title: Myoglobin microplate assay to evaluate prevention of protein peroxidation.
abstract: The current therapeutic strategies are based on the design of multifunctional drug candidates able to interact with various disease related targets. Drugs that have the ability to scavenge reactive oxygen species (ROS), beyond their main therapeutic action, may prevent the oxidative damage of biomolecules. Therefore, analytical approaches that monitor in a continuous mode the ability of drugs to counteract peroxidation of physiologically relevant biotargets are required. In the present work, a microplate spectrophotometric assay is proposed to evaluate the ability of selected cardiovascular drugs, including angiotensin-converting enzyme (ACE) inhibitors, β -blockers and statins to prevent protein peroxidation. Myoglobin, which is a heme protein, and peroxyl radicals generated from thermolysis of 2,2'-azo-bis(2-amidinopropane) dihydrochloride at 37 °C, pH 7.4 were selected as protein model and oxidative species, respectively. Myoglobin peroxidation was continuously monitored by the absorbance decrease at 409 nm and the ability of drugs to counteract protein oxidation was determined by the calculation of the area under the curve upon the myoglobin oxidation. Fluvastatin (AUC₅₀=12.5 ± 1.2 μM) and enalapril (AUC₅₀=15.2 ± 1.8 μM) showed high ability to prevent myoglobin peroxidation, providing even better efficiency than endogenous antioxidants such as reduced glutathione. Moreover, labetalol, enalapril and fluvastatin prevent the autoxidation of myoglobin, while glutathione showed a pro-oxidant effect.
authors.affiliation: UCIBIO, REQUIMTE, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira, 228, Porto 4050-313, Portugal.
authors.forename: Sara S
authors.initials: SS
authors.lastname: Marques
authors.affiliation: UCIBIO, REQUIMTE, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira, 228, Porto 4050-313, Portugal. Electronic address: luismagalhaes@ff.up.pt.
authors.forename: Luís M
authors.initials: LM
authors.lastname: Magalhães
authors.affiliation: UCIBIO, REQUIMTE, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira, 228, Porto 4050-313, Portugal.
authors.forename: Ana I P
authors.initials: AI
authors.lastname: Mota
authors.affiliation: UCIBIO, REQUIMTE, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira, 228, Porto 4050-313, Portugal.
authors.forename: Tânia R P
authors.initials: TR
authors.lastname: Soares
authors.affiliation: UCIBIO, REQUIMTE, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira, 228, Porto 4050-313, Portugal.
authors.forename: Barbara
authors.initials: B
authors.lastname: Korsak
authors.affiliation: UCIBIO, REQUIMTE, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira, 228, Porto 4050-313, Portugal.
authors.forename: Salette
authors.initials: S
authors.lastname: Reis
authors.affiliation: UCIBIO, REQUIMTE, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira, 228, Porto 4050-313, Portugal.
authors.forename: Marcela A
authors.initials: MA
authors.lastname: Segundo
mesh_terms: D000806:Angiotensin-Converting Enzyme Inhibitors / Q000737:chemistry; D000975:Antioxidants / Q000737:chemistry*; D001681:Biological Assay; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D004656:Enalapril / Q000737:chemistry; D000431:Ethanol / Q000737:chemistry; D005229:Fatty Acids, Monounsaturated / Q000737:chemistry; D000077340:Fluvastatin; D005609:Free Radicals / Q000737:chemistry; D005978:Glutathione / Q000737:chemistry; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000737:chemistry; D007211:Indoles / Q000737:chemistry; D007700:Kinetics; D009211:Myoglobin / Q000737:chemistry*; D016877:Oxidants / Q000737:chemistry; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010100:Oxygen / Q000737:chemistry; D010545:Peroxides / Q000737:chemistry*; D012997:Solvents / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D013654:Taurine / Q000737:chemistry; D013696:Temperature
chemical_list: D000806:Angiotensin-Converting Enzyme Inhibitors; D000975:Antioxidants; D005229:Fatty Acids, Monounsaturated; D005609:Free Radicals; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D007211:Indoles; D009211:Myoglobin; D016877:Oxidants; D010545:Peroxides; D012997:Solvents; D013654:Taurine; C049375:perhydroxyl radical; D000431:Ethanol; D000077340:Fluvastatin; D004656:Enalapril; D006861:Hydrogen Peroxide; D005978:Glutathione; D010100:Oxygen; D004121:Dimethyl Sulfoxide
keywords: Microplate; Myoglobin; Peroxyl radical; Protein oxidation; Statins; β-blockers