Reactive Species Detection in Biology. From Fluorescence to Electron Paramagnetic Resonance Spectroscopy

335 Pages·2015·21.126 MB·English

by  Frederick A. Villamena

REACTIVE SPECIES
DETECTION IN BIOLOGY
REACTIVE SPECIES
DETECTION IN BIOLOGY
From Fluorescence to Electron Paramagnetic
Resonance Spectroscopy
FREDERICK A. VILLAMENA
Department of Biological Chemistry and Pharmacology
TheOhio State University, OH, United States
AMSTERDAM(cid:129)BOSTON(cid:129)HEIDELBERG(cid:129)LONDON(cid:129)NEWYORK(cid:129)OXFORD
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DEDICATION
To Prof. DeLanson R. Crist, my mentor, father, and friend
And to my parents, Prisco and Tess,
for all your love and sacrifices.
PREFACE
Life is a sea of electrons that when in the state of equilibrium thrives but causes havoc
when perturbed. But this oversimplistic analogy leaves a multitude of unanswered
questions and is ridden with complexities. It is encouraging that current advancements
in biochemistry and biological chemistry have allowed modern-day biomedical inves-
tigators to understand some of these complexities and get ever closer to unraveling
some of the most fundamental questions in disease development. Most often over-
looked are the analytical tools employed to probe these mysteries. A research area by
itself, it owes its modern progress to chemists, biologists, engineers, and biomedical
researchers for the development of innovative molecular probes and imaging agents,
efficient methodologies, and state-of-the-art instrumentation. This book is therefore a
tribute to them: without their tireless dedication, our understanding of disease patho-
genesis would not be in the state it is today. At the heart of this perturbation of a sea
of electrons is the reduction and oxidation chemistry that involves a variety of players,
the most important of which is molecular oxygen; when not properly metabolized, it
results in the production of reactive species. These reactive species are like an army of
destructive forces that destroy, inactivate, or cause only partial functioning of key bio-
molecular systems essential for normal functions of living systems. Knowledge of reac-
tive species’ location, their origin, and their identity as well as their target molecules
and molecular trails they leave as biomarkers are critical and can only be accomplished
through the use of both earlier and modern sophisticated analytical tools. Reactive
species detection is therefore important for identifying specific molecular and cellular
pathways leading to oxidative stress that could lead the way to the development of
genetic, molecular, and pharmacological approaches, as well as diagnostic tools to pre-
vent or ameliorate free radical(cid:1)mediated diseases.
This book is basically divided into four major parts: introduction, chemistry of
reactive species, their biology, and their modes of detection. Chapter 1, Introduction,
provides a historical account of how oxygen and its reactive metabolites have been
implicated in the pathogenesis of diseases. It also describes how important discoveries
of the last century were aided by the use of analytical tools to characterize reactive
species as important biological mediators and how bioassays can complement such
findings. Chapter 2, Chemistry of Reactive Species, will only present biologically rel-
evant reactions of reactive species in an effort to give readers a sense of their relative
reactivity, specificity, and selectivity to various biomolecules. In Chapter 3, Reactive
Species in Biological Systems, shows various sources of reactive species in biological
settings, either from exogenous sources or as generated from enzymes, and it will
ix
x Preface
discuss various sources of reactive species production from cellular compartments.
Lastly, Chapters 4, Fluorescence Technique; Chapter 5, EPR Spin Trapping;
Chapter 6, UV(cid:1)Vis Absorption and Chemiluminescence Techniques; Chapter 7,
Electrochemical, Mass Spectroscopic, Immunochemical, and Nuclear Magnetic
Resonance Techniques; showcase conventional and modern analytical techniques that
are often if not widely employed in the detection of biological reactive species such as
fluorescence, electron paramagnetic resonance (EPR) spin trapping, UV(cid:1)Vis light
and chemiluminescence, and electrochemical, mass spectrometric, immunochemical,
and other magnetic resonance techniques with the exception of some techniques that
are too specialized (e.g., EPR and positron emission tomography imaging) or redun-
dant in their principles and chemistry to those already discussed here.
I hope readers will find this first edition a one-stop kind of reference material for
fundamental principles, limitations, and applications of the various analytical tech-
nique used in reactive species detection. Since this book will only cover so much
information, readers are encouraged to consult the references cited herein for more
detailed discussions and descriptions of the topics discussed.
This book would not have been possible without the dedication and passion of
investigators working in the field of free radical research, whether in the synthesis of
new molecules or the development of methodologies and instrumentations, as well as
biomedical researchers who continuously validate these tools for their effective
application in biological systems.
Frederick A. Villamena
Columbus, OH, United States
October, 2016
CHAPTER 1
Introduction
Respirationhasbeencommonlyknownforcenturiesasanessentialprocessfor survival
because it provides the “fuel” for the normal functioning of animal organs. This fuel
found in air was first known as dephlogisticated air on its discovery in the 1770s by
scientists Joseph Priestly and Carl Wilhelm Scheele.1,2 Antoine Lavoisier later coined
the term oxygen and extended the theoryof combustion to introduce the idea of respi-
rationasabiologicalprocessinwhichinhaledoxygenisusedintheoxidationofcarbon
andhydrogenfromfoodtogivecarbonicacid,thusrelating respirationasacombustion
process.3,4 Studies from 1940s and 1950s on the enzymatic metabolism of oxygen
provided molecular bases for oxygen’s diverse biological functions through enzyme-
catalyzedtransferofoxygenatomtoasubstrate,orelectron-transfer reactionstooxygen
leading to the formation of reactive oxygen species (ROS) or water.5 This enzyme-
catalyzed reduction of oxygen was later found to have beneficial as well as detrimental
effects on cellular function. Without a doubt, the progress made inunderstanding oxy-
genmetabolismowesadebttothedevelopmentofelectrochemicaltechniquesforana-
lyzing oxygen in biological fluids and tissues as well as whole animals. Oxygen sensor
developmentwasdescribed6datingasfarbackas1938whenthefirstbiologicalapplica-
tion of platinum electrodeswas demonstrated for the purpose of monitoring oxygen to
study photosynthesis, and this was followed by the use of a Clark-type electrode that
allowed for the measurement of oxygen tension (pO ) in an invivo system, air, blood,
2
andcellcultures,whichthenledtofurther innovationthatexhibitedhighaccuracy.
The absorption of oxygen and its transformation to carbonic anhydride (carbon
dioxide) during respiration has thus been the paradigm of oxygen metabolism.7 In the
early 1900s, the relationship between oxygen and disease was suggested by Todd,8
whereby the human body is in a state of chemical equilibrium between the processes
of oxidation and reduction; when this equilibrium shifts toward the formation of
more reduced species than oxidized ones, the body could lose resistance to diseases,
and hence resupplying the body with oxygen in the form of ozonized air or oxidized
oils is used as a therapeutic means of counteracting diseases such as tuberculosis or
Bright’s disease. Oxygen therapy was employed for a variety of diseases; e.g., patients
with respiratory disease such as pneumonia exhibited excellent therapeutic effect
when such therapy was introduced soon after diagnosis.9 It also became apparent that
ReactiveSpeciesDetectioninBiology r2017ElsevierInc.
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DOI:http://dx.doi.org/10.1016/B978-0-12-420017-3.00001-3 Allrightsreserved.
2 ReactiveSpeciesDetectioninBiology
oxygen exhibits toxicity against bacterial pneumococcus type I10 as well as in protozo-
ans,11 brain respiration,12,13 or in whole animals causing pulmonary damage.14 In
humans, oxygen results in the reduction of blood-flow rate to the brain when inhaled
at high atmospheric pressure,15 causing cerebral complications as well as diminished
overall cardiac output and changes in alveoli that result in edema, transudation, and
fibrinous deposits.16
The formation of hydroxyl radicals from water under ionizing radiation had long
been implicated for the radicals’ biological actions and toxicity.17 Soon thereafter,
chemical agents that have radiation-like properties were implicated in the initiation18
of cancer or tumor inhibition via chromosome alteration through formation of free
radicals.19 Evidence supported the idea that free radicals formed radiolytically were
toxic because they were found to diffuse inside the cells when generated extracellu-
larly.20 Moreover, it was also demonstrated that x-ray radiation inhibited glutathione
metabolism inside the cells, and this inhibition was decreased at low oxygen concen-
tration and on addition of catalase, which suggested the involvement of oxygen-
derived reactive species such as H O .21 The link between radiation and oxygen levels
2 2
on their cellular toxicity had become more apparent by their inactivation of T bacte-
2
riophage and by the observation that thiol compounds such as thiourea could com-
pete with oxygen-derived radicals, thereby protecting the phages from radiation.22
Using electron paramagnetic resonance (EPR), radiation damage to DNA or RNA
was reported to produce paramagnetic nucleic acids at 77K.23 This finding was consis-
tent with the increased effect of radiation on DNA inactivation in the presence of
oxygen and protection in the presence of the thiol cysteamine24,25 with mutations
successfully induced in the cell nucleus of onion rootlets, e.g., by hydroxyl radical and
x-irradiation.26
While the effect of irradiation-mediated free radical formation on nucleotides
was not as pronounced as in proteins and peptides, it became clear that free radicals
formed enzymatically could have profound consequences on protein function.
Metabolic hydroxylation of aromatic amino acids has long been suspected as a
biosynthetic process for the conversation of phenylalanine to tyrosine, tyrosine to
3,4-dihydroxyphenylalanine, kynurenine to 3-hydroxykynurenine, and tryptophan
to 5-hydroxytryptophan.27 In the 1950s, metabolic hydroxylation of aromatic com-
pounds such as N-2-fluorenylacetamide in guinea pigs and rats was demonstrated
and believed to be a detoxification mechanism.28 This conversion was duplicated in
cell-free in vitro studies involving ferrous ion, ascorbic acid, oxygen, and a chelating
agent (ethylenediaminetetraacetic acid) under physiological conditions, showing that
hydroxylation of aromatic compounds could indeed be mediated by free radial
reaction, specifically that of hydroxyl radicals.29 Not long after, it was proposed that
biological hydroxylation occurs via activation of oxygen by peroxidase30,31 and
by other hydroxylating systems found in liver microsomes that require
Introduction 3
triphosphopyridine nucleotides and oxygen for their activity.32 Altogether,
Harman33 proposed the role of oxygen, metals ions, oxidative enzymes, and
radiation on the development of age-related and degenerative diseases through gen-
eration of reactive oxygen species, and these propositions became the foundation of
today’s widely accepted free radical theory of aging. Subsequently, the link between
free radicals and the development of atherosclerosis,34,35 cancer,36 and neurodegen-
eration37 was proposed, and the role of proper nutrition and lower metabolic
demand were seen as essential for the slower progression of free radical(cid:1)mediated
reactions in the body.38,39 While free radicals such as semiquinones were identified
using EPR as integral components of the mitochondrial respiratory chain,40,41 evi-
dence for the production of ROS such as superoxide and hydrogen peroxide by
mitochondria through electron transfer had become more compelling.42(cid:1)44
Although the existence of superoxide as an inorganic species is known dating as far
back as the late 1890s, its paramagnetic character was not established until the
1930s.45 For the next four decades, studies on superoxide were mostly focused on
their chemistry with metals and nonmetals and, for the first time, its characterization
through EPR spectroscopy.46 Only in the late 1960s did the idea become
acceptable that superoxide could also be generated in biological system, an idea helped
by the discovery of superoxide generation from enzymatic systems. The idea was first
introduced by McCord and Fridovich,47 whose seminal study demonstrated the
production of superoxide from xanthine oxidase and xanthine; they found this formed
species capable of reducing cytochrome c and initiating the sulfite oxidation reaction.
In further support of this evidence, superoxide formation from xanthine oxidase was
confirmed using EPR spectroscopy at pH 10; signal intensity was shown to be depen-
dent on oxygen concentration and not the enzyme itself.48 The oxygen origin of
superoxide from xanthine oxidase was unequivocally confirmed using 17O-labeled O
2
and EPR spectroscopy giving the 11-line or 6-line EPR spectra for the formed
17O (cid:129)(cid:1) or 17O16O(cid:129)(cid:1), respectively.49 Generation of superoxide from oxygen was
2
achieved through electrolytic reduction of oxygen in aqueous solution.50 In addition,
electrochemically generated chemiluminescence of lucigenin showed evidence of
superoxide-mediated light emission, paving the way for the development of chemilu-
minescence probes for superoxide.51 Excited-state oxygen resulting from the oxidation
of xanthine by xanthine oxidase can also induce chemiluminescence via recombina-
tion of ROS, probably that of superoxide,52 and this finding was further supported
by evidence showing that singlet oxygen(cid:1)sensitized fluorescence from organic
compounds is mediated by superoxide, which suggests the possible enzyme-mediated
formation of singlet oxygen.53
The chemistry of superoxide enzymatic formation and decomposition then
became of interest to investigators who wanted to know whether the mechanism is
ligand mediated or metal mediated. It was demonstrated that superoxide is
4 ReactiveSpeciesDetectioninBiology
decomposed by erythrocuprein and ferricytochrome c and is formed during the
oxidation of reduced flavin54 rather the iron heme of flavoproteins.55 This was later
supported by studies showing that the one-electron reduction of oxygen by reduced
flavins and quinones results in the formation superoxide.56 However, the formation of
superoxide from the reaction of oxygen with reduced iron-sulfur proteins from plant
ferredoxins that are flavin free was also reported,57 indicating that oxygen reduction
can occur via electron transfer not only from organic radicals but also from low-valent
metal ions.56 The enzyme superoxide dismutase (SOD) was then proposed to catalyze
the dismutation of superoxide to oxygen and hydrogen peroxide and had been a gold
standard as a competitive inhibitor for the investigation of superoxide-mediated
reactions such as the oxidation of epinephrine to adrenochrome by xanthine oxidase
and the reduction of ferricytochrome c or tetranitromethane.58 The reduction of
ferricytochrome c was found to be augmented by electron carriers such as flavin
adenine dinucleotide, menadione, or flavin mononucleotide.59 The characterization
of SOD in bovine heart and its ubiquity in mammalian tissues suggest the important
role SOD plays in the regulation of ROS in biological systems.60 It became clearer
that the cause of oxygen toxicity is not oxygen itself but the oxygen-derived reactive
species such as superoxide, hydrogen peroxide, and hydroxyl radicals.
Detection of reactive species in biological systems could date as far back as the late
1800s and early 1900s through visual observations of color changes in test tubes or
spot plates. For example, the detection of hydrogen peroxide in plants and milk
employed the use of various reagents that impart color changes with oxidation.61(cid:1)63
The introduction of commercial ultraviolet visible spectrum (UV vis) spectrophot-
ometers in the early 1940s by Arnold O. Beckman64 allowed for the detection of
oxygen-derived radicals (e.g., superoxide and hydroxyl) in biological systems. Since
then, ROS detection focused on the use of spectrophotometric techniques that allow
detection at the UV region and providing a more accurate, reproducible, time-saving,
and reduced sample size for ROS analysis. Detection of superoxide became possible
through superoxide dismutation by superoxide dismutase or reduction by ferricyto-
chrome c.56,59,65 Several other techniques has been employed for the analysis of H O
2 2
and superoxide radical such as electrochemistry,66 fluorescence,67 chemilumines-
cence,68 and EPR spin trapping.69 Hydroxyl radical detection involved the analysis of
gaseous substances such as methane on hydroxyl radical reaction with dimethyl sulfox-
ide from human phagocytes as analyzed by mass spectroscopy70 or 14C-carbon dioxide
from the decarboxylation of [14C]benzoic acid on oxidation by hydroxyl radicals, also
from human ganulocytes, using an ionization chamber-electrometer.71 Concurrently,
the hydroxyl radical was identified through hydroxylation of 5,5-dimethyl-pyrroline
N-oxide (DMPO) by EPR spin trapping in respiring rat heat mitochondria44
or through hydroxylation of salicylate as measured by colorimetric or gas chro-
matographic (GC) assays.72