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Selective On/Off-Nitroxides as Radical Probes to Investigate Non-radical Enzymatic Activity by Electron Paramagnetic
Indranil Duttagupta1, Natacha Jugniot2, Gérard Audran1
1Aix Marseille Univ, CNRS, ICR, UMR 7273, Case 551, Avenue Escadrille Normandie-Niemen, 13397, Marseille Cedex 20, France.
Abstract:
A nitroxide carrying a peptide specific to the binding pocket of the serine proteases chymotrypsin and cathepsin G is prepared. This peptide is attached as an enol ester to the nitroxide. Upon enzymatic hydrolysis of the peptide, the enol ester moiety is transformed into a ketone moiety. This transformation affords a difference of 5 G in phosphorus hyperfine coupling constant between the electronic paramagnetic resonance (EPR) signals of each nitroxide. This property is used to monitor the enzymatic activity of chymotrypsin and cathepsin G by EPR. Michaelis constants were determined and match those reported for conventional optical probes.
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