COX) Redox-dependent interaction Mimic interface Association/dissociation rate constant 

Supplementary material

216_2016_9586_MOESM1_ESM.pdf (476 kb)
ESM 1(PDF 476 kb)

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  1. 1.College of Chemistry and Chemical EngineeringCentral South UniversityChangshaChina

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