Cytochrome c Mutants for Superoxide Biosensors

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• 作者：Franziska Wegerich ; Paola Turano ; Marco Allegrozzi ; Helmuth Mhwald ; Fred Lisdat
• 刊名：Analytical Chemistry
• 出版年：2009
• 出版时间：April 15, 2009
• 年：2009
• 卷：81
• 期：8
• 页码：2976-2984
• 全文大小：257K
• 年卷期：v.81,no.8(April 15, 2009)
• ISSN：1520-6882

文摘

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.