[5l D E O X Y R I B O S E ASSAY F O R H Y D R O X Y L R A D I C A L S 57

[5] D e o x y r i b o s e A s s a y for D e t e c t i n g H y d r o x y l R a d i c a l s
By OKEZIE I. ARUOMA

Introduction

The mechanisms by which biological systems generate reactive oxygen

species (ROS), a general term often used to refer to the radicals superoxide
(O27), hydroxyl (.OH), and peroxyl (ROO.) and the nonradicals hydrogen
peroxide (H202), hypochlorous acid (HOC1), singlet oxygen (102), and
ozone (O3), and their potential toxicity have been discussed extensively
in the literature and in this volume. In any branch of science, it is important
for unanimity in methodology to prevail. Figure I attempts to summarize
the various methods currently in use in studying free radical reactions in
biological systems, l This chapter discusses the deoxyribose assay, which
was first introduced in 1981,2,3and the practical application of the method. 4
The highly reactive .OH radical is generated in living systems by
exposing the system to ionizing radiation5 and by having reduced forms
of several transition metal ions come into contact with H20 2. Under certain
circumstances, the reaction between NO. and 027 might also produce
•O H . 6 The iron salt-dependent decomposition of H202 is often called the
Fenton reaction. 7 Indeed, iron promoters of the Fenton reaction seem to
be available within cells. Fenton reactions can be accelerated by the
addition of reducing agents, thus causing more damage to the biological
molecule. For example, the superoxide radical, ascorbate, and paraquat
radical can accelerate the iron-catalyzed Haber-Weiss reaction by gener-
ating the reduced form of the iron complex, for example,
Fe3÷-chelate + 027 --" Fe2÷-chelate + 02
Fe3÷-chelate + ascorbate ~ Fe2+-chelate + ascorbate radical
which will decompose hydrogen peroxide to generate the damaging
species.

1 0 . I. Aruoma, in " F r e e Radicals in Tropical Diseases" (O. I. Aruoma, ed.), Chap. 11.
Harwood Academic Publ., London, 1993.
z j. M. C. Gutteridge, FEBS Lett. 128, 434 (1981).
3 B. Halliwell and J. M. C. Gutteridge, FEBS Lett. 12,1t, 347 (1981).
4 B. Halliwell, M. Grootveld, and J. M. C. Gutteridge, Methods Biochem. Anal. 33, 59 (1987).
5 G. Scholes, Br. J. Radiol. 56, 221 (1983).
6 j. S. Beckman, T. W. Beckman, J. Chen, P. A. Marshall, and B. A. Freeman, Proc.
Natl. Acad. Sci. U.S.A. 87, 1620 (1990).
7 C. Wailing, Acc. Chem. Res. 8, 125 (1975).

Copyright © 1994by Academic Press, Inc.

METHODS IN ENZYMOLOGY, VOL. 233 All rights of reproduction in any form reserved.
58 GENERATION,
DETECTION,AND CHARACTERIZATION [5]

, Deoxyribose assay
1
Bleomycin assay
I Measurement of
damage to protein

Lipid
peroxidation

Elastase assay I
Assays for [
hydrogen peroxide

Spectrophotometric
assay for HOCI
Free radical reaction
In biological
systems

Aromatic hydroxylation Assays for superoxide

Pulse radiolysis

Electron spin resonance

Measurement of
DNA damage
Assays for singlet
oxygen

FIG. 1, Methods in free radical research. (From AruomaJ)

Deoxyribose Assay
The hydroxyl radical (see Scheme I) formed in the reaction between
iron(III)-EDTA and H2Oz in the presence of ascorbic acid attacks the
sugar deoxyribose to form products that on heating with thiobarbituric
[5] DEOXYRIBOSE ASSAY FOR HYDROXYL RADICALS 59

(Deoxyribose) .~

(Other biomolecules)

I
(Proteins)
(Scavengers)

(DNA) <

Fe3+ - EDTA + H202 + Ascorbate Ionizing Radiation

SCHEME I

acid (TBA), at low pH, yield a pink chromogen. The chemistry of the
reaction is complex. Hydroxyl radicals are involved in the reaction. For
example, studies using gas chromatography-mass spectrometry with se-
lected ion monitoring (GC/MS/SIM) on DNA show that the pattern of
the base damage by ionizing radiation, where the base modification has
been shown to be unequivocally due to .OH generated in free solution is
similar to that produced by the Fe3+-EDTA/H2OJascorbate system. 8
The hydroxyl radical that escapes scavenging by the EDTA will become
available to attack the deoxyribose molecule, and any compound present
in the reaction mixture will compete for the hydroxyl radical.
The rate at which hydroxyl radical reacts with the target depends on
the rate constants for that reaction (Scheme I) and on the concentrations
of molecules presented as targets. In proteins and in DNA, the constituent
amino acids and bases will have different rate constants for reaction with
the hydroxyl radical.
In performing the assay, the reaction mixture may contain, in a final
volume of 1.2 ml, the following reagents at the final concentrations stated:
s O. I. Aruoma, B. Halliwell, E. Gajewski, and M. Dizdaroglu, J. Biol. Chem. 264,
20509 (1989).
60 GENERATION
DETECTION,
, AND CHARACTERIZATION [5]

deoxyribose (2.8 mM), FeCI3 (25 mM), EDTA (100 /~M) [EDTA and
iron(III) ions are premixed at the ratio given prior to the addition of
deoxyribose], H202 (2.8 mM), KH2PO4/KOH buffer at pH 7.4 (10 mM),
compounds to be tested at a concentration up to 20 mM depending on
solubility, and ascorbate (100/xM). The ascorbate is added to start the
reaction. Unfortunately, compounds not soluble in aqueous media cannot
be used in the deoxyribose assay. Some compounds are dissolved in alkali
solution and the pH then readjusted to 7.4, but high concentrations of
sodium carbonate (final concentrations greater than 20 mM) interfere with
the assay probably because the carbonate ions scavenge .OH.
After incubation at 37 ° for 1 hr, 1 ml of 1% (w/v) thiobarbituric acid
(TBA) in 50 m M NaOH and 1 ml of 2.8% (w/v) trichloroacetic acid (TCA)
are added to the reaction mixture, which is then heated to develop color
due to the malondialdehyde (MDA)-likeproduct of deoxyribose damage.
This can be conveniently done in the plastic test tubes used for the assay.
The tubes are placed in a hot water bath maintained at 80° for up to 20
min. At the end of this period, remove the tubes and allow the contents
to cool. If turbidity is experienced, the resulting pink color may be
extracted into l-butanol. This is done by adding an equal volume of
1-butanol, mixing, and separating the layers of centrifugation. Measure-
ment of the absorbance at 532 nm, either in the heated solution or, when
1-butanol extraction is used, in the upper (organic) layer, gives a measure
of the deoxyribose damage.
The compound to be tested might also interfere with the color
development stage of the assay. This can be checked by adding a given
concentration of the compound to the TBA/TCA reagent containing
tetraethoxypropane (TEP). A concentration of the compound to be
tested that does not interfere with this important stage of the assay
can be evaluated. One additional problem is iron contamination in the
reagents used for the assay (this may not be a problem if the assay
is just used to measure .OH scavenging, where excess iron ions are
added). It has been calculated that approximately 1 /zM iron ions are
present in the reaction mixture without the "added" iron ions. Careful
use of apoproteins in the assay will minimize the high background caused
by the contaminating iron ions. For example, use of approximately 0.5
/.tM apotransferrin will minimize the effect of the contaminating iron
ions. Iron correctly bound onto transferrin does not participate in
formation of hydroxyl radicals. Alternatively, contaminating iron ions
can be removed from the reagents by careful use of Chelex resin
(sodium form, mesh 200 Bio-Rad, Richmond, CA) and by use of sacks
of conalbumin placed in the reagents. 4 Iron contamination is very
[5] DEOXYRIBOSE ASSAY FOR HYDROXYL RADICALS 61

critical, for example, in the bleomycin assay ~ (discussed in [7] in

this volume).
Calculation of Rate Constant of Reaction with Hydroxyl Radical in
Deoxyribose Assay
The deoxyribose assay can be used to calculate the rate constant for
the reaction of a scavenger molecule with .OH. The absolute rate constants
are usually best determined by pulse radiolysis9 (readers may wish to refer
to Chapters 1-3 in this volume.) How is the rate constant of a reaction
calculated? If it is assumed that the initial attack of .OH on deoxyribose
(DR) is the rate-determining step in the formation of a product(s) that
leads to MDA [Eqs. (1) and (2)], then analysis of the results in terms
Fe3+-EDTA + ascorbate ---> Fe2÷-EDTA + oxidized ascorbate (1)
Fe2÷-EDTA + H20:---> Fe3÷-EDTA + .OH + OH- (2)
of a simple competition between scavenger and deoxyribose (detector
molecule should allow calculation of rate constant for reaction of the
scavenger with .OH [Eqs. (3) and (4)]. Let us also assume that the rate
•OH + DR ---> fragment ---> MDA (3)
2 TBA + MDA ---> chromogen (4)
of attack of "OH on deoxyribose is consistent with time and that the
absorbance obtained at the end of the experiment is a measure of the rate
of attack of .OH on deoxyribose. Then, in a reaction mixture containing
deoxyribose (DR) and another molecule (S) capable of reacting with .OH,
Rate of reaction of DR with .OH = kDR['OH][DR] (5)
Rate of reaction of S with .OH = ks[-OH][S] (6)
where kDRand ks are the respective second-order rate constants for deoxy-
ribose and the scavenger molecule reacting with hydroxyl radical.
The absorbance A obtained at the end of the experiment, taken as a
measure of the rate of reaction, is also given by Eq. (5). The absorbance
A ° in the absence of IS] is given by
A ° = kDR['OH][DR] + ks['OH][S] (7)
From Eq. (5), [.OH] = A/kDrc[DR]. Substituting into Eq. (7), we have
A ° - AkDR[DR] + Aks[S] (8)
kDR[DR] kDR[DR]

Aks[S]
= A + - -
kDR[DR]
9 M. Anbar and P. Neta, Int. J. Appl. Radiat. Isot. 18, 493 (1967).
62 GENERATION, DETECTION, AND CHARACTERIZATION [5]

14

0" 6 1'2
Concentration (mM)
FIG. 2. Scavenging of hydroxyl radicals in the deoxyribose assay, illustrated with hypotau-
fine, which has been proposed as an antioxidant. The rate constant (k s) for reaction with
•OH (in the presence of EDTA) was calculated from the equation ks = slope x kDR[DR]A°,
where kDa is 3.1 x 109 M -1 sec -t. A ° is the absorbance in the absence of hypotaurine or
any added scavenger. [DR] is the concentration of deoxyribose in the reaction mixture. The
calculated rate constant for hypotaurine is 5.0 x 109 M -t sec -~. D, EDTA absent; II, EDTA
present. (From Aruoma et alJ °)

ff we divide Eq. (8) by A and then by A °, we have an expression for 1/A

[Eq. (9)]. Thus a plot of the reciprocal of As32,m, obtained in the presence
1 1 ks[S]
= ~ + kDR[DR]A0 (9)

of EDTA, against the scavenger concentration will be linear (Fig. 2) l°'H

and would have a slope equal to ks/kDR[DR]A °.
ks (the rate constant of reaction of the test compound with .OH) can
be calculated given kDR. The value for kDR obtained by pulse radiolysis is
3.1 x 109 M -1 sec -~. Thus, in situations where a pulse radiolysis facility
is not readily available, it is still possible to determine rate constants of
reactions between hydroxyl radicals and the test compound in aqueous
solutions. Table I shows that most rate constants for reactions of .OH

l00. I. Aruoma, B. Halliwell, B. M. Hoey, and J. Butler, Biochem. J. 256, 251 (1988).
ll B. Halliwell, J. M. C. Gutteridge, and O. I. Aruoma, Anal. Biochem. 165, 215 (1987).
[5] DEOXYRIBOSE ASSAY FOR HYDROXYL RADICALS 63
TABLE I
SECOND-ORDER RATE CONSTANTS FOR REACTIONS OF REAGENTS WITH HYDROXYL
RADICALS USING DEOXYRIBOSE ASSAY AND PULSE RADIOLYSIS a

Rate c o n s t a n t (M -~ sec -l)

C o m p o u n d tested Deoxyribose assay Pulse radiolysis

Mannitol (1.0-2.0) x 109 (1.0-1.8) × 109

Histidine (2.3-3.0) × 109 3.0 × 109
l-Butanol (2.0-3.2) x 109 2.2 x 109
Ethanol (1.0-1.5) x 109 (0.7-1.1) x 109
2-Propanol (1.0-1.5) x 109 (1.1-1.7) x 109
Dimethyl sulfoxide (0.70-1.0) x 101° 7.0 x 109
Urea N o scavenging detected <7.0 x 105
Allopurinol (0.84-2.86) × 109 (1.45 ± 0.24) x 109
Oxypurinol (3.0-7.1) x 109 (4.95 +- 0.84) x 109
HEPES (1.7-2.0) x 109 5.1 x 109
Citrate (1.0-1.3) × 108 3.0 × 107
O-Acetylsalicylic acid (aspirin) (6.0-9.6) x 109 (5.0-10.0) x 109
AMP (1.6-2.0) × 109 (1.8-3.3) × 109
Tricine (1.0-1.1) x 109 1.6 x 109
MOPS (2.0-2.6) x 109 --
MES (2.0-3.0) x 109 --
ATP (2.5-3.0) x 109 --
ADP (2.5-2.8) x 109 --
Carnosine 4.0 × 109 --
Homocarnosine 2.6 × 109 --
Anserine 5.2 x 109 --
L-Alanine 8.1 × 107 --
L-Histidine 7.1 × 109 --
Imidazole 4.8 x 109 --
Histamine 5.0 x 109 --
Mannitol 2.7 × 109 --
Glucose 1.0 x 109 --
Hypotaurine 5.00 x 109 1.15 x 10 I°
Cysteamine N.D. 5.90 × 109
Taufine 1.40 × 107 2.42 × 106
DL- or L-Cysteic acid 1.60 x 108 5.30 × 107
Cysteinesulfinic acid 3.20 × 109 N.D.

o D a t a were selected f r o m e x p e r i m e n t s performed at p H values equal or close to 7.4.

T h e range o f values given in t h e d e o x y r i b o s e a s s a y for e a c h c o m p o u n d r e p r e s e n t s the
results obtained in at least three experiments. W h e r e n e c e s s a r y (e.g., with A T P and
H E P E S ) , s u b s t a n c e s were adjusted to p H 7.4 before addition to the reaction mixtures.
C y s t e a m i n e w a s n o t studied in the d e o x y r i b o s e s y s t e m b e c a u s e its ability to react with
H202 interferes with the a s s a y . N . D . , n o t determined. Data compiled from the published
w o r k s of the author and from Ref. 9.
64 GENERATION, DETECTION, AND CHARACTERIZATION [5]

obtained using a wide range of different molecules by the test-tube method

are similar to those produced by the pulse radiolysis technique.

Ability to Chelate Iron

Another feature of the deoxyribose method is that it is possible to
obtain information on the likelihood that the compounds to be tested
could chelate iron in a way that prevents that iron from catalyzing .OH
formation. Iron ions bound to the deoxyribose molecule will stimulate
damage in the presence of ascorbate and H2Ov Compounds able to chelate
the catalytic iron ions will prevent t h e " site-specific" reaction only if they
inhibit the reaction of Fe 3+ with ascorbate or H202 ~2-~4(EDTA chelates
iron but does not block deoxyribose degradation). Performing the deoxyri-
bose assay as described in the absence of EDTA would produce the typical
profile (Fig. 2) for compounds that are able to chelate iron and perhaps
direct the damage to themselves, thereby protecting the target (deoxyri-
bose). The effects of EDTA on Fe(II)-dependent deoxyribose degradation
are complicated. EDTA concentrations lower than Fe(II) ion concentra-
tions slightly stimulate Fe(II)-dependent deoxyribose degradation,
whereas EDTA concentrations higher than Fe(II) concentrations are
slightly inhibiting. 4 This is why the assay described makes use of Fe(III)
ions and a reducing agent.
If excess EDTA is present in the reaction mixture, the iron ions will
be bound to it and not to the deoxyribose. There are therefore two scenar-
ios (Fig. 2): (1) deoxyribos¢ degradation produced by site-specific .OH
generation and the effects of scavengers (iron bound to the targe03) and
(2) deoxyribose degradation produced by attack of .OH free in solution
and the effects of scavengers (iron bound to EDTA, see Scheme I). Thus,
the ability of a scavenger molecule to inhibit deoxyribose damage in a
site-specific manner depends additionally on its ability to chelate iron and
hence direct the damage to itself. 4'~2-z5 If, having performed the deoxyri-
bose assay, a scavenger molecule produces a typical curve (Fig. 2), it is
recommended that a complementary assay involving the iron chelating
agent desferrioxamine be performed to gain further evidence regarding
the ability of a compound to chelate iron. Figure 3 shows a typical example.
The experiment can be conveniently conducted using a spectrophotom-
eter equipped with a repeat-scan facility. Figure 3a shows evidence consis-
12j. M. C. Gutteridge, Biochem. J. 2/,4, 761 (1984).
13 O. I. Aruoma, M. Grootveld, and B. Halliwell, J. lnorg. Biochem. 29, 289 (1987).
z40. I. Aruoma and B. Halliwell, Xenobiotica 18, 459 (1988).
15 C. P. Moorhouse, B. Halliwell, M. Grootveld, and J. M. C. Gutteridge, Biochim. Biophys.
Acta 843, 261 (1985).
[5] DEOXYRIBOSE ASSAY FOR HYDROXYL RADICALS 65

1.0

0.8

8
-e 0.6
0

0,4

0.2

20O 300 400 500 600 700

;k/nm
0.6
b

.3
,dr
<

16o 26o
time (min)
F1G. 3. (a) Spectral changes observed on mixing Fe(III), desferrioxamine (DFX), and
deoxyribose. Reaction mixtures were as described in (b). The reaction was started by adding
DFX (final concentration 250/~M). The first spectrum was run after 5 rain, the second after
13 rain, and subsequent spectra at 8-rain intervals. The scan speed was 120 nm/min. (b)
Rate of formation of an Fe(III)-desferrioxamine complex (ferrioxamine) in the presence of
deoxyribose. Reaction mixtures contained, in a final volume of 1.00 ml, 50 mM HEPES
buffer, pH 6.0, 0.2 M NaC1 (to maintain ionic strength), 250/~M FeCI3, and, where indicated,
18.2 mM deoxyribose. The reaction was started by adding desferrioxamine at a final concen-
tration of 250 t~M, and formation of ferrioxamine was followed at 420 rim. Fe(III) and
deoxyribose solutions were equilibrated at laboratory temperature for 30 rain. 0 , No deoxyri-
bose; &, deoxyribose present. (From Aruoma et al. ~3)
66 GENERATION, DETECTION, AND CHARACTERIZATION [5]

tent with the formation of an Fe(III)-deoxyribose complex. 13 Binding of

Fe(III) ions to desferrioxamine gives an orange-red 1 : 1 complex (ferriox-
amine, hm~ 425 nm). Premixing the Fe(III) with excess deoxyribose mark-
edly retards the rate of color formation (Fig. 3b), suggesting that the
deoxyribose is restricting the availability of Fe(III) to the desferrioxamine.
Compounds that produce curves of the type shown by circles in Fig. 2
will have a similar curve as that for the deoxyribose in Fig. 3b. Hence,
this is a very useful experimental tool.

Conclusion
Numerous comments have been expressed in the literature concerning
the deoxyribose method. 4,m3,tS-18 It has been suggested that iron ions
might play a role in the deoxyribose assay via formation of other species
such as the ferryl or perferryl radical, whose properties are different from
that of .OH. However, no real evidence has been found that ferryl or
the perferryl species exist in Fenton systems at physiological pH. 19 The
mechanism by which the oxidation of deoxyribose occurs in the present
discussion remains valid if the assay conditions as described are applied.
Use of Fe(III)-EDTA in the presence of a reducing agent such as ascorbate
or the superoxide radical from the xanthine-hypoxanthine system will
mimic the conditions of ionizing radiation as described in Scheme I. The
deoxyribose assay has been adapted to assess the ability of food additives
and nutrient components to act as prooxidants and hence mediate damage
to the food matrix. 1,20-22
The deoxyribose method remains an easy-to-use laboratory tool. When
performed with an understanding that the inherent mechanisms involved
are complex, the assay allows for (1) measuring rate constants for reactions
with .OH and (2) assessing the iron-binding ability of the compound be-
ing tested.

Acknowledgments
I thank the U.K. Medical Research Council (MRC) and the U.K. Ministry of Agriculture,
Fisheries and Food (MAFF), and Nestec SA, Switzerland for research support.

16 C. C. Winterbourne and H. C. Sutton, Arch. Biochem. Biophys. 244, 272 (1986).

IT C. C. Winterbourne, Free Radical Biol. Med. 11, 353 (1991).
18 B. Tadolini and L. Cabrini, Biochem. J. 253, 931 (1988).
19 B. Halliwell and J. M. C. Gutteridge, FEBS Lett. 307, 108 (1992).
2~ O. I. Aruoma, B. Halliwell, R. Aeschbach, and J. Loliger, Xenobiotica 22, 257 (1992).
2J M. J. Laughton, B. Halliwell, P. J. Evans, and J. R. S. Hoult, Biochem. Pharmacol. 38,
2859 (1989).
22 O. I. Aruoma and B. Halliwell, "Free Radicals and Food Additives." Taylor and Francis,
London, 1991.