DETERMINING THE MECHANISM
FOR PHOTOSENSITIZED OXIDATIONS
Jeffrey R. Kanofsky
261 E Adelia Street
Elmhurst, IL 60126
Photosensitizers absorb light, generating electronically excited states.
Here S is a photosensitizer in the ground state, 1
S* is the photosensitizer in an
excited singlet state and 3
S* is the photosensitizer in an excited triplet state.
Generally, an excited singlet state is produced first, but most efficient
photosensitizers will then undergo an intersystem crossing to produce an excited
triplet state that is relatively long-lived. Excited states with long lifetimes are more
likely to interact with other molecules to initiate photochemistry.
As shown by Equations 3, 4 and 5, respectively, an excited photosensitizer may
loose its excitation energy via fluorescence, via phosphorescence or via non-
radiative transitions that ultimately produce heat.
When energy loss occurs by any of these mechanisms, no photochemistry is
Photochemistry occurs when an excited photosensitizer interacts with other
molecules. Most commonly, these molecules are oxygen, a solvent molecule or a
target molecule added to the solvent. The excited photosensitizer may transfer
either energy or charge during interactions with other molecules. The various
possibilities are shown in Figure 1.
Figure 1. Mechanisms of photosensitized reactions. Here 3S* is a photosensitizer in an excited triplet state, 3SH* is another representation of the same photosensitizer, but with a potentially transferable proton indicated, X is a target molecule, and XH+ is a substrate or solvent molecule that has accepted a proton.
The mechanisms responsible for photosensitized oxidations have been classified
into two categories, called Type I and Type II photochemistry. There is general
agreement that reactions generating free-radicals from a target molecule or the
solvent should be classified as Type I photochemistry. Similarly, there is
agreement that singlet-oxygen generation should be classified as Type II
Authors differ on how the other mechanisms shown in Figure 1 should be
classified. Some authors classify photochemical mechanisms based on the initial
molecule with which the excited photosensitizer interacts (1). Photosensitizer
interactions with either solvent or target molecules are called Type I
photochemistry. Photosensitizer interactions with oxygen are called Type II
photochemistry. Consequently, these authors would classify the formation of an
electronically excited target molecule as Type I photochemistry, and superoxide
generation as Type II photochemistry. Other authors feel that the distinction
between Type I and Type II photochemistry should be based on whether charge
transfer or energy transfer occurs (2). All charge-transfer reactions are classified
as Type I photochemistry, while all energy-transfer reactions are classified as
Type II photochemistry. Consequently, these authors would classify the
superoxide production as Type I chemistry, and the formation of an electronically
excited target molecule as Type II chemistry.
There are relatively few molecules with electronically excited states that are
sufficiently low-lying so that these molecules can accept energy from commonly-
used photosensitizers. Thus, the formation of an electronically-excited target or
solvent molecule is an uncommon means of initiating photochemistry, particularly
in biological systems. For some molecules with low-lying electronic states, such
as carotenoids, the energy transferred to the molecule is often ultimately
dissipated as heat rather than used to initiate a chemical reaction.
Throughout this chapter, the term singlet-oxygen will refer to the 1g
exists a more energetic singlet state of oxygen, the 1g+
However, the 1g+
state rapidly relaxes to the 1g
state. The lifetime of the 1g+
state is so short that
it does not appear to initiate any photochemistry (3).
Considerable effort has been devoted to the development of methods that can
identify the various mechanisms of photosensitized oxidations. There are many
assays for singlet-oxygen generation with various degrees of sensitivity and
specificity. Likewise, there are multiple tests that can detect the formation of free
radicals, including superoxide. Several excellent reviews of this topic have been
published previously (4-10). The major emphasis in this chapter will be on assays
for singlet oxygen.
Assays for Singlet Oxygen
Assays for singlet oxygen include the measurement of singlet-oxygen
phosphorescence at 1270 nm, the detection of specific singlet-oxygen products
using various chemical traps, the inhibition of product formation using singlet-
oxygen quenchers, and the use of the deuterium-isotope solvent effect.
Singlet oxygen is an electronically excited molecule and consequently can emit
light. The measurement of singlet-oxygen phosphorescence at 1270 nm is one of the
most specific methods for detecting singlet oxygen. The high specificity results
from the empiric observation that other emission sources with peaks close to
1270 nm are uncommon. This is probably due to the fact that very few molecules
have low-lying electronically excited states with transition energies similar to
oxygen. Since the spin restriction rule of quantum mechanics does not allow the
transition from singlet oxygen (1g) to ground-state oxygen, the intensity of the singlet-
oxygen phosphorescence is generally very weak. The low intensity of the
emission limits the sensitivity of the method. Studies of singlet-oxygen
phosphorescence can be carried out using either time-resolved or steady-state
In time resolved-studies, the photosensitizer is usually excited with a pulsed
laser. A block diagram of a typical apparatus is shown in Figure 2. The
phosphorescence is measured at right angles to the laser beam, using filters that
transmit 1270-nm radiation, but prevent scattered laser light and fluorescence
from reaching the near-infrared detector. The most commonly used near-infrared
detectors are germanium photodiodes, indium gallium arsenide photodiodes and
photomultipliers with indium phosphide/indium gallium arsenide phosphide
photocathodes. All of these detectors are sensitive to the 1270-nm radiation.
One advantage of the time-resolved method over the steady-state method is that the lifetime of the singlet-oxygen emission is measured in the time-resolved method. The measured emission lifetime can be compared with the theoretical lifetime of singlet oxygen under the conditions being studied. Agreement between the measured lifetime and the theoretical lifetime provides additional confirmation that the emission actually comes from singlet oxygen.
Figure 2. Apparatus for time-resolved measurements of singlet-oxygen phosphorescence.
In a homogeneous photochemical system, the formation and decay of singlet
oxygen are described by Equations 6 and 7.
is the rate constant for the reaction of the excited triplet photosensitizer,
S*, with ground-state oxygen, ki
is the total singlet-oxygen quenching constant
(physical + chemical) for quencher, Mi
are the oxidation products of Mi
n is the number of different substances in the system (including the solvent). If
we assume that the concentrations of all Mi
and of ground-state oxygen remain
constant, Equation 8 will describe the singlet-oxygen kinetics.
The kinetics of the excited triplet photosensitizer will be given by
If we now assume that a quantity of excited triplet photosensitizer is produced
instantaneously (i.e., via a very short laser pulse), then the solution to Equation 9
is given by
is the initial concentration of excited triplet photosensitizer
generated. The time dependence of the excited triplet photosensitizer given by
Equation 10 can then be substituted into Equation 8.
The intensity of the singlet-oxygen phosphorescence is proportional to the
concentration of singlet oxygen in the system. Thus, the solution to Equation 8,
multiplied by a constant, will describe the kinetics of the singlet-oxygen emission. The emission kinetics are given by Equation 11,
where I is the intensity of the emission, A is a constant that depends on various
instrument parameters and on the solvent,
is the emission lifetime, ß is the
emission rise time, and t is time (11). Under conditions where the k1
greater than k2
, the emission rise time, ß, will be determined by the rate of
reaction of the excited photosensitizer with oxygen, and the emission lifetime,
will be determined by the lifetime of singlet oxygen. Under conditions where
] is less than k2
, the emission lifetime will be determined by the rate of
reaction of the excited photosensitizer with oxygen, while the emission rise time
will be determined by the lifetime of singlet oxygen (11).
The yield of singlet oxygen from a photosensitized reaction is proportional to the
time-integrated singlet-oxygen phosphorescence intensity divided by the singlet-oxygen lifetime,
where Y is the yield of singlet oxygen,
is the lifetime of singlet oxygen in the
system, and t is time. Combining Equations 11 and 12 gives (11):
In Equation 13, we assume that the singlet-oxygen lifetime is longer than the
lifetime of the excited triplet photosensitizer (i.e.,
It is important to remember that Equation 13 cannot be used to compare the
singlet-oxygen yields in different solvents. At an identical concentration of singlet
oxygen, the intensity of the phosphorescence in various solvents will differ,
because the radiative lifetimes of singlet oxygen vary in different solvents (12).
Thus, the constant, A, will differ in different solvents.
The fluorescence of some photosensitizers extends out into the near-infrared to
at least 1270 nm. An advantage of the time-resolved method over the steady-state method is that the fluorescence can sometimes be cleanly separated from
the singlet-oxygen phosphorescence. In time-resolved studies, this fluorescence
appears as a sharp peak coincident with the laser pulse. The apparent lifetime of
the fluorescence peak is usually determined by the response time of the near-infrared detector. Large fluorescence peaks can overload near-infrared detectors
or their associated electronics. This instrumental artifact limits the energy of the
laser pulse that can be used, and consequently limits the sensitivity of the time-resolved method for photosensitizers with a large fluorescence at 1270 nm.
Simulated data for a typical time-resolved experiment are shown in Figure 3. Note
how the singlet-oxygen emission is nicely separated from the fluorescence.
Figure 3. Data set simulating time-resolved emission from a photosensitizer in
deuterium oxide. The rapidly decreasing emission at the left of the figure is due
to fluorescence. The rate of increase in emission is determined by the rate at
which the excited triplet photosensitizer reacts with oxygen. The decay rate is
determined by the lifetime of singlet oxygen in deuterium oxide, about 68 µs.
Steady-state measurements of singlet-oxygen phosphorescence have also
proved to be useful for determining mechanisms of photooxidation. The spectral
resolution may be achieved with a series of interference filters, a conventional
monochromator or an interferometer-based monochromator (13). ). A typical apparatus for the measurement of steady-state singlet oxygen phosphorescence is shown in Figure 4.
Figure 4. Apparatus for the measurement of steady-state singlet-oxygen phosphorescence.
Photosensitizer fluorescence in the near-infrared can potentially obscure singlet-
oxygen phosphorescence in steady-state measurements. In the interpretation of
emission spectra from steady-state experiments, investigators generally take
advantage of the fact that photosensitizer fluorescence tends to decrease
monotonically with increasing wavelength, and generally does not have any
structure in the spectral region near 1270 nm. In contrast, singlet-oxygen
phosphorescence shows a clear emission peak near 1270 nm. Thus, the
fluorescence signal usually appears as a sloping baseline under the singlet-
oxygen emission peak (13).
Singlet-oxygen traps are compounds that react with singlet oxygen to produce
specific products, not generated by any other oxidants. Many traps for singlet
oxygen have been developed. They differ in sensitivity, selectivity and solubility.
Cholesterol is one of the most specific traps for singlet oxygen. The reaction of
singlet oxygen with cholesterol produces three specific products, 5
-cholesterol hydroperoxide and 6ß-cholesterol hydroperoxide
(14-16). Generally, the 5
-cholesterol hydroperoxide is the most abundant
product (14). In contrast, radical oxidation of cholesterol produces 7
7ß-cholesterol hydroperoxide (14-17). However, the
significance of two
7-cholesterol hydroperoxides is ambiguous, since 5
-cholesterol hydroperoxide, one of the oxidation products generated by singlet
oxygen, can undergo allylic rearrangement to form 7
(14-17). Subsequently, 7
-cholesterol hydroperoxide can epimerize to form 7ß-cholesterol hydroperoxide (see Figure 5)
Figure 5 A. Cholesterol oxidation products resulting from singlet oxygen.
Figure 5B. Cholesterol oxidation products resulting from oxidant radicals.
Figure 5C. Cholesterol oxidation products that result from
the allylic rearrangement
and epimerization of
The various cholesterol hydroperoxides can be separated with either thin-layer
chromatography or HPLC. Sensitive detection methods for the cholesterol
hydroperoxides have been developed that use radiolabeling of the cholesterol,
chemiluminescence or electrochemical reduction with a mercury drop electrode
Cholesterol trapping has two principal limitations. First, the sensitivity of
cholesterol trapping is limited due to its relatively slow rate of reaction with singlet
oxygen (20). Second, cholesterol is not soluble in highly polar solvents, such as
water. The solubility limitation can be overcome by preparing small beads with a
thin coating of cholesterol, and placing the beads in the polar solvent during the
photosensitized oxidation (21).
The reaction of singlet oxygen with various anthracene derivatives also provides
a relatively specific trapping system. As shown in Figure 6, the specific product
formed is an endoperoxide. Singlet oxygen reacts more rapidly with anthracenes
than cholesterol, making the anthracenes more sensitive traps than cholesterol.
Derivatives of anthracene have been prepared that are soluble in a wide variety
of solvents including water. Thin deposits of anthracenes on the surface of small
beads, or on surface of flat sheets, have also successfully been used as singlet-
oxygen detectors in biological studies (22-23). One caution in the use of
anthracenes is that anthracenes are photosensitizers. Thus, anthracenes cannot
be used in photochemical studies at wavelengths where the anthracenes absorb
Figure 6. Singlet oxygen reacts with anthracenes to produce endoperoxides.
A singlet-oxygen quencher is a molecule that rapidly interacts with singlet oxygen
either physically, producing ground-state oxygen and heat, or chemically,
producing oxidation products.
Examples of molecules that are commonly used as
quenchers include azide anion, 1,4-diazabicyclo[2.2.2]octane (DABCO), histidine
and various carotenoids, such as ß-carotene. The principal limitation of
quenchers is a lack of specificity. Most quenchers are easily oxidized substances
and thus will react with many types of oxidants.
The specificity of a singlet-oxygen quencher can be improved by comparing the
decrease in oxidation products caused by the addition of the quencher with the
theoretical reduction expected. A formula to calculate the theoretical reduction is
easily derived. Consider a photochemical system undergoing constant irradiation.
The system is composed of solvent, photosensitizer, target molecules for singlet oxygen
and multiple other components. Assume that the concentrations of all system
components remain constant. The following two equations will apply:
where k is the rate constant for energy transfer between the excited triplet
photosensitizer and ground state oxygen, and
is a system component, such as the solvent, a substrate or an added
is the total singlet-oxygen quenching constant (chemical plus
physical) for system component i, and Pi
is an oxidation product. From Equations
14 and 15, it follows that
where n is the number of system components. Making the steady-state
assumption that d[1
]/dt = 0 gives
From inspection of Equation 17, we see that the steady-state concentration of
singlet oxygen will be directly proportional to the concentration of excited triplet
photosensitizer, and inversely proportional to the total singlet-oxygen quenching
The denominator of Equation 17 can be rewritten to provide better clarity.
Quenching due to the solvent is generally expressed in terms of the singlet-
oxygen lifetime in the neat solvent rather than as the product of the concentration
of solvent molecules and the singlet-oxygen quenching constant for the solvent.
Also, we will separate the quenching component due to the added quencher from
the rest of the sum. This gives,
is the lifetime of singlet oxygen in the neat solvent, kq
is the total singlet-oxygen quenching constant for the added quencher, and cq
is the concentration of
the added quencher.
The rate of product formation should be directly proportional to the steady-state
concentration of singlet oxygen. From Equation 18, it follows that the ratio of
product formed in the absence of the added quencher (P0
) to the product formed
in the presence of the added quencher (P) is given by,
From the inspection of Equation 19, it follows that plots of P0
/P against the concentration of added quencher, cq
, will be linear. These are called Stern-Volmer plots. It is important to point out that a linear Stern-Volmer plot can also be seen with Type I photochemistry. The only requirement for a linear Stern-
Volmer plot is that there be an intermediate that can either be inactivated by the
quencher or go on to produce the product being measured (6). The intermediate
does not have to be singlet oxygen. Agreement between the slope of a Stern-Volmer plot of experimental data and the slope calculated from known singlet-
oxygen quenching constants is strong evidence favoring a singlet-oxygen
Singlet-oxygen lifetimes in many solvents are known (24). Thus, Equation 19 can often be evaluated from data in the literature to calculate a theoretical value
for the slope of a Stern-Volmer plot. Obtaining Stern-Volmer plots with more than
one quencher will further improve the specificity of the method.
Deuterium-isotope solvent effect.
The lifetime of singlet oxygen is considerably longer in solvents where deuterium
has been substituted for hydrogen than the lifetime in unadulterated solvents
(25). For example, the lifetime of singlet oxygen is 68 µs in deuterium oxide, but
only 3.1 µs in water (26-27). Thus, under continuous irradiation, the steady-state
concentration of singlet-oxygen should be higher in deuterated solvents and
consequently, the rate of production of oxidation products should be higher.
Thus, in very dilute solutions, the deuterium-isotope solvent effect is substantial.
In dilute aqueous solutions, the deuterium-isotope solvent effect can be as large
However, caution is needed in interpreting the effect of deuterium substitution in the solvent, deuterium substitution can perturb photochemical systems in other ways. In some systems, the deuterium-substituted solvent will exchange deuterium atoms with hydrogen atoms in target molecules. The kinetics of a photooxidation being studied may then be changed due to primary or secondary kinetic isotope effects that can occur at any step in a complex photooxidation mechanism.
A primary kinetic isotope effect occurs when the deuterium-hydrogen exchange
occurs at a bond that is either broken or formed during the oxidation. Primary
kinetic isotope effects can be large. At 25° C, substitution of deuterium for
hydrogen can reduce reaction rates by factors of up to 6.9, 9.2 and 11.5, for
carbon-hydrogen bonds, nitrogen-hydrogen bonds and oxygen-hydrogen bonds,
respectively (28). These ratios are comparable in magnitude to the ratios in
singlet-oxygen lifetimes seen between deuterated solvents and non-deuterated
solvents. Secondary kinetic isotope effects occur when the substitution of
deuterium for hydrogen occurs at bonds that are not broken or made during the
photooxidation. These effects are generally much smaller, but in water, the
secondary solvent isotope effect can be large, due to changes in acidity (29).
Substituting two deuterium atoms for two hydrogen atoms in the water molecule
significantly changes its chemical properties (29).
When using the deuterium isotope solvent effect as a mechanistic test, it is best
to select conditions where the reactions being studied go to completion. In some
cases, this will reduce the perturbations caused by kinetic isotope effects.
However, in systems, where two or more alternative reactions compete, kinetic
isotope effects can still cause significant changes in the oxidation-product
distribution even when the reactions are carried to completion.
The specificity of the deuterium-isotope solvent effect can be improved if one
requires quantitative agreement between the amount of enhanced photooxidation
and a theoretical value for the expected increase in singlet-oxygen lifetime. The
expected increase in oxidation products caused by deuterium substitution in the
solvent is given by Equation 20,
is the amount of oxidation product formed in deuterated solvent, PH
the amount of oxidation product formed in non-deuterated solvent, H
lifetime of singlet oxygen in non-deuterated solvent, D
is the lifetime of singlet
oxygen in deuterated solvent, and n is the number of components in the system
other than the solvent. The definitions of the other symbols are the same as
those given in Equation 19. In Equation 20, we assume that no exchange of
deuterium for hydrogen occurs between the solvent and the substrates. The
numerator of Equation 20 represents the total singlet-oxygen quenching rate of
the system being studied using a non-deuterated solvent. The denominator
represents the total singlet-oxygen quenching rate for the system using the
analogous deuterated solvent.
The derivation of Equation 20 is analogous to the derivation of Equation 19. The
student may wish to derive Equation 20 as an exercise.
From Equation 20, it follows that in some photochemical systems, the deuterium
isotope solvent effect will be very small. This phenomenon is shown in Figure 7.
When the rate of quenching of singlet oxygen by the various substrates present
in the system is much greater than the rate of quenching by solvent, deuterium
substitution in the solvent will not significantly affect the total quenching rate, and
consequently there will be only a small effect on the lifetime of singlet oxygen.
Figure 7. Blunting of the deuterium solvent effect by added quenching agents to an
aqueous photochemical system. The singlet-oxygen lifetime shown on the x-axis is the result of quenching by both water and added quenching agents.
Substitution of deuterium oxide for water in cell cultures is particularly
problematic. The effect of this solvent change on the complex biochemistry of
cells is incompletely understood. Data from experiments of this type are
particularly difficult to interpret when one uses a biological assay of cell damage,
such as apoptosis, as opposed to a simple chemical assay, such as
hydroperoxide formation. Further, there is uncertainty about the lifetime of
singlet oxygen within the cell. Some studies suggest that the lifetime of singlet
oxygen is less than 1 µs and that most of the singlet oxygen is quenched by the
high concentration of biomolecules present within the cell rather than by water
(30-33). If these studies are correct, one would expect a very small deuterium
solvent effect. However, other studies suggest that the lifetime of singlet oxygen
within the cell is close to that of water and that there is little quenching of singlet
oxygen by biomolecules present within the cell (34). If these latter studies are
correct, one would expect a very large deuterium solvent effect in cells.
Photooxidations based on the generation of singlet oxygen are clearly oxygen
dependent. In contrast, radical oxidations may or may not have an oxygen
dependence. Very often free-radical chemistry is mediated by chain reactions,
such as those shown by Equations 21 and 22.
Even though no oxygen may be required for the initial photochemical production
of the radical R.
, oxygen is required for the radical propagation steps. Thus, the
exclusion of oxygen from a photochemical system, with free-radical propagation
steps such as those shown by Equations 21 and 22, may decrease or eliminate
the formation of oxidation products.
Assays for Free Radicals Including Superoxide Anion
Now we will consider the assays for the detection of radical-mediated
photochemistry. Electron paramagnetic spin resonance (ESR) detection of spin-trap adducts is one of the most commonly used assays and is useful for most free radicals. Photochemistry that does not have a dependence on oxygen also provides strong evidence favoring free-radical mechanism. In some cases, time-resolved transient-absorption spectroscopy can be used to detect cationic or
anionic photosensitizer radicals. Finally, cytochrome-c reduction that is inhibited
by superoxide dismutase is a widely used assay for superoxide.
ESR spin trapping.
Some free radicals have distinctive, narrow-lined ESR spectra and are relatively
stable. Free radicals with these properties can often be directly detected by ESR.
In photochemical oxidations, most free radicals of interest, such as hydroxyl
radical or superoxide cannot be directly detected with ESR. Detection of these
radicals depends on the use of spin traps (35-36). Spin traps are compounds that
react with unstable radical species to form relatively stable radical adducts that
have distinctive ESR spectra.
Most of the commonly used spin traps are nitrone or nitroso derivatives. One of
the most popular spin traps for aqueous solutions is 5,5-dimethyl-1-pyrroline N-
oxide (DMPO). This trap is useful for the identification of hydroxyl radicals, of
various peroxyl radicals, and of superoxide.
Superoxide reacts with DMPO relatively slowly (35-36). As a result, very high
concentrations of DMPO, on the order of 100 mM, are needed to efficiently trap
superoxide in biological studies. These very high concentrations of DMPO can
potentially be toxic to cells. The DMPO-OOH adduct can occasionally be
confused with adducts formed from the reaction of peroxyl radicals with DMPO.
As a consequence, definitive identification of superoxide requires that the DMPO-OOH adduct decrease with the addition of enzymatically-active superoxide
dismutase, but not superoxide dismutase that has been denatured. An additional
problem with the DMPO-OOH adduct is that it may decompose to produce the
DMPO-OH adduct. Formation of this later adduct is usually taken as evidence for
Hydroxyl radicals react with DMPO to give the DMPO-OH adduct with a rate
constant on the order of 109
. However, generation of the DMPO-OH
adduct lacks specificity. The DMPO-OH adduct may result from the
decomposition of the DMPO-OOH adduct or from the interaction of singlet
oxygen with DMPO (37-38). Thus, additional studies are required to confirm the
presence of free hydroxyl radicals. Generally, hydroxyl radical traps are used to
carry out this confirmation. Ethanol, formate and dimethyl sulfoxide are common
hydroxyl radical traps. These compounds react rapidly with hydroxyl radicals,
successfully competing with DMPO. The products of the reactions of hydroxyl
radicals with all of these three traps are carbon-centered radicals that
subsequently may be trapped by DMPO. Thus, the addition of a hydroxyl-radical
trap to the system should decrease the amount of DMPO-OH formed and replace
it with the DMPO adduct of a carbon-centered radical. The specificity of the
method is further improved if more than one hydroxyl-radical trap is used.
Oxygen dependence of products.
As was discussed previously, the absence of oxygen dependence is a strong
argument favoring radical-mediated photochemistry. In contrast, the presence of
a dependence on the oxygen concentration has an ambiguous interpretation that
can occur with both Type I and Type II photochemistry.
Time-resolved transient absorption spectroscopy.
When charge is transferred from the photosensitizer to an acceptor molecule,
either a cationic or anionic photosensitizer radical is produced. In some cases,
these radical species have absorption spectra significantly different from the
ground-state photosensitizer and from the excited triplet photosensitizer. In such
cases, these radical species may be detected using time-resolved transient
absorption spectroscopy (39). Generally the photosensitizer is excited with a
pulsed laser. Time-dependent changes in the absorption spectra are then
measured at right angles to the laser beam.
Superoxide-dismutase inhibitable reduction of cytochrome c.
This simple assay requires only a spectrometer (40-41). Cytochrome c is readily
reduced by superoxide, but cytochrome c is also readily reduced by many other
agents. Superoxide dismutase is an enzyme that specifically catalyzes the
destruction of superoxide. The difference between the rate of reduction of
cytochrome c in the presence of active superoxide dismutase and in the
presence of denatured superoxide dismutase gives the rate of production of
superoxide. Measurements of both DMPO spin trapping and cytochrome c
reduction may be used together to provide additional confirmation of the
production of superoxide.
Determining the mechanisms of photochemical oxidations has proven to be a
challenging problem. A wide variety of assays are available for both singlet
oxygen and for free radicals including superoxide. In photochemical oxidations,
singlet-oxygen generation often competes with free-radical generation. The
fraction of the phooxidation that proceeds by each route will depend upon the
conditions. High oxygen concentrations favor the formation of singlet oxygen or
of superoxide. In contrast, high target-molecule concentrations and low oxygen
concentrations favor the formation of other free radicals.
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