[53] Assays for the chlorination activity of myeloperoxidase

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[53] A s s a y s f o r t h e C h l o r i n a t i o n A c t i v i t y o f M y e l o p e r o x i d a s e By ANTHON¢ J. KETTLE and CHRISTINE C. WINTERBOURN Introduction Myeloperoxidase (donor: hydrogen-peroxide ' oxidoreductase, EC 1. I 1.1.7) is the most abundant protein in neutrophils and is also found in monocytes. ~,2 It contains two heme prosthetic groups and is a unique peroxidase that catalyzes the conversion of hydrogen peroxide and chloride to hypochlorous acid. Hydrogen peroxide is formed from the spontaneous dismutation of superoxide, which is produced by an NADPH oxidase in the cell membrane. Hypochlorous acid is the major strong oxidant produced by neutrophils. It has powerful antimicrobial activity, and it is extremely reactive with biological molecules. It inactivates enzymes and oq-antitrypsin, cross-links proteins, and reacts with unsaturated fatty acids to form chlorohydrins, which may destabilize cell membranes. 1-3 Given this broad spectrum of reactivity, hypochlorous acid is an obvious candidate for causing much of the damage mediated by neutrophils in inflammatory diseases. Reaction Mechanism of Myeloperoxidase The ferric or native enzyme (MP 3+) reacts with hydrogen peroxide (H202) to form the active redox intermediate compound I [Fig. 1, reaction (1)], which oxidizes chloride (CI-) to hypochlorous acid (HOC1) [Fig. I, reaction (2)]. 1,2 These reactions are termed the chlorination cycle. Compound I also oxidizes bromide, iodide, and the pseudohalide thiocyanate to the corresponding hypohalous acids. In addition to its chlorination activity, myeloperoxidase oxidizes numerous phenols, anilines, and /3-diketones to the respective free radicals via the classic peroxidation cycle [Fig. 1, reactions (I), (3), and (4)]. 1 The relative concentrations of chloride and the reducing substrate (AH) will determine whether myeloperoxidase uses hydrogen peroxide for chlorination or peroxidation. Poor peroxidase substrates that react readily with compound I, but reduce s. J. Klebanoff, in "Inflammation: Basic Principles and Clinical Correlates" (J. I. Gallin, I. M. Goldsteia, and R. Snyderman, eds.), p. 391. Raven, New York, 1988. : C. C. Winterbourn, in "OxygenRadicals: SystemicEvents and DiseaseProcesses" (D. K. Das and W. B. Essman, eds.), p. 31. Karger, Basel, 1990. C. C. Winterbourn, J. J. M. van den Berg, E. Roitman, and F. A. Kuypers,Arch. Biochem. Biophys. 296, 547 (1992). METHODS IN ENZYMOLOGY, VOL. 233

Copyright © 1994by Academic Press, Inc. All rights of reproduction in any form reserved.

[53]

CHLORINATION ASSAYS FOR MYELOPEROXIDASE

HOC1

503

C1-

Mpa+ + H~02

(1) ~ Compound I

(6)~

(a)

0z

+

Compound III ~

0S ~. (7)

l

HzO2

H +

Compound II

Ii

08

Fic. 1. Reaction mechanismof myeloperoxidase[reactions (1)-(7)]. compound II slowly, are good inhibitors of hypochlorous acid production because they trap the enzyme as compound 11.4 However, they are unable to competitively inhibit peroxidation activity, because peroxidation is dependent on the turnover of compound II [Fig. 1, reaction (4)]. In the neutrophil, myeloperoxides operates in the presence of superoxide, which reacts with the enzyme to modulate its chlorination activity. 5 Under conditions where compound II accumulates, superoxide enhances activity by reducing compound II back to the native enzyme [Fig. 1, reaction (5)]. When compound II does not accumulate, superoxide inhibits chlorination by converting the enzyme to oxymyeloperoxidase or compound HI [Fig. 1, reaction (6)]. Compound llI is not a dead-end intermediate, but is converted to compound II by hydrogen peroxide [Fig. 1, reaction (7)]. Chlorination Assays for Myeloperoxidase Assays for myeloperoxidase measure either the chlorination or peroxidation activity of the enzyme. These activities are affected differently by pH, superoxide, chloride, hydrogen peroxide, and inhibitors of myeloperoxidase. 1,6For example, the pH optimum for the peroxidation of 3,3',5,5'tetramethylbenzidine is 5.4, 7 whereas that of hypochlorous acid produc4 A. J. Kettle and C. C. Winterbourn, Biochem. Pharmacol. 41, 1485 (1991). 5 A. J. Kettle and C. C. Winterbourn, Biochem. J. 252, 529 (1988). 6 A. J. Kettle and C. C. Winterbourn, Biochem. J. 263, 823 (1989). 7 K. Suzuki, H. Ota, S. Sasagawa, T. Sakatani, and T. Fujikura, Anal. Biochem. 132, 345 (1983).

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tion is sear neutral. 6 Thus, it is imperative to make the distinction between the two activities of the enzymes, and choose the appropriate assay for the activity of interest. In this chapter we focus on chlorination assays for myeloperoxidase. Assays for peroxidation activity have been described previously in this series, a There are four instances when it is essential to measure the chlorination activity of myeloperoxidase. To verify that the peroxidase under investigation is myeloperoxidase, it must be shown to oxidize chloride to hypochlorous acid. Because the function of myeloperoxidase is to produce hypochlorous acid, conditions that may affect it in vivo should be tested against this activity rather than in a peroxidation assay. Similarly, potential inhibitors of myeloperoxidase must be tested in a chlorination assay because they may have different effects on the peroxidation activity. Finally, chlorination assays are used to measure hypochlorous acid production by neutrophils and monocytes. Peroxidation assays are perfectly adequate, however, for determining the amount of myeloperoxidase present in neutrophil extracts or inflammatory exudates. We consider that the best method for continuously measuring chlorination activity, and for providing rates of reaction, is to monitor the loss of hydrogen peroxide with a hydrogen peroxide electrode. 6 We favor this assay because it measures chlorination activity directly without the need to add detector compounds that may react with the enzyme. Also, myeloperoxidase is unambiguously identified by showing that chloride greatly accelerates the loss of hydrogen peroxide (Fig. 2). In the presence of chloride, the formation of hypochlorous acid accounts completely for the loss of hydrogen peroxide. Ifa hydrogen peroxide electrode is unavailable, or if it is unnecessary to monitor chlorination activity continuously, then the most appropriate method is the taurine chloramine assay. 9 Unlike many other molecules that detect hypochlorous acid, taurine is unreactive toward the enzyme intermediates of myeloperoxidase and, therefore, does not influence enzyme activity. This assay gives results comparable to those obtained with the hydrogen peroxide electrode (Table I). It is also the most suitable assay for measuring hypochlorous acid production by neutrophils and monocytes. General Considerations

Human myeloperoxidase is purified from peripheral blood neutrophils as described previously in this series.l° The enzyme can also be isolated 8 p. C. Andrews and N. I. Krinsky, this series, Vol. 132, p. 132, p.369. 9 S. J. Weiss, R. Klein, A. Slivka, and M. Wei, J. Clin. Invest. 70, 598 (1982). I0 N. R. Matheson, P. S. Wong, and J. Travis, Biochemistry 20, 325 (1981).

[53]

CHLORINATION ASSAYS FOR MYELOPEROXlDASE i

505

i

+ MP'O 100

75

O

50 25

0

2

4 6 Time (rain)

8

FIG. 2. Effects of chloride and monochlorodimedon on the loss of hydrogen peroxide catalyzed by myeloperoxidase. The loss of hydrogen peroxide by 20 nM myeloperoxidase was monitored in 50 mM phosphate buffer, pH 7.4, in the absence or presence of 100 mM chloride and 100/zM monochlorodimedon.

from bovine spleen.11 The concentration of myeloperoxidase is determined by measuring A430 (e430 91,000 M -1 cm -1 per hemel2). Myeloperoxidase produces hypochlorous acid over a wide pH range, with the pH optimum decreasing as the ratio of hydrogen peroxide concentration to chloride concentration increases. In the presence of a superoxide-generating system, and at physiological concentrations of chloride and hydrogen peroxide, the chlorination activity is optimal between pH 6.5 and 7.0. 6 We therefore recommend that the chlorination activity be measured near neutral pH. It is best to use phosphates buffers. Tris and Good buffers must be avoided because the amino groups scavenge hypochlorous acid. Substrates required for chlorination are chloride and hydrogen peroxide. The concentration of chloride should be about 100 raM. To confirm that any loss of hydrogen peroxide is due to myeloperoxidase, controls should be performed without chloride. Reagent hydrogen peroxide can be used, or it can be generated continuously with either xanthine oxidase or glucose oxidase. At high concentrations, hydrogen peroxide irreversibly inactivates myeloperoxidase) 3 To minimize inactivation, it is prudent to 11 M. Ikeda-Saito, J. Biol. Chem. 2,611, 11688 (1985). t2 T. Odajima and I. Yamazaki, Biochem. Biophys. Acta 21)6, 71 (1970). 13 j. W. Naskalski, Biochim. Biophys. Acta 485, 291 (1977).

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TABLE I ASSAYS FOR CHLORINATING ACTIVITY OF MYELOPEROXIDASEa Rate of reaction

Assay

No addition

+Methionine (5 mM)

+Dapsone (5/zM)

Hydrogen peroxide electrode Rate over first 10 sec t#M H202/min) Rate over first 30 sec (/~MH~O2/min) Taurine chloramine(/zMHOCl/min) Monochlorodimedon0zM HOCl/min) Ascorbate QzMHOC1/min) Tetramethylbenzidine(A65s/min)

-47.0 -23.2 21.0 0.9 34.2 0.904

-47.0 -23.0 0.0 0.0 0.0 0.920

-3.1 -2.1 4.2 n.d. 13.2 0.916

a Reactions were carried out at 37° and started by adding 20 nM myeloperoxidase to 50 /xM hydrogen peroxide in 10 mM phosphate buffer, pH 7.4, containing 140 mM sodium chloride. Concentrations of detectors were as follows: taurine, 10 mM; monochlorodimedon, 50 /zM; ascorbate, 100 /zM; and 3,3',5,5'-tetramethylbenzidine, 1.5 mM. Except where indicated, rates were calculated over the first 30 sec of the reaction. All assays were performed in duplicate and agreed within 10% of one another, n.d., not determined.

keep the concentration at 50 t~M or less, either by continuous infusion or adding it incrementally. Reactions are stopped by adding a 10-fold molar excess of catalase over myeloperoxidase. Effects of superoxide are best assessed using xanthine oxidase and 20 mM of freshly distilled acetaldehyde. Hypoxanthine and xanthine should not be used because they are oxidized to uric acid which masks effects of superoxide. 5 When using xanthine oxidase reactions should be started by adding acetaldehyde and stopped with catalase and 100/xM allopurinol.

Hydrogen Peroxide Electrode Assay The loss of hydrogen peroxide catalyzed by myeloperoxidase in the presence of chloride is continuously monitored 6 using a YSI Model 25 oxidase meter fitted with a YSI 25 I0 oxidase probe (Yellow Springs Instrument Co., Yellow Springs, OH). To protect the electrodes and define a diffusion path to them, the probe is covered with a membrane made from either collagen film, Nucleopore, or Cuprophane. Interference from compounds that produce a current at the anode can be eliminated by placing a cellulose acetate membrane between the collagen film and the probe. The membrane excludes compounds with a molecular weight of greater than 150, including ascorbate, acetaminophen, uric acid, glutathi-

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one, cysteamine, xanthine, and NADPH. 14 Exclusion membranes are made by dissolving cellulose acetate (4%, w/w) in cyclohexanone and pouring it onto distilled water. 15,16The resulting membrane that forms on the surface of the water is removed with a polyethylene sheet, dried, and fitted to the probe. The cellulose acetate membranes cannot be stored, so they must be prepared when required. It is best to allow the probe to stabilize overnight at the desired reaction temperature with constant stirring. To maintain stability it should be operated continuously. The probe may lose sensitivity after several days owing to deterioration of the cellulose acetate membrane, which should be renewed and the system allowed to restabilize for at least 3 hr. The meter is operated in the variable sensitivity mode and calibrated with concentrations of hydrogen peroxide up to 100 txM (8240 43.6 M - l cm-117). The response should be linear. It is imperative that solutions be stirred continuously; otherwise, there is a marked drop in the hydrogen peroxide signal. The probe consumes negligible amounts of hydrogen peroxide and should give a stable reading. If not, it should be cleaned with a 200 txM hypochlorite solution to inactivate any bound enzymes that catabolize hydrogen peroxide, then rinsed thoroughly with the reaction buffer. It is also necessary to clean the reaction cell with 50% nitric acid. This cleaning procedure must be followed because myeloperoxidase sticks to the probe and glass surfaces. Reagents to be used in the assay should be tested for interference with the hydrogen peroxide signal. Taurine and acetaldehyde cause an initial change in response but reach a new steady state, which can be used as the baseline for hydrogen peroxide detection. Taurine (10 raM), or methionine (500 txM), should be included in the reaction buffer to scavenge hypochlorous acid and prevent it from inactivating myeloperoxidase. Taurine is converted to taurine chloramine, and methionine to methionine sulfoxide. These products can be used to determine the stoichiometry of conversion of hydrogen peroxide to hypochlorous acid. 6,9 The probe is placed in buffer containing all the reagents except hydrogen peroxide and myeloperoxidase, and the meter and recorder are set at zero. A known amount of hydrogen peroxide is added, and when the signal is steady the meter and recorder are set at 100% deflection. The reaction is started by adding myeloperoxidase, and the rate is determined by drawing a tangent to the initial part of the curve 14 S. T. Test and S. J. Weiss, this series, Vol. 132, p. 401. 15 Instruction manual, YSI Model 25 oxidase meter and YSI oxidase probe, YSI Co., Yellow Springs, Ohio. 16 p. j. Taylor, E. Kmetec, and J. M. Johnson, Anal. Chem. 49, 789 (1977). 17 R. J. Beers and I. W. Sizer, J. Biol. Chem. 195, 133 (1952).

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for hydrogen peroxide loss (Fig. 2). At pH 7.4 with 100 mM chloride and 50/.tM hydrogen peroxide, the turnover number of myeloperoxidase over the first 10 sec of the reaction is about 2500 per minute. When using xanthine oxidase or glucose oxidase systems, the meter must first be calibrated against a known concentration of hydrogen peroxide. The chlorination activity is determined by calculating the difference between the rates of accumulation of hydrogen peroxide in the absence and presence of myeloperoxidase.

Taurine Chloramine Assay The taurine chloramine assay is based on the reaction of hypochlorous acid with taurine (TauNHz) to produce taurine chloramine (TauNHCI) [reaction (8)], which is measured by reacting it with 5-thio-2-nitrobenzoic acid (TNB) [reaction (9)]. 9 Yellow TNB (~412 14,100 M -I cm -118) is oxidized to colorless 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). This is an extremely sensitive assay and can accurately measure concentrations of hypochlorous acid as low as 5/xM. HOCI + TauNHz--* TauNHC1 + HzO TauNHCI + 2TNB ~ DTNB + CI- + TauNHE

(8) (9)

A 1 mM solution of TNB is prepared by dissolving 2 mM DTNB (Sigma Chemical Co., St. Louis, MO) in 50 mM phosphate buffer, pH 7.4. The solution of DTNB is titrated to pH 12 with sodium hydroxide to promote its hydrolysis, and after 5 min the pH is brought back to 7.4 with hydrochloric acid. TNB is light sensitive and undergoes slow oxidation in air, so it should be prepared weekly and kept under nitrogen at 4 ° in a brown glass bottle containing 1 mM EDTA. Hypochlorous Acid Production by Isolated Myeloperoxidase. When determining hypochlorous acid production by the purified enzyme or extracts containing myeloperoxidase, reactions are carried out in the required buffer containing 10 mM taurine. Reactions are started by adding hydrogen peroxide to myeloperoxidase and chloride. After the reactions have been stopped, an excess of TNB is added over the amount of hypochlorous acid produced. TNB is also added to a blank without hydrogen peroxide. To ensure complete and rapid mixing, the TNB should be added on a vortex mixer. It is preferable that this mixture have a pH of 7-7.4. Tubes are then placed in the dark and left for 5 min for reaction (9) to go to completion. Finally the absorbances at 412 nm are recorded using the buffer system as the reference. The concentration of hypochlorous acid ~s p. W. Riddles, R. L. Blakeley, and B. Zerner, this series, Vol. 91, p. 49.

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CHLORINATION ASSAYS FOR MYELOPEROXIDASE

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produced is calculated by determining the loss in A412compared to the blank, multiplying it by the dilution factor, and dividing by 28,200. At pH 7.4, with 100 mM chloride and 50/xM hydrogen peroxide, the turnover number of myeloperoxidase should be about 1200 per minute over the first 30 sec of the reaction. When reagents are added to the reaction system to determine their effects on the chlorination activity of myeloperoxidase, it should first be verified that at the concentrations used they do not compete with taurine for hypochlorous acid, or react with TNB or DTNB. Hypochlorous Acid Production by Neutrophils. Neutrophiis are prepared by standard techniques. 19 The most satisfactory stimuli are opsonized zymosan ~4and phorbol myristate acetate, but the method is generally applicable. Neutrophils (1 x 106) are suspended in 500 ill of 10 mM phosphate buffer, pH 7.4, with 138 mM sodium chloride, 10 mM potassium chloride, 0.5 mM magnesium chloride, 1 mM calcium chloride, 1 mg/ml of glucose, and 20 mM taurine. After preincubating the cells at 37° for l0 rain, they are stimulated with 50 ng of phorbol myristate acetate or 0.5 mg of opsonized zymosan. To ensure that the cells are kept in suspension and fully aerated, reaction mixtures are shaken every 5 min. Reactions are stopped after 30 min by adding 20/~g/ml catalase and placing the tubes in melting ice for l0 min. Cells are then pelleted (5 rain at 14,000 g, 4°), and the supernatants are added to TNB. The concentration of hypochlorous acid is then calculated as described above, using the reaction buffer as a reference. Under the reaction conditions outlined, neutrophils stimulated with either phorbol myristate acetate or opsonized zymosan generate about 50 nmol of hypochlorous acid in 30 min. 9 Negligible amounts are produced by unstimulated cells. To check that the oxidant produced by the neutrophils is hypochlorous acid, its formation should be inhibited by 100/~M azide, 20 txg/ml catalase, and I mM methionine, but not by 10 mM mannitol. Comparison of Chlorination Assays Assays based on the chlorination of monochlorodimedon and on oxidation of ascorbate and tetramethylbenzidine have been described for measuring the chlorination activity of myeloperoxidase. We have compared them with the hydrogen peroxide electrode and the taurine chloramine assays (Table I) and have found that each has features that limit their usefulness for investigating chlorination. All conditions, except for the concentration of detector, were kept constant. Methionine was added as a scavenger of hypochlorous acid that does not react with the enzyme, 19 A. Boyum, Scand. J. Clin. Lab. Invest. 21 (Suppl. 97), 77 (1%8).

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to confirm that the assays measure chlorination activity. We also added dapsone because it inhibits chlorination by converting the enzyme to compound If. It is therefore useful to check if detectors interact with myeloperoxidase and reduce compound lI. Using the hydrogen peroxide electrode, it can be seen that myeloperoxidase catalyzes the same rate of loss of hydrogen peroxide in the presence of methione or taurine. This confirms that these compounds do not affect enzyme activity. The rate of loss of hydrogen peroxide decreases with time (Fig. 2 and Table I) because, above 20 /.tM, hydrogen peroxide converts some of the enzyme to compound If. 6 Myeloperoxidase is almost totally inhibited when it is trapped as compound II by dapsone. When monitored over the same time period, the rate of hypochlorous acid production as measured by the taurine chloramine assay is equal to the rate of hydrogen peroxide loss. This verifies that hydrogen peroxide is stoichiometrically converted to hypochlorous acid. Detection of hypochlorous acid in the taurine chloramine assay is confirmed by complete inhibition by methionine and almost complete inhibition by dapsone.

Monochlorodimedon Assay Chlorination of monochlorodimedon by hypochlorous acid to form dichlorodimedon, with an associated loss of A290 (~290 19,000 M - 1 cm- 1), has been routinely used as a specific assay for myeloperoxidase. 2° Although this method is useful for detecting hypochlorous acid, as shown by its complete inhibition by methionine, it grossly underestimates the chlorinating activity of myeloperoxidase (Table I and Fig. 2). Activity is diminished because, like dapsone, monochlorodimedon inhibits chlorination by reacting with compound I and trapping myeloperoxidase as compound 11.20 At pH 5 monochlorodimedon is efficiently oxidized by the peroxidation cycle so that the assay loses its specificity for hypochlorous acid. We recommend that monochlorodimedon not be used to measure the activity of myeloperoxidase.

Ascorbate Assay The rapid reaction of hypochlorous acid with ascorbate (e2ss.5 15,000 M-1 cm-1) has been used as the basis for assaying the chlorination activity of myeloperoxidase. 21The 10s s of ascorbate is attributable to hypochlorou s acid because it is totally inhibited by methionine (Table I). However, results obtained with the ascorbate assay must be interpreted with caution 2o A. J. Kettle and C. C. Winterbourn, Biochim. Biophys. Acta 957, 185 (1988). 21 j. A. Chesney, J. R. Mahoney, and J. W. Eaton, Anal. Biochem. 196, 262 (1991).

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CHLORINATION ASSAYS FOR MYELOPEROXIDASE

5I 1

since ascorbate readily reduces compounds I and II. 22 This is evident in Table I, where the ascorbate assay gave a rate midway between those obtained with the hydrogen peroxide electrode for first I0 and 30 sec of chlorination. The lower activity at 10 sec is explained by ascorbate diverting some of the enzyme from the chlorination cycle. In contrast, the higher activity at 30 sec is attributed to ascorbate preventing accumulation of compound II caused by hydrogen peroxide. The reaction of ascorbate with compound II is also seen when dapsone is added to the ascorbate assay. In this assay dapsone is a far less effective inhibitor than it is when using either the hydrogen peroxide electrode or the taurine chloramine assay. Ascorbate also reacts with compound 111.23 Therefore, the assay is unsuitable for investigating inhibitors of myeloperoxidase that act by converting the enzyme to compound II or compound III. it will also mask reactions of superoxide with myeloperoxidase, since they involve compounds I| and III, and therefore it has limitations for investigating production of hypochlorous acid by neutrophils. However, there are situations where the ascorbate assay can be used as a sensitive and continuous method for the determination of hypochlorous acid production.

Tetramethylbenzidine Assay Oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) by myeloperoxidase to produce a blue product with an absorbance maximum at 655 nm is an extremely sensitive method for determining the activity of the enzyme. 7 The same product is formed ifTMB is oxidized via the peroxidation cycle or by hypochlorous acid. 24 Therefore, TMB has been used to detect and quantitate the total utilization of hydrogen peroxide by myeloperoxidase. z5 However, under the conditions of the assay, the lack of inhibition by methionine indicates that the peroxidation activity completely swamps the chlorination activity. Dapsone has no effect in this assay because the peroxidation activity is dependent on the rate at which substrates reduce compound II to the ferric enzyme [reaction (4), Fig. 1]. Because dapsone has a limited capacity to reduce compound II, it would be unable to compete with TMB and inhibit myeloperoxidase. This result illustrates how assays for peroxidation activity can be quite inappropriate for determining how effectively compounds inhibit hypochlorous acid production by myeloperoxidase. Although the TMB assay is the most sensitive for quantifying the concentration of myeloperoxidase, and can easily distin22 B. G. J. M. Bolscher, G. R. Zoutberg, R. A. Cuperus, and R. Wever, Biochem. Biophys. Acta 784, 189 (1984). 23 L. A. Marquez and H. B. Dunford, J. Biol. Chem. 265, 6074 (1990). 24 p. C. Andrews and N. I. Krinsky, Anal. Biochem. 127, 346 (1982). 25 p. C. Andrews and N. I. Krinsky, J. Biol. Chem. 257, 13240 (1982).

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guish between myeloperoxidase, eosinophil peroxidase, and lactoperoxidase, 26 it does not measure the chlorination activity of the enzyme. Acknowledgments This work was supported by the Health Research Council of New Zealand. 26 p. M. Bozeman, D. B. Learn, and E. L. Thomas, J. Immunol. Methods 126, 125 (1990).

[54] O x i d a t i v e Stress to L e n s Crystallins B y JESSICA JAHNGEN-HODGE, ALLEN TAYLOR, F U SHANG, L I L l HUANG, a n d CASILDA MURA

Introduction There is keen interest in age- and environment-related damage to lens proteins because accumulation of such proteins during aging is associated with lens opacification or senile cataract. This disease involves precipitation of long-lived proteins from the normally clear lens milieu. The notion that cataract-related damage results from (photo)oxidation during a lifetime of exposure to light and various forms of damaging oxygen is derived from human, animal, and cell-free experiments. Patients exposed to hyperbaric oxygen developed lens nuclear opacities) Epidemiological reports indicate that people exposed to higher levels of light 2-4 or other forms of radiation5 have a greater risk of developing cataracts. Rabbit lenses exposed to hyperbaric oxygen6 and mice exposed to hyperoxia7 also develop cataracts. The age-related oxidative insults to lens proteins can be modeled in cell-free systems as well. 8-~1 Taken together, these data indicate roles for oxygen and light in the damage of lens proteins. t B. Palmquist, B. Philipson, and P. Barr, Br. J. Ophthalmol. 68, 113 (1984). 2 H. R. Taylor, S. K. West, F. S. Rosenthal, B. Munoz, H. S. Newland, H. Abbey, and E. A. Emmett, N. Engl. J. Med. 319, 1429 (1988). 3 F. Hollows and D. Moran, Lancet 2, 1249 (1981). 4 R. Hiller, R. D. Spurduto, and F. Ederer, Am. J. Epidemiol. 118, 239 (1983). 5 j. Harding, "Cataract." Chapman & Hall, London, 1991. 6 V. Padgaonkar, F. J. Giblin, and V. N. Reddy, Exp. Eye Res. 49, 887 (1989). 7 S. S. Schocket, J. Esterson, B. Bradford, M. R. Michaelis, and R. D. Richards, Isr. J. Med. 8, 1596 (1972). s j. Blondin, V. J. Baragi, E. Schwartz, J. Sadowski, and A. Taylor, Free Radical Biol. Med. 2, 275 (1986).

METHODS IN ENZYMOLOGY, VOL. 233

Copyright © 1994 by Academic Press, Inc. All rights of reproduction in any form reserved.