Osmium tetroxide, 2,2'-bipyridine: electroactive marker for probing accessibility of tryptophan residues in proteins
Language English Country United States Media print-electronic
Document type Journal Article, Research Support, Non-U.S. Gov't
PubMed
18473484
DOI
10.1021/ac800527u
Knihovny.cz E-resources
- MeSH
- 2,2'-Dipyridyl chemistry MeSH
- Avidin chemistry MeSH
- Biotin chemistry MeSH
- Electrochemistry MeSH
- Electrodes MeSH
- Electrons * MeSH
- Molecular Structure MeSH
- Osmium Tetroxide analysis chemistry MeSH
- Proteins analysis chemistry MeSH
- Cross-Linking Reagents chemistry MeSH
- Tryptophan analysis chemistry MeSH
- Carbon chemistry MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- 2,2'-Dipyridyl MeSH
- Avidin MeSH
- Biotin MeSH
- Osmium Tetroxide MeSH
- Proteins MeSH
- Cross-Linking Reagents MeSH
- Tryptophan MeSH
- Carbon MeSH
A complex of osmium tetroxide with 2,2'-bipyridine (Os,bipy) has been applied as a chemical probe of DNA structure as well as an electroactive DNA label. The Os,bipy has been known to form covalent adducts with pyrimidine DNA bases. Besides the pyrimidines, electrochemically active covalent adducts with Os,bipy are formed also by tryptophan (W) residues in peptides and proteins. In this paper we show that Os,bipy-treated proteins possessing W residues (such as avidin, streptavidin, or lysozyme) yield at the pyrolytic graphite electrode (PGE) a specific signal (peak alphaW) the potential of which differs from the potentials of signals produced by free Os,bipy or by Os,bipy-modified DNA. No such signal is observed with proteins lacking W (such as ribonuclease A or alpha-synuclein). Subpicomole amounts of W-containing proteins modified with Os,bipy can easily be detected using adsorptive transfer stripping voltammetry with the PGE. Binding of biotin to avidin interferes with Os,bipy modification of the protein, in agreement with the location of W residues within the biotin-binding site of avidin. These Ws are accessible for modification in the absence of biotin but hidden (protected from modification) in the avidin-biotin complex. The Os,bipy-modified avidin is unable to bind biotin, and its quarternary structure is disrupted. Analogous effects were observed with another biotin-binding protein, streptavidin. Our results demonstrate that modification of proteins with Os,bipy under conditions close to physiological, followed by a simple electrochemical analysis, can be applied in the microanalysis of protein structure and interactions.
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