Free Radical and Antioxidant Protocols

455 Pages·1998·15.63 MB·English

by  Donald Armstrong

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Oxygen Consumption Methods
Xanthine Oxidase and Lipoxygenase
lstvan Stadler
1. Introduction
A biochemical paradox that has been observed for many years is now
becoming understood: 02, essential for the aerobic life forms, can be inappro-
priately metabolized, becoming toxic to an organism. Mammals derive most of
their cellular adenosine triphosphate (ATP) from the controlled four-electron
reduction of 02, to form HZ0 by the mitochondrial electron-transport system.
Approximately 98% of all O2 consumed by cells enters the mitochondria, where
it is reduced by a terminal oxidase, such as cytochrome oxidase.
However, O2 can accept less than four electrons to form a reactive 0;
metabolite, which may be toxic to cells. The reactions producing various
reduced species are illustrated on the following system:
O2 + e > 0, (superoxide)
O2 + 2e- + 2H+ > H202 (hydrogen peroxide)
02 + H202 + H+ > O2 + HZ0 + *OH (hydroxyl radical)
O2 + 4e- + 4H+ > 2 H20 (water)
The spin restriction on 02, limits the speed of molecular O2 reactions.
Organic compounds exist m a single state, meaning the outer orbital is closed,
with one electron. O2 however, exists in a triplet diradical state, with three
electrons in the outer orbital capable of activity. Spin restriction can be over-
come if enough energy of activation is available to excite OZ. If this occurs, a
spin inversion or pairing of outer electrons is possible. A singlet O2 (Oi), is
From Methods m Molecular Bology, vol 108 Free Radmal and Anbox/dant Protocols
Edlted by D Armstrong 6 Humana Press Inc , Totowa, NJ
3
4 Staciler
Table 1
Forms of Injury-Associated Free Radical Production
Injury Damage
Ischemm/reperfusion Lipidperoxidation (enzymatic and non)
Thermal injuries Breaking of DNA, proteins
Trauma, toxins, radiation exposure Metal ion released, stimulating free-
radical formation
Infection, excess exercise Interference with natural anttoxidants
I
S ATP Fe3’ + OH* + OH-
C & T
H ADP Fenton reaction
E 4. ?
M AMP
I A xanthine dehydrogenase Fe2+
A Adenine & ?
& xanthine oxidase
Hypoxanthine - Urate + 02* + Hz02
REPERFUSION (oxygenation)
Fig 1 Sources for oxygen-derived free radicals
produced, which is a potent oxidant with an extremely short lifetime. Once thts
oxidative agent is formed, it can cause severe tissue damage (1,2).
The productron of toxic oxygen radicals is associated with various forms of
inJury and/or trauma to an organisms system. Table 1 highlights some of these
injuries and their potential consequences. Few possibilities exist to serve as
sources for oxygen derived free radicals (2,3). The pathways illustrated in
Fig. 1, depict the precise reactions that are necessary.
The reactive oxygen metabolites that form within an organism must contend
with the system’s natural defense measures. For tracmg this quite complicated
mechanism, we should examine an element of that system, such as oxygen
consumptton. Momtormg the oxygen consumption provides a good indication
of activity, and the progression of events in this chain reaction. Oxygen con-
sumption is the disappearance of dissolved oxygen from the system, and can
be determined by the followmg three methods:
Oxygen Consumption Methods 5
1 Electrochemical measurements of dissolved oxygen by the Clark electrode.
2. Spectrophotometric detection of oxygen consumption in the presence of mchca-
tor compounds like cytochrome C.
3. Spectrophotometric detectlon of oxygen consumption by the determmation of
xanthine-oxidase activity
Various enzymatic processes, that utilize oxygen may be measured by
applying the methods that monitor oxygen consumption:
1. Enzymes that produce oxygenated compounds, including toxic ones (e.g.,
hpidperoxldase and cyclooxyganase).
2. Oxygen-derived free radical-producing enzyme xanthine oxidase (X0)
3. The free-radical scavenger enzymes, i.e., superoxide-chsmutases (SODS), cata-
lases, and glutathione peroxidases.
4. The mitochondrlal respiration enzymes, such as the cytochrome oxidases
2. Materials
2.1. Electrochemical Measurements
2.1. I. Oxygen (PO,) Electrode (Clark Electrode)
1. The application of Clark electrode is a good example of amperometry. The mea-
suring system contains a thermostable cuvet of approx 2-3 mL, m which one
wall is replaced by an oxygen-permeable polypropylene or teflon membrane. The
membrane separates the sample from the supportmg electrolyte (see Note 1).
Behmd this membrane, a platinum microelectrode serves as a cathode and a
alver-silver chloride electrode serves as an anode. The supportmg electrolyte 1s
0.1 M phosphate buffer, pH 7.5, saturated with silver chloride. The electrode pair
is charged with +0.65 V. The amount of oxygen entering from the sample through
the membrane to the electrolyte wrll be proportional to the oxygen concentratron
of the original sample. The entered oxygen ~11 be reduced in the following man-
ner: O2 + HZ0 + 4 e- > 4 OH- The current flow that results from this reaction IS
directly proporhonal to the partial pressure of oxygen in the sample. The sensi-
tivity of this reaction 1s: AI = pOZ = 10e4 A/Pa
2 Before using the electrode, the system 1s calibrated with a known concentration
of dissolved oxygen m an applied buffer system. The buffer system 1s saturated
with a mixture of gases containing known oxygen concentrations (for example:
15% oxygen, 5% carbon dioxide, and 80% mtrogen). Applying Dalton’s law, the
partial pressure m the system 1s
p(amb) = PO;! + pCOZ + pN2 + pHzO
pHz0 at 37°C = 47 mm Hg
6 Stadler
3. Taking a barometrtc reading for actual atmospheric pressure, the partial pressure
of oxygen is calculated. Once the partial pressure for oxygen is known, Henry’s
Law may be applied:
c d0 = a x p0 where c = mol/L of dissolved O2 (do)
a = Henry constant
4 Once the concentration of dissolved gas (oxygen) is calculated, the mstrument 1s set
Zero oxygen concentration is produced by adding sodium thiosulfate (a few crys-
tals) to the cuvets, and the zero lure is adjusted on the output device Oxygen con-
sumption (@4) is calculated based on a signal change dunng the applied reactton
5 Output device: The Clark electrode should be attached to an output device, such
as a potentiometrtc recorder or PC via interface.
2.2. Specfrophofomefric Methods
2.2.1. Indirect Methods
1. An ultraviolet-visible spectrophotometer, with ttme-course registration (kinetic)
capacity (The SHIMADZU UV-VIS Spectrophotometer-1601 wtth cuvets IS
suitable ) (See Note 2.)
2 50 mM Carbonate buffer with 0.1 mM ethylenediamme tetraacetic acid (EDTA),
pH 10.275 mL of 0.1 M Na,COs and 225 mL of 0.1 M NaHCOs, 23.4 mg EDTA,
add 400 mL type I water, adJust pH to 10.0 with 2.0 N HCl or NaOH, adJust the
volume to 1000 mL with type I water
3. Xanthme (2,6 dihydroxypurme) stock solution: Dissolve 15.7 mg xanthme in
30 mL type I water, add 2 drops 1 A4 NaOH if xanthine shows difficulty going
into solution (mix 10 mm).
4. X0 solution (grade III from buttermtlk, Sigma, St. Louis, MO, cat. no. X-4500):
0.12-0.16 U/mL X0 m 50 mM carbonate buffer (approx lo-20 U/mL of enzyme
stock solution needed for 50 mL)
5. Cytochrome C solution (from horse heart; Sigma, cat. no. C-2506): 10.5 pA4 m
50 mM carbonate buffer, 3000 U/mg standard SOD-lyophilized powder from
bovine erythrocytes (Sigma, cat. no S-25 15).
6. Working solution: 28 mL of 50 mM carbonate buffer; 1 mL of xanthme solution,
1 mL of cytochrome C solution. Absorbance of this working solution at 550 nm
should be approx 0.200.
2.2.2. Direct Methods
1. Preparation of the substrate (see Note 3)
a. Dissolve 20 Mg polyunsaturated fatty acid (Lmolenic acid, 9,12,15-
octadecatrtenoic acid Sigma, cat. no. L-2376) m 0.5 mL 96% ethanol.
b. Convert the polyunsaturated fatty acid to sodmm-lmolenate, by adding an
equivalent amount of sodium hydroxide (1 M>.
Oxygen Consumption Methods 7
c. Adjust the volume to 20 mL yielding a solution of 1.0 mg/mL sodmm
lmolenate in 0.2 M borate buffer, pH 9.0.
2. Borate buffer preparation: Add 6.18 g bortc acid to 300 mL deionized H20,
adjust pH to 9 0 using 50% NaOH, and adjust volume to 500 mL using deion-
ized H,O.
2.2.3. Direct Method Using X0
1. Xanthme solutton: Add 17.4 mg of xanthme (Sigma, cat. no X 2502) to
10 mL of type I H20 Boll 5 min to dissolve components completely.
2. X0 solution: Use 250 U/mL of X0 (Sigma, cat. no. X 4500) in 0.1 A4 phosphate
buffer, pH 7.4. This X0 solution will serve as the standard.
3. Methods
3.1. Electrochemical Measurements of Dissolved Oxygen Using
the Clark Electrode
3.1.7. Oxygenase activity with C/ark Electrode
1. Add 1.9 mL of 10 mg/mL of lipoxygenase enzyme solution (soybean
hpoxygenase Sigma or potato hpoxygenase [4]), m 1 M borate buffer, pH 9 0.
Transfer to a measuring cuvet. One wall of the cuvet consists of a Clark
electrode-specific membrane. Thereafter, cuvet is hermetically sealed and equili-
brated for 10 mm at 37°C.
2. 0.100 mL of 1.0 mg/mL sodrum arachtdonate (see Note 4). Inject into
the cuvet through the rubber stopcock; start output device (recorder) at
same time. Record oxygen consumption for 15 mm The typical curve 1s depicted
m Fig. 2
3.1.2. Potato Lipoxygenase Activity
1. Potato hpoxygenase (a model for 5-ltpoxygenase)* Prepared accordmg to
O’Flaherty (4).
2. Enzyme activity is measured with same instrument as previously described, by
applying 0.1 A4 phosphate buffer at pH 6.5. Enzyme concentration is 5.0 mg/mL
based on a protein assay.
3.1.3. Cyclooxygenase Activity
Partially purified cyclooxygenase (from sheep vesicula seminahs) (4), is
prepared for this measurement. 1.9 mL of 10 mg/mL protein extract is applied
as described in Subheading 3.1.1.