Fluorescence quenching of dipyridamole associated to peroxyl radical scavenging: a versatile probe to measure the chain breaking antioxidant activity of biomolecules.
Dypiridamole is a highly efficient chain breaking antioxidant (Iuliano et al., Free Radic. Biol. Med. 18 (1995) 239-247) with an aromatic ring system responsible for an intense absorption band in the 400-480-nm region and for an intense fluorescence. Dipyridamole fluorescence is quantitatively quenched upon reaction with peroxyl radicals. In the presence of a flux of peroxyl radicals generated by thermal dissociation of azo-initiators, dipyridamole fluorescence decays linearly, showing a first-order reaction with respect to peroxyl radicals, and zero-order with respect to dipyridamole. The pH optimum for the fluorescence quenching is in the 7-8 range, from pH 7 to 6, the decay of fluorescence rapidly decreases to became negligible below pH 5.5. Dipyridamole consumption is blocked in the presence of an added chain breaking antioxidant for a time that is proportional to the antioxidant concentration. This effect is shown for ascorbic acid, trolox, vitamin E, uric acid, and N, N'-diphenyl-p-phenylenediamine. The slope of the linear correlation relative to trolox allows calculation of the bimolecular rate constant for a given molecule and peroxyl radicals. Comparison of data obtained by the dipyridamole consumption are comparable to values obtained by the oxygen consumption method.
Iuliano
L
L
Institute of Clinical Medicine I, University La Sapienza, via del Policlinico, 00185, Rome, Italy. iuliano@uniroma1.it
Piccheri
C
C
Coppola
I
I
Praticò
D
D
Micheletta
F
F
Violi
F
F
D000975:Antioxidants / Q000494:pharmacology*; D004176:Dipyridamole / Q000494:pharmacology*; D005456:Fluorescent Dyes / Q000737:chemistry*; D016166:Free Radical Scavengers / Q000494:pharmacology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015227:Lipid Peroxidation; D010545:Peroxides / Q000737:chemistry*; D013050:Spectrometry, Fluorescence
D000975:Antioxidants; D005456:Fluorescent Dyes; D016166:Free Radical Scavengers; D010545:Peroxides; C049375:perhydroxyl radical; D004176:Dipyridamole