Myoglobin microplate assay to evaluate
prevention of protein peroxidation
Luís Miguel Andrade de Magalhães
DISSERTAÇÃO DE MESTRADO INTEGRADO EM MEDICINA 2016
Myoglobin microplate assay to evaluate prevention of
protein peroxidation
Tese para obter o grau de Mestre em Medicina
Artigo Original
Luís Miguel Andrade de Magalhães
Orientador:
Doutora Isabel Fonseca
Coorientador:
Prof.ª Doutora Luísa Lobato
Here, I would like to acknowledge to some of the people who had some contribution to this work
and also to those that support to achieve this goal, Master in Medicine.
To my supervisor Prof. Isabel Fonseca, for her comprehension, enthusiasm and for being always
available when I needed.
To my co-supervisor Prof. Luísa Lobato, my special thanks for guiding me to the right person,
specialized in oxidative stress.
To Prof. Henrique José Cyrne Carvalho and to Prof. António Martins da Silva for providing me
the possibility to change the title and modality of this dissertation.
To all co-authors of this manuscript for their contribution to the work and for their approval to use
this work as dissertation theme.
Thanks to two friends meet in this course, Pedro Rodrigues and Miguel Ferreira, for their
friendship and valuable support during all these years.
To my parents, all my thanks.
Abstract 1 Resumo 2 1. Introduction 3 2. Objectives 7 3. Original paper 8 4. General conclusions 16 5. References 17 Supplementary data 19 Figure S1 20 Figure S2 21 Figure S3 22
Abstract
Introduction: The current therapeutic strategies are based on the discovery and developing 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.
Objectives: A microplate spectrophotometric assay is tested and proposed to evaluate the ability
of selected cardiovascular drugs to prevent protein peroxidation.
Methods: The scavenging capacity of several drugs was tested and compared with two
endogenous antioxidants (taurine and reduced glutathione). The studied drugs included a
angiotensin-converting enzyme inhibitor (enalapril), several β-blockers (atenolol, labetalol and
propranolol) and two statins (pravastatin and fluvastatin). 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.
Results and Conclusions: 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 autooxidation of myoglobin, while glutathione showed a pro-oxidant effect.
Resumo
Introdução: As estratégias terapêuticas atuais residem na descoberta e desenvolvimento de
fármacos multifuncionais que sejam capazes de intervir em diferentes alvos terapêuticos. Os
fármacos que possuem a capacidade de sequestrar espécies reativas do oxigénio (ERO), para além
do seu principal efeito terapêutico, podem dessa forma prevenir o dano oxidativo das
biomoléculas. Neste contexto, torna-se necessário dispor de metodologias que monitorizem em
modo contínuo a capacidade dos fármacos impedirem a peroxidação dos alvos biológicos.
Objetivos: Testar e propor um método espetrofotométrico em microplaca para avaliar a
capacidade de fármacos usados na área cardiovascular para prevenirem a peroxidação proteica.
Métodos: A capacidade antioxidante de vários fármacos foi testada e comparada com a de dois
antioxidantes endógenos (taurina e glutationa reduzida). Os fármacos estudados incluíram um
inibidor da enzima de conversão da angiotensina (enalapril), vários β–bloqueadores (atenolol,
labetalol, propanolol) e duas estatinas (pravastatina e fluvastatina). Como proteína modelo foi
selecionada a mioglobina, proteína heme, enquanto que os radicais peroxilo foram gerados a partir
da termólise a 37ºC, pH 7,4 do 2,2'-azo-bis(2-amidinopropano) dicloridrato como espécie
oxidativa. A peroxidação da mioglobina foi monitorizada continuamente pelo decréscimo de
absorvência a 409 nm e a capacidade dos fármacos impedirem a oxidação proteica foi determinada
pelo cálculo da área sob a curva da oxidação da mioglobina.
Resultados e Conclusões: A fluvastatina (AUC50 = 12,5 ± 1,2 µM) e o enalapril (AUC50 = 15,2
± 1,8 µM) foram os que demonstraram maior capacidade para impedir a peroxidação da
mioglobina, que foi inclusivamente superior à de antioxidantes endógenos tais como a glutationa
reduzida. O labetalol, enalapril e a fluvastatina previnem a auto-oxidação da mioglobina, enquanto
a glutationa demonstrou um efeito pro-oxidante.
Palavras-chave: Mioglobina; Radical peroxilo; Oxidação proteica; Microplaca; Estatinas;
1. Introduction
Redox reactions are characterized by chemical mechanisms that involve the oxidation (loss of
electrons) of one substance and the reduction (acceptance of electrons) of another substance [1].
These reactions are essential for numerous biochemical pathways and cellular chemistry,
biosynthesis and regulation of cell signaling [2]. In biological systems, pro-oxidant is a substance
that undergoes reduction by inducing oxidative damage to various biological targets such as
nucleic acids, lipids and proteins. On the other hand, an antioxidant is a substance that can
efficiently counteract the activity of a pro-oxidant, reducing oxidation and inducing the
concomitant formation of products having no or low toxicity. In 1995, Halliwell et al. [3] suggested
that an antioxidant is “any substance that when present at low concentrations, compared to those
of an oxidizable substrate significantly delays or prevents oxidation of that substrate”. In this
context, not all reductants involved in a chemical reaction are antioxidants, only those compounds
which are capable of protecting the biological target (oxidizable substrate).
The pro-oxidants are referred as reactive oxygen species (ROS) such as alkoxyl radicals (RO•),
peroxyl radicals (ROO•), hydroxyl radical (HO•), superoxide anion radical (O2•–), singlet oxygen
(1O
2), hydrogen peroxide (H2O2) and hypochlorous acid (HOCl). Reactive nitrogen species (RNS)
are also produced such as nitric oxide radical (NO•), nitrogen dioxide radical (NO2•) and
peroxynitrite anion (ONOO–) [4, 5]. These reactive species, can be easily formed from exposure
of organisms to exogenous sources (UV-irradiation, drugs, pollutants, toxins, pesticides and
herbicides) and much more important to endogenous sources that is a continuous process such as
from mitochondrial electron-transport chain and from activity of some enzymes as nitric oxide
synthases and xanthine oxidase [6]. Moreover, activated phagocytes produce a variety of reactive
species that play an important role in the mechanism of defense against infectious agents [7].
Depending on the site and the concentration generated, these reactive species are well recognized
prevent the oxidative damage mediated by these species, living cells have developed a complex
defense system composed of enzymatic (superoxide dismutase, catalase and glutathione
peroxidase) and non-enzymatic antioxidants (uric acid, glutathione, bilirubin and albumin) that
convert them to harmless species. Dietary antioxidants such as ascorbic acid (vitamin C),
α-tocopherol (vitamin E), carotenoids and polyphenolic compounds have also an important role in
protecting essential biological targets against ROS/RNS action [4].
The state called ‘oxidative stress’ occurs when this balance can be shifted towards the pro-oxidant
agents due to an overproduction of ROS/RNS and/or to a decrease of antioxidant protection [9].
This can be triggered by several factors such as genetic, diet, lifestyle, environmental conditions
and diseases [6]. The oxidative stress has been implicated in the pathogenesis and development of
several human diseases, including cardiovascular diseases [10], cancer [11], diabetes mellitus [12],
inflammatory diseases [13], ischemia/reperfusion injury [14-16] and neurodegenerative disorders
[17].
In this context, the interest in antioxidant research has become a topic of increasing attention in
the last two decades, especially within biological, medical and nutritional fields. In the case of
biological samples (e.g. plasma, serum, urine), measurement of antioxidant status may be helpful
for diagnostic and treatment monitoring, especially during supplementation trials for boosting
plasmatic antioxidant levels [18]. However, a validated in vitro assay that can reliably measure the
total antioxidant capacity of biological samples is not yet available. This situation is due to the fact
that the term total antioxidant capacity is a broader definition that covers: i) inhibition of generation
and scavenging capacity against ROS/RNS; ii) reducing capacity; iii) metal chelating capacity; iv)
activity of antioxidative enzymes; v) and inhibition of oxidative enzymes. Moreover, different
antioxidants act by different mechanisms and even the same compound can have different ways
of actuation [19]. For that reason, the evaluation of overall antioxidant capacity is complex and requires multiple assays to generate an “antioxidant profile” [20].
Drugs that scavenge reactive oxygen species (ROS) and/or inhibit the protein and lipid
peroxidation have beneficial clinical effects in the prevention and/or treatment of oxidative-stress
related diseases, usually named as pleotropic effects [21]. For instance, statins, namely
3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) inhibitors, are the first line therapy
for lowering cholesterol levels but various cholesterol-independent beneficial properties, such as
reduction of endothelial dysfunction, stabilization of atherosclerotic plaques and decrease of
oxidative stress have been indicated, that may potentiate their clinical effects [22]. Actually,
current therapeutic strategies are based on the design of multifunctional drug candidates that are
able to interact with multiple disease related targets, with the advantages of reduced molecularity,
absence of drug-drug interactions and improved pharmacokinetics and pharmacodynamics [23].
The analytical methods that have been applied to measure the scavenging capacity of drugs are
not adequate because the assays are based on the use of synthetic radical, such as
2,2-diphenyl-1-picrylhydrazyl radical (DPPH•) and 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulphonate) radical
cation (ABTS•+) and/or on competitive assays that uses target molecules (e.g., fluorescein, luminol
and pyrogallol in Oxygen Radical Absorbance Capacity, named as ORAC assay) that do not
represent any reactive species and biotarget, respectively, found in living organisms [24].
The research work presented in this thesis was directed to the development of a novel analytical
approach to determine the scavenging capacity of cardiovascular drugs using reactive species and
oxidizable substrates found at biological milieu. Among the ROS formed in pathophysiological
conditions, peroxyl radicals were selected as oxidizing species because they are responsible for
the propagation step of lipid and protein peroxidation [8]. Hence, the peroxyl radicals generated
from thermo-decomposition of 2,2´-azobis (2-methylpropionamidine) dihydrochloride (AAPH)
and an endogenous protein (myoglobin) were used as models of reactive species and biotarget,
Figure 1. Schematic representation of myoglobin (Mb) oxidation mediated by peroxyl radicals (ROO•) generated from thermolysis of 2,2´-azobis (2-methylpropionamidine) dihydrochloride (AAPH) and its prevention by drugs.
The myoglobin oxidation induced by peroxyl radicals was monitored by the absorbance decrease
at 409 nm under reaction conditions (pH, temperature) similar to those found in vivo (Fig. 1). The
analytical protocol was implemented in a microplate format in order to obtain a high-throughput
procedure. The capacity of cardiovascular drugs and endogenous antioxidants to prevent protein
oxidation was determined by the increase of the area under the curve (AUC) and the antioxidant
effectiveness of drugs were characterized by their IC50 values, which represents the drug
concentration that causes 50% inhibition of myoglobin oxidation. Finally, the scavenging capacity of several drugs, such as β-blockers (atenolol, labetalol, propanolol), statins (pravastatin,
fluvastatin) and angiotensin-converting enzyme (ACE) inhibitors (enalapril) was compared to that
obtained for endogenous antioxidants (reduced glutathione and taurine).
Mb Mb oxidized AAPH ROO • Drug pH 7.4 37 C 409 nm 409 nm 409 nm
2. Objectives
The main aims of the present work were:
i) to develop a high-throughput microplate approach to determine the ROS scavenging
capacity of drugs applying relevant biological reactive species (peroxyl radicals) and targets
(myoglobin);
ii) to monitor in a continuous mode the myoglobin peroxidation by the absorbance decrease at
409 nm under reaction conditions (pH, temperature) similar to those found in vivo;
iii) to determine the role of ethanol and DMSO used as cosolvents upon the oxidation kinetics
of myoglobin;
iv) to determine the prevention of autoxidation of myoglobin by cardiovascular drugs;
v) to assess the capacity of drugs to prevent protein peroxidation by the increase of the area
under the curve (AUC);
vi) to compare the scavenging capacity of several drugs used for cardiovascular pathologies,
such as several ACE inhibitors, β-blockers and statins with endogenous antioxidants.
Finally, at the end of this work it is expected that the results obtained may elucidate the relevance
3. Original paper*
Myoglobin microplate assay to evaluate prevention of protein peroxidation
Sara S. Marques, Luís M. Magalhãesa, Ana I.P. Mota, Tânia R.P. Soares1, Barbara Korsak2, Salette Reis, Marcela A. Segundo
UCIBIO, REQUIMTE, Department of Chemical Sciences, Faculty of Pharmacy, University of
Porto, Rua de Jorge Viterbo Ferreira, 228, Porto 4050-313, Portugal
a Corresponding author.
Luís M. Magalhães (e-mail: [email protected] or [email protected])
Present address:
1 REQUIMTE, Lab. of Pharmacology, Department of Drug Sciences, Faculty of Pharmacy,
University of Porto, Porto, Portugal. 2 Department of Chemistry, University of Aveiro, Aveiro,
Portugal
* Acknowledgment: Reprinted from Journal of Pharmaceutical and Biomedical Analysis, 114,
Sara S. Marques, Luís M. Magalhães, Ana I.P. Mota, Tânia R.P. Soares, Barbara Korsak, Salette Reis, Marcela A. Segundo, Myoglobin microplate assay to evaluate prevention of protein peroxidation, 305–311, Copyright (2015), with permission from Elsevier.
4. General conclusions
The analytical approach developed using biomimetic system represents a step forward for
physiologically relevant antioxidant assays and for the assessment of ability of drugs to protect
biotargets from oxidative damage. The microplate protocol allows monitoring in a continuous
mode the myoglobin peroxidation providing kinetic information about the ability of drugs to
prevent protein oxidation. Drugs with low solubility in buffer media could be tested at 10% (v/v)
in ethanol with minor modifications in the heme environment of myoglobin, while dimethyl
sulfoxide (DMSO) as cosolvent should be avoided. Moreover, for comparison purposes the
antioxidant standard compound (Trolox) should also be tested under the same conditions.
The autoxidation process of myoglobin, which is an important role in pathophysiology of
vasospasms following subarachnoid hemorrhages and renal dysfunction after rhabdomyolisis, was
inhibited to 2.5–5.0% by Trolox (100 µM), labetalol (100 µM), enalapril (100 µM) and fluvastatin
(37.5 µM), while taurine (500 µM), atenolol (500 µM), propranolol (100 µM) and pravastatin (100
µM) did not prevent autoxidation. On the other hand, for reduced glutathione a pro-oxidant effect
was observed since the autoxidation of myoglobin increased up to 30% after 4h of reaction
monitoring. This took place because the interaction of thiols with reactive radicals can generate
thiyl radicals which have a pro-oxidant effect, especially in iron-rich environments, which is the
case of iron atoms release during Mb autoxidation.
Among the drugs tested, fluvastatin (12.5 ± 1.2 μM), enalapril (15.2 ± 1.8 μM) and propranolol (21.0 ± 2.2 μM) were those that presented higher ability to prevent myoglobin oxidative damage,
even higher than that determined for reduced glutathione (232 ± 10 μM). These results indicate
that these drugs may have pleiotropic effects by preventing oxidative damage of biomolecules,
such as myoglobin. Finally, the proposed microplate protocol allowed the assessment of
the estimated cost per analysis for each drug is about 0.90 €. And this is particularly important
since health care costs is one of the most important challenges of our time.
5.
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Supplementary Data
Myoglobin microplate assay to evaluate prevention of protein peroxidation
Journal of Pharmaceutical and Biomedical Analysis*
Sara S. Marques, Luís M. Magalhãesa, Ana I.P. Mota, Tânia R.P. Soares1, Barbara Korsak2, Salette Reis, Marcela A. Segundo
UCIBIO, REQUIMTE, Department of Chemical Sciences, Faculty of Pharmacy, University of
Porto, Rua de Jorge Viterbo Ferreira, 228, Porto 4050-313, Portugal
1 REQUIMTE, Lab of Pharmacology, Department of Drug Sciences, Faculty of Pharmacy,
University of Porto, Porto, Portugal. 2 Department of Chemistry, University of Aveiro, Aveiro,
Portugal
a Corresponding author.
Luís M. Magalhães (e-mail: [email protected] or [email protected])
* Acknowledgment: Reprinted from Journal of Pharmaceutical and Biomedical Analysis, 114,
Sara S. Marques, Luís M. Magalhães, Ana I.P. Mota, Tânia R.P. Soares, Barbara Korsak, Salette Reis, Marcela A. Segundo, Myoglobin microplate assay to evaluate prevention of protein peroxidation, 305–311, Copyright (2015), with permission from Elsevier.
Figure S1. Representation of the 96-well microplate scheme applied for monitoring of myoglobin oxidation from
peroxyl radicals and its protection by drugs: AAPH, 100 μl of AAPH (180 mM) + 200 μl phosphate buffer (pH 7.4, 50 mM,); Mb, 100 μl of myoglobin (75 μg/mL) + 200 μl of phosphate buffer; A6 or B6, 100 μl of the highest concentration of drug A or B + 200 μl phosphate buffer; AA6 or AB6, 100 μl of AAPH (180 mM) + 100 μl of the highest concentration of drug A or B + 100 μl phosphate buffer; MA6 or MB6, 100 μl of myoglobin (75 μg/mL) + 100 μl of the highest concentration of drug A or B + 100 μl phosphate buffer; Ct, 100 μl of myoglobin (75 μg/mL) + 100 μl of AAPH (180 mM) + 100 μl phosphate buffer; DAi, 100 μl of myoglobin (75 μg/ml) + 100 μl of AAPH (180 mM) + 100 μl of drug A with increasing concentrations; DBi, 100 μl of myoglobin (75 μg/ml) + 100 μl of AAPH (180 mM) + 100 μl of drug B with increasing concentrations. Each experiment was performed in quadruplicate and in three different days. A B C D E F G H 1 2 3 4 5 6 7 8 9 10 11 12 Mb Mb Mb Mb Mb Mb Mb Mb Ct Ct Ct Ct Ct Ct Ct Ct DA1 DA1 DA1 DA1 DB1 DB1 DB1 DB1 DA2 DA2 DA2 DA2 DB2 DB2 DB2 DB2 DA3 DA3 DA3 DA3 DB3 DB3 DB3 DB3 DA4 DA4 DA4 DA4 DB4 DB4 DB4 DB4 DA5 DA5 DA5 DA5 DB5 DB5 DB5 DB5 DA6 DA6 DA6 DA6 DB6 DB6 DB6 DB6 AAPH AAPH AAPH AAPH AAPH AAPH AAPH AAPH A6 A6 A6 A6 B6 B6 B6 B6 AA6 AA6 AA6 AA6 AB6 AB6 AB6 AB6 MA6 MA6 MA6 MA6 MB6 MB6 MB6 MB6
Figure S2. UV–Vis spectra of myoglobin solution (90 µg/mL) in the presence of AAPH solution (60 mM) obtained
at different reaction times (0, 30, 60, 90 and 120 min). The absorbance peak (409 nm) of native myoglobin decrease throughout reaction time due to protein oxidation induced by peroxyl radicals. Intrinsic absorbance of AAPH was subtracted from the absorbance decay of myoglobin.
0 0.2 0.4 0.6 350 400 450 500 550 600 A bsorba nce λ (nm) t = 0 t = 120 min
Figure S3. Molecular structures of cardiovascular drugs, endogenous antioxidants (glutathione and taurine) and
standard antioxidant (Trolox) tested.
Enalapril maleate salt
Reduced glutathione (±)-Propranolol hydrochloride
Labetalol hydrochloride
Taurine
Atenolol
Pravastatin sodium salt Fluvastatin sodium salt
