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Cytochrome c mutants for superoxide biosensors

Authors :
Wegerich, Franziska
Turano, Paola
Allegrozzi, Marco
Mohwald, Helmuth
Lisdat, Fred
Source :
Analytical Chemistry. April 15, 2009, Vol. 81 Issue 8, p2976, 9 p.
Publication Year :
2009

Abstract

The effect of introducing positive charges (lysines) in human cytochrome c (cyt c) on the redox properties and reaction rates of cyt c with superoxide radicals was studied. The mutated forms of this electron-transfer protein are used as sensorial recognition elements for the amperometric detection of the reactive oxygen radical. The proteins were prepared by site-directed mutagenesis focusing on amino acids near the heme edge. The 11 mutants of human cyt c expressed in the course of this research have been characterized by UV--vis spectroscopy, circular dichroism, and NMR spectroscopy to verify overall structure integrity as well as axial coordination of the heme iron. The mutants are investigated voltammetrically using promoter-modified gold electrodes with respect to redox activity and formal redox potential. The rate constants for the reaction with superoxide have been determined spectrophotometrically. Four mutants show a higher reaction rate with the radical as compared to the wild type. These mutants are used for the construction of superoxide sensors based on thiol-modified gold electrodes and covalently fixed proteins. We found that the E66K mutant-based electrode has a clearly higher sensitivity in comparison with the wild-type-based sensor while retaining the high selectivity and showing a good storage stability.

Details

Language :
English
ISSN :
00032700
Volume :
81
Issue :
8
Database :
Gale General OneFile
Journal :
Analytical Chemistry
Publication Type :
Academic Journal
Accession number :
edsgcl.198849461