The deoxyribose method: A simple “test-tube” assay for determination of rate constants for reactions of hydroxyl radicals

ANALYTICAL

BIOCHEMISTRY

1652

15-2 19 ( 1987)

The Deoxyribose Method: A Simple “Test-Tube” Assay for Determination of Rate Constants for Reactions of Hydroxyl Radicals BARRY

HALLIWELL,*

JOHN M. C. GUTTERIDGE,~

AND OREZIE

I. ARUOMA*

*Department of Biochemistry, University of London Kings College, Strand Campus, London WC2R 2LS, and tNationa1 Institute for Biological Standards and Control, Holly Hill, Hampstead, London NW3 6RB, United Kingdom Received February 18, I987 Hydroxyl radicals, generated by reaction of an iron-EDTA complex with HzOz in the presence of ascorbic acid, attack deoxyribose to form products that, upon heating with thiobarbituric acid at low pH, yield a pink chromogen. Added hydroxyl radical “scavengers” compete with deoxyribose for the hydroxyl radicals produced and diminish chromogen formation. A rate constant for reaction of the scavenger with hydroxyl radical can be deduced from the inhibition of color formation. For a wide range of compounds, rate constants obtained in this way are similar to those determined by pulse radiolysis. It is suggested that the deoxyribose assay is a simple and cheap alternative to pulse radiolysis for determination of rate constants for reaction of most biological molecules with hydroxyl radicals. Rate constants for reactions of ATP, ADP, and Good’s buffers with hydroxyl radicals have been determined by this method. o 1987 Academic

Press, Inc.

The role of oxygen-derived species, such as superoxide (0;) and H202, in producing tissue damage in various human diseases is becoming increasingly recognized ( 1). There is considerable interest in the possibility that 0: and H202 mediate some of their toxicity by becoming converted into the highly reactive hydroxyl radical, ‘OH, in reactions that need metal ions (2,3). Various “hydroxyl radical scavengers” are often used to study the role of ‘OH in biological systems (3,8). It has also been suggested that a number of agents of therapeutic use, such as allopurinol (4,5), anti-inflammatory drugs (6), and amygdalin (7), might exert some of their beneficial effects by scavenging ‘OH radicals. In assessing such claims, or in using scavengers, it is important to know the secondorder rate constant for reaction of the molecule in question with ‘OH (6,8). Such rate constants are best determined by the technique of pulse radiolysis (9), and extensive tables of rate constants have been published (9-l 1). Pulse radiolysis is also useful for in215

vestigations of the mechanism of reactions involving ‘OH (9,12). However, pulse radiolysis facilities are expensive to set up and operate and are not available to many scientists interested in free radical reactions. There is need for a simple “test-tube” assay that can be used to determine approximate second-order rate constants for reactions of ‘OH with molecules of potential therapeutic use or newly introduced scavengers of ‘OH. The sugar deoxyribose (2-deoxy-D-ribose) is degraded on exposure to hydroxyl radicals generated by irradiation (13) or by Fenton systems (14). If the resulting complex mixture of products is heated under acid conditions, malonaldehyde (MDA)’ is formed (14) and may be detected by its ability to react with thiobarbituric acid (TBA) to form a ’ Abbreviations used: Hepes, 4-(2-hydroxyethyl-1 -piperazineethanesulfonic acid; Tricine, N-[Tris(hydroxymethyl)methyl]glycine; Mes, 4-morpholineethanesulfonic acid; Mops, 4-morpholinepropanesulfonic acid; MDA, malonaldehyde; TBA, thiobarbituric acid. 0003-2697187

$3.00

CopyrigJn 0 1987 by Academic Press, Inc. AI1 rights of reproduction in any form reserved.

216

HALLIWELL,

GUTTERIDGE,

pink chromogen ( 14,19). Indeed, deoxyribose has often been used in this way to measure the formation of ‘OH radical in biochemical systems ( 14- 17,19). If an Fez+-EDTA chelate is incubated with deoxyribose in phosphate buffer at pH 7.4, ‘OH radicals are formed ( 14). Any such radicals that escape scavenging by the EDTA itself (18) can react with deoxyribose: Fe2+-EDTA

+ 0

2*

Fe3+-EDTA

+ 0;

20; + 2H+ + H202 + 02 Fe2+-EDTA

[ 1]

PI

+ H202 --* OH- + ‘OH + Fe3+-EDTA

[3]

‘OH + deoxyribose --* fragments=

MDA

[4]

acid

2TBA + MDA --, chromogen.

[51

The rate of deoxyribose degradation may be increased by including a reducing agent, such as ascorbic acid ( 16), in the reaction mixture: Fe3+-EDTA

+ ascorbate +

Fe2+-EDTA

+ oxidized ascorbate. [6]

Addition of extra H202 may also accelerate the rate (16,17). Any other molecule added to the reaction mixture that is capable of reacting with ‘OH should compete with deoxyribose for ‘OH to an extent depending on its rate constant for reaction with ‘OH and its concentration relative to deoxyribose; hence, it will decrease the rate of deoxyribose degradation. If it is assumed that the initial attack of ‘OH on deoxyribose is the rate-determining step in formation of the product(s) that leads to MDA, then analysis of the results in terms of a simple competition between scavenger and deoxyribose should allow calculation of the rate constant for reaction of the scavenger with ‘OH. In the present paper, deoxyribose has been used in this way to determine rate con-

AND

ARUOMA

stants for comparison with those obtained by pulse radiolysis methods. MATERIALS

AND

METHODS

Reagents. 2-Deoxy-D-ribose was from Sigma Chemical Co. (Poole, Dorset). All other chemicals used were of the highest purity available from BDH Chemicals Ltd. Degradation of deoxyribose by Fd+EDTA, ascorbic acid, and H202. Reaction mixtures contained, in a final volume of 1.O ml, the following reagents at the final concentrations stated: deoxyribose (variable concentration), KH2P04-KOH buffer, pH 7.4 (20 mM), FeC4 (100 PM), EDTA (104 PM), Hz02 (1 mM), and ascorbate (100 PM). Solutions of FeC13 and ascorbate were made up immediately before use in deaerated water. Reaction mixtures were incubated at 37°C for 1 h, and color developed as described above. The rate of deoxyribose degradation was constant over the l-h incubation period. RESULTS

Hydroxyl radicals are generated by a mixture of Fe3+, ascorbate, and H202 in the presence of a slight molar excess of EDTA over the Fe3+ salt (14,19). The ‘OH radicals attack the deoxyribose and set off a series of reactions that eventually result in formation of MDA, measured as a pink MDA-TBA chromogen. Since the amount of color formation (as A532) in reaction mixtures subsequently heated with TBA is proportional to the time of incubation before adding TBA, it may be assumed that the rate of attack of ‘OH on deoxyribose is also constant with time and that the absorbance obtained at the end of the experiment is a measure of the rate of ‘OH attack on deoxyribose (this assumption is examined under Discussion). If both deoxyribose (DR) and another molecule (S) capable of reacting with ‘OH are present in the reaction mixture, then Rate of reaction of DR with ‘OH =

HYDROXYL

RADICALS

AND DEOXYRIBOSE

217

DEGRADATION

Rate of reaction of S with ‘OH =

k[‘OHIM.

PI

The absorbance (A) obtained at the end of the experiment, taken as a measure of the rate of reaction, is given by A = k,,,[‘OH][DR]

191

The absorbance (A’), as a measure of the rate that would be obtained in the absence of S, is given by A0 = k,,[‘OH][DR]

+ ks[‘OH][S].

These equations may be combined 1 -=A

1 A0

[lo]

to give

[Ill

where A is the absorbance in the presence of a scavenger S at concentration [S] and A0 is the absorbance in the absence of a scavenger. Hence, a plot of l/A against [S] should give a straight line of slope ks/knrJDR~o with an intercept on the y-axis of l/A’, and the rate constant for reaction of S with ‘OH can be obtained from the slope of the line. It is obviously essential to have an accurate value of kDR. Extensive pulse radiolysis studies by Butler and Hoey at the Christie Hospital in Manchester (personal communication) have obtained a value of 3.1 X lo9 M-’ s-l using both the thiocyanate and iodide competition methods (9). Figure 1 shows an experiment in which the scavenger was ethanol; a linear plot was obtained and a rate constant of 1.47 X lo9 M-I s-’ was obtained from this experiment. Linear plots were also obtained in many similar experiments with other scavengers; Table 1 summarizes the results obtained. The rate constants obtained from the slopes of the lines were unaffected by the exact concentrations of deoxyribose (if 22.8 mM), Fe3+EDTA, ascorbic acid, phosphate buffer, and H202 used. Although different rates of ‘OH generation are obtained under these different experimental conditions ( 17,19), this does not alter the competition analysis (Eq. [ 111).

1

1 10 Concentratiin

20 mM

FIG. 1. Hydroxyl radical scavenging by ethanol: determination of a rate constant. Deoxyribose degradation in the presence of various concentrations of ethanol was followed as described under Materials and Methods using a final deoxyribose concentration of 2.8 mM in the reaction mixture. The rate constant was determined from the slope of the line (k = slope X kDR X [DR] X A’), as described in the text, giving a value of 1.47 X lo9 M-’ s-i in this experiment.

Table 1 shows that most rate constants for reactions of ‘OH obtained, using a wide range of different molecules, by this simple test-tube method are very similar to those produced by pulse radiolysis techniques, except that the value for citrate was found to be somewhat higher than claimed in (11). The results in Table 1 confirm the report (22) that the Good’s (23) buffers Hepes and Tricine are good scavengers of hydroxyl radicals, and they show that the same is true of Mes and Mops. Table 1 also shows values for the rate constants for reactions between hydroxyl radical and ATP or ADP as determined by the deoxyribose method. DISCUSSION

Pulse radiolysis will obviously remain the method of choice for accurate reference determination of rate constants for reactions of hydroxyl radicals. However, the deoxyribose assay ( 14) used in this paper is simple and cheap and seems to give an equally accurate result in almost all of the cases examined. We recognize that the chemistry by which the initial products of-OH attack on deoxyri-

218

HALLIWELL,

GUTTERIDGE, TABLE

AND ARUOMA 1

SECONDORDERRATECONSTANTSFORREACTIONSOFREAGENTSWI~HYDROXYLRADICALS:ACOMPARISON OFRESULTSOBTAINEDBYTHEDEOXYRIBOSEASSAYANDBYPULSERADIOLYSIS Rate constant Compound tested Mannitol Histidine Butan- l-01 Ethanol Propan-2-01 Dimethyl sulfoxide Urea Allopurinol Oxypurinol Hepes Citrate O-Acetylsalicylic acid (aspirin) AMP Tricine Mops Mes ATP ADP

Deoxyribose assay

Pulse radiolysis

(1.0-2.0) x lo9 (2.3-3.0) x lo9 (2.0-3.2) x lo9 (1.0-1.5) x 109 (1.0-1.5) x log (0.70-1.0) x 1O’O No scavenging detected (0.84 -2.86) x 10’ (3.0-7.1) x IO9 (1.7-2.0) X lo9 (1.0-1.3) x 108 (6.0-9.6) X lo9 (1.6-2.0) x lo9 (1.0-1.1) x 109 (2.0-2.6) X 10’ (2.0-3.0) x lo9 (2.5-3.0) X 10’ (2.5-2.8) x lo9

(1.0-1.8) x lo9 (20) 3.0 x 109(11) 2.2 x 109(11) (0.7-1.1) x 109(11) (1.1-1.7) x 109(11) 7.0 x 109 (21) 17.0 x 105(11) (1.45 k 0.24) x lo9 (5) (4.95 k 0.84) x lo9 (5) 5.1 x lo9 (22) 3.0x lO’(l1) (5.0-10.0) X 10’ (6) (1.8-3.3) x 109(11) 1.6 x lo9 (22) -

Note. References to pulse radiolysis experiments are given in parentheses. To the extent possible, the data were selected from experiments performed at pH values equal or close to 7.4. The range of values given in the deoxyribose assay for each compound represents the results obtained in at least three experiments. Where necessary (e.g., with ATP and Hepes), substances were adjusted to pH 7.4 before addition to the reaction mixtures.

bose are converted into the final TBA-reactive material is very complex (13,14) and that the iron-EDTA complex may be involved not only in the generation of ‘OH but also in later stages of the reaction (16,19). However, the fact that linear competition plots are obtained with such a wide range of compounds (Table 1) suggests that the assumptions made in the kinetic analysis are valid, i.e., that the initial attack of ‘OH on deoxyribose is the rate-determining step for the whole reaction under our experimental conditions. The use of EDTA in our reaction systems is essential. Inhibition of iron-dependent deoxyribose degradation in the absence of EDTA depends not only on the ability of a scavenger to react with ‘OH, but also on its ability to form complexes with iron ions (19,25,16).

We hope that the deoxyribose assay, used in this way, will provide a simple screening method for ‘OH radical scavengers. It should be noted that the assay is inapplicable to certain compounds. For example, strong metalchelating agents that can withdraw iron from EDTA could not be tested in this assay. Perhaps the slightly anomalous result with citrate is related to its metal-binding ability. A reagent that reacts with Hz02 might diminish the rate of ‘OH generation (Eq. [3]), but this will probably be revealed in a nonlinear competition plot, as in the case of thiourea (24). Reaction of ‘OH radicals with molecules such as ethanol generates secondary radicals that might themselves reduce or oxidize iron. However, inclusion of ascorbic acid in the reaction mixture seems to overcome this problem. For example, rate con-

HYDROXYL

RADICALS

AND

stants for reaction of ethanol and propan-201 with ‘OH, determined by the method in the present paper, are within the expected range (Table 1). However, if iron is added to the reaction mixture as Fe*+ in the absence of ascorbate (19), the inhibitory effects of ethanol and propan-2-01 are much less than expected (data not shown), perhaps because the alcohol-derived radicals can reduce Fe3+ and increase the basal rate of ‘OH generation.

DEOXYRIBOSE

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ACKNOWLEDGMENTS We are very grateful to Drs. F rIoey and J. Butler for their pulse radiolysis determination of the deoxyribose rate constant and to the Arthritis and Rheumatism Council for financial support. B.H. is a Lister Institute Research Fellow.

DEGRADATION

343-346.

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