New Approach for the Detection of Peptide- and Protein-Based Radicals Using a Pre-Fluorescent Probe

A novel application for pre-fluorescent probes in the detection of peptide- and protein-based radicals is proposed. Pre-fluorescent probes combine a fluorescent moiety labeled with a paramagnetic nitroxide that acts as a fluorescence quencher. Trapping of a radical by the nitroxide group restores the fluorescence properties. The increase in fluorescence intensity with time reflects the formation and quenching of free radicals and can be employed for the quantitative evaluation of yields and kinetics. In this test system, the pre-fluorescent probe 4-(9-acridinecarbonate)-2,2,6,6-tetramethylpiperidinyl-1-oxyl radical (Ac-Tempo), in which an acridine moiety was labeled with 2,2,6,6-tetramethylpiperidinyl-1-oxy (Tempo), was employed to probe peptide- and protein-based radicals. Peptide-based radicals were generated through the reaction between horseradish peroxidase (HRP)/H₂O₂ and a derivative of tyrosine. Protein-based radicals were generated through the reaction between myoglobin (Mb) and H₂O₂. In both cases the Ac-Tempo was found, using a combination of high-performance liquid chromatography (HPLC) and mass spectrometry, to be converted into fluorescent acridine (Ac)-piperidine (4-(9-acridinecarbonate)-2,2,6,6-tetramethylpiperidine).