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[38] Protein oxidative damage
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By EMILY SHACTER Introduction Proteins can undergo numerous covalent changes on exposure to oxidants. 1'2 Some of these changes result from direct attack of a free radical on the protein molecule whereas others are incurred indirectly such as through covalent attachment of oxidation by-products. One mode of direct oxidative attack on a protein derives from site-specific metal-catalyzed oxidation (MCO) in which the reduced form of a protein-bound transition metal (e.g., Fe 2+, Cu +) reduces H 2 0 2 to form a reactive intermediate (e.g., O H . , ferryl radical) in the immediate proximity of amino acid side chains. 3'4 Radical-mediated oxidation induces the formation of amino acyl carbonyl groups (aldehydes and ketones) in amino acids, especially in Lys, Arg, Pro, Thr. 4'5 A metal-catalyzed attack on His and Tyr residues leads to the formation of oxo-His 6 and dityrosine, 7 respectively. The sulfhydryl moiety of Cys is highly prone to oxidative attack by several mechanisms, leading to the formation of disulfide bonds and thiyl radicals. 8'9 Similarly, numerous oxidative pathways lead to the oxidation of Met residues, leading to the formation of methionine sulfoxide. TMVal, Leu, and Tyr residues are oxidized to hydroxy and hydroperoxy derivatives on exposure to y-irradiation in O2.U Reaction of myeloperoxidase-derived HOC1 with Tyr, Trp, Lys, and Met residues leads to the formation of chlorotyrosine, chloramines, aldehydes, and methionine sulfoxide, a2-15 Peroxynitrite, generated from the
t R. T. Dean, S. Fu, R. Stocker, and M. J. Davies, Biochem. J. 324, 1 (1997). 2 B. S. Berlett and E. R. Stadtman, J. Biol. Chem. 272, 20313 (1997). 3 A. Samuni, J. Aronovitch, D. Godinger, M. Chevion, and G. Czapski, Eur. J. Biochem. 137, 119 (1983). n E. R. Stadtman, Free Radic. Biol. Med. 9, 315 (1990). 5 A. Amici, R. L. Levine, L. Tsai, and E. R. Stadtman, J. Biol. Chem. 264, 3341 (1989). 6 S. A. Lewisch and R. L. Levine, Anal Biochem. 231, 440 (1995). 7 C. Giulivi and K. J. A. Davies, Methods Enzymol. 233, 363 (1994). 8 M-L. Hu, Methods Enzymol. 233, 380 (1994). 9 B. Kalyanaraman, Biochem. Soc. Syrup. 61, 55 (1995). t0 W. Vogt, Free Radic. Biol. Med. 18, 93 (1995). u S. L. Fu and R. T. Dean, Biochem. J. 324, 41 (1997). 12 L. J. Hazell, J. J. M. van den Berg, and R. Stocker, Biochem. J. 302, 297 (1994). 13 C.-Y. Yang, Z.-W. Gu, H.-X. Yang, M. Yang, A, M. Gotto, and C. V. Smith, Free Radic. Biol. Med. 23, 82 (1997).
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interaction of nitric oxide and superoxide, causes nitrosylation of tyrosine residues and oxidative modification of other amino acid residues, including Cys, Trp, Met, and Phe, 16 but does not induce protein carbonyls (R. L. Levine, personal communication). Indirect modification of protein amino acyl side chains occurs through adduct formation. For example, lipid peroxidation breakdown products such as hydroxynonenal (HNE) and malondialdehyde (MDA) bind covalently to Lys, His, and Cys residues, leading to the addition of aldehyde moieties to the protein. 17,18Glutathiolation of Cys residues 19 and addition of p-hydroxyphenylacetaldehyde to Lys residues 2° also occur under conditions of oxidative stress. Carboxymethyllysine represents a form of protein modification generated by oxidation products of sugars and lipids. 2'21 A summary of well-characterized protein amino acyl oxidative modifications is provided in Table I, and a list of assays commonly employed to measure these modifications is given in Table II. Note that ozone can induce a similar spectrum of protein oxidative modifications as MCO systems and has a particularly strong ability to induce protein carbonyl groups. 22'23 Singlet oxygen generated by exposure to UV light or by photochemical methods has a greater tendency to attack Trp, His, Tyr, Met, and Cys residues and a lesser capacity to induce carbonyls. 24-26 This article focuses on the detection of carbonyl groups as markers of oxidative protein modification. Protein carbonyls become elevated under various conditions of oxidative stress and are generated readily under experimental conditions in vitro, such as through exposure to iron/ascorbate, 5
14 S. L. Hazen and J. W. Heinecke, J. Clin. Invest. 99, 2075 (1997). 15 A, J. Kettle, FEBS Lett. 379, 103 (1996). 16 H. Ischiropoulos and A. B. A1-Mehdi, FEBS Lett. 364, 279 (1995). 17 K. Uchida and E. R. Stadtman, Methods Enzymol. 233, 371 (1994). 18 j. R. Requena, M. X. Fu, M. U. Ahmed, A. J. Jenkins, T. J. Lyons, J. W. Baynes, and S. R. Thorpe, Biochem. J. 322, 317 (1997). 19 C.-K. Lii, Y.-C. Chai, W. Zhao, J. A. Thomas, and S. Hendrich, Arch. Biochem. Biophys. 308, 231 (1994). 20 S. L. Hazen, J. P. Gaut, F. F. Hsu, J. R. Crowley, A. d'Avignon, and J. W. Heinecke, J. Biol. Chem. 27, 16990 (1997). 21 T. P. Degenhardt, S. R. Thorpe, and J. W. Baynes, Cell. Mol. Biol. 44, 1139 (1998). 22 C. E. Cross, A. Z. Reznik, L. Packer, P. A. Davis, Y. J. Suzuki, and B. Halliwell, Free Radic. Res. Commun. 15, 347 (1992). 23 B. S. Berlett, R. L. Levine, and E. R. Stadtman, J. Biol. Chem. 271, 4177 (1996). 24 D. Balasubramanian, X. Du, and J. S. Zigler, Photochem. PhotobioL 52, 761 (1990). z5 H. R. Shen, J. D. Spikes, P. Kopecekova, and J. Kopecek, J. Photochem. PhotobioL B 34, 203 (1996). 26 M. L. Hu and A. L. Tappel, Photochem. Photobiol. 56, 357 (1992).
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TABLE I EXAMPLE OF OXIDATIVE PROTEIN MODIFICATIONS
Modification Disulfides, mixed disulfides (e.g., glutathiolation) Methionine sulfoxide Carbonyls oxo-Histidine Dityrosine Chlorotyrosine Nitrotyrosine Tryptophanyl modifications [e.g., (N-formyl)kynurenine] Hydro(pero)xy derivatives Chloramines, deamination Lipid peroxidation adducts (MDA, HNE, acrolein) Glycoxidation adducts Amino acid oxidation adducts Cross-links, aggregates, fragments
Amino acids modified Cys Met All (especially Lys, Arg, Pro, Thr) His Tyr Tyr Wyr Trp
Oxidizing sources" All, ONOO (with GSSG) All, ONOOAll y-ray, MCO, I o 2 y-ray, MCO, 10 2 HOC1 ONOOy-ray, IO2, MCO, ozone
Val, Leu, Tyr, Trp Lys Lys, Cys, His
y-ray HOC1 y-ray, MCO (not HOC1)
Lys Lys, Cys, His several
Glucose HOCI All
a MCO, metal-catalyzed oxidation; all = y-ray, MCO, HOC1, 102,
ozone.
ionizing radiation, 27 singlet oxygen, 2s and o z o n e ) 2 Their chemical stability m a k e s t h e m suitable targets for laboratory m e a s u r e m e n t . Early techniques for the detection of protein carbonyls were based either on i n c o r p o r a t i o n of tritium t h r o u g h reduction with tritiated b o r o h y d r i d e 29 or on incorporation of a stable dinitrophenyl ( D N P ) a d d u c t t h r o u g h reaction of the carbonyls with 2,4-dinitrophenylhydrazine ( D N P H ) . 3° Because D N P absorbs light at 370 nm, the c a r b o n y l groups can be m e a s u r e d spectrophotometrically. While reliable and quantitative, these two techniques suffer f r o m three significant technical drawbacks: (a) T h e y require the use of a substantial a m o u n t of purified protein ( ~ 1 - 2 mg); (b) the derivitizing reagents have to be r e m o v e d prior to analysis in o r d e r to reduce b a c k g r o u n d levels sufficiently to allow detection of p r o t e i n - b o u n d label (either H 3 or D N P ) ; and (c) they are unable to differentiate oxidized f r o m nonoxidized proteins in cell or tissue extracts. 27E. Shacter, J. A. Williams, and R, L. Levine, Free Radic. Biol. Med. 18, 815 (1995). 28j. A. Silvester, G. S. Timmins, and M. J. Davies, Arch. Biochem. Biophys. 3511,249 (1998). 29A. Lenz, U. Costabel, S. Shaltiel, and R. L. Levine, Anal, Biochem. 177~419 (1989). 30R. L. Levine, D. Garland, C. N. Oliver, A. Amici, I. Climent, A. Lenz, B. Ahn, S. Shaltiel, and E. R. Stadtman, Methods Enzymol. 186, 464 (1990).
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TABLE II METHODSFORDETECTIONOF OXIDATIVEPROTEINMODIFICATIONS a Modification Disulfides Thiyl radicals Glutathiolation Methionine sulfoxide Carbonyls
2-oxo-His Dityrosine Chlorotyrosine Nitrotyrosine Tryptophanyl Hydroperoxides Lipid peroxidation adducts Amino acid oxidation adducts Cross-links, aggregates, fragments
Methods of detection
References b
SDS-gel electrophoresis (_+2-ME); DTNB Electron spin resonance spectroscopy RP-HPLC/mass spectroscopy; IEF; S35-Cys/Chx/SDS-PAGE CNBr cleavage/amino acid analysis DNPH/Western blot/ELISA/ immunocytochemistry/HPLC/ A370; reduction with Naborotritide Amino acid analysis Fluorescence; proteolysis or hydrolysis/HPLC Hydrolysis/nitroso-napthol/HPLC; HBr hydrolysis-GC/MS Immunoassay; hydrolysis/HLPC; HPLC/electrochemical detection Fluoroscence spectroscopy; amino acid analysis (alk. hydrolysis) KI/Ig/NaBH4/hydrolysis/OPAHPLC NaBH4/hydrolysis/OPA-HPLC; hydrolysis-GC/MS; DNPH; immunoassays Reduction/hydrolysis/NMR SDS-gel electrophoresis; HPLC
41 9 19,42,43 44,45 This article and 30,35,36,46 29,47 6 7,48 14,15 49-53
11 17,18,54,55 20 12,56,57
"DTNB, dithiobisnitrobenzoate; IEF, isoelectric focusing; DNPHI, dinitrophenylhydrazine; CNBr, cyanogen bromide; Chx, cyclohexamide; OPA, o-pthaldialdehyde. b References for some methods currently in use. Other methods are also available.
W e s t e r n Blot I m m u n o a s s a y for P r o t e i n C a r b o n y l s Several years ago, we described a new m e t h o d for detection of p r o t e i n carbonyls. 31 T h e m e t h o d takes advantage of the fact that D N P groups are i m m u n o g e n i c and that a n t i - D N P antibodies are available commercially. By derivatizing proteins with D N P H in an s o d i u m dodecyl sulfate (SDS) buffer, oxidized protein can be detected by p e r f o r m i n g S D S - p o l y a c r y l a m i d e gel electrophoresis ( S D S - P A G E ) , electroblotting to nitrocellulose (Western blotting), and assaying with a n t i - D N P antibodies (immunoassay). 3aE. Shacter, J. A. Williams, M. Eim, and R. L. Levine, Free Radic. Biol. Med. 17, 429 (1994).
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This method has significant advantages in that it requires very little protein (as little as 50 ng of a 50-kDa protein oxidized to the extent of 0.3-0.5 mol carbonyl/mol protein) and it identifies which proteins in a complex mixture are oxidized and which are not. 31 The sensitivity of the method for detecting minor oxidized proteins in a cell can be enhanced greatly by preparing subcellular fractions prior to derivatization and performance of the Western blot immunoassay. 32'33 The carbohydrate groups in glycoproteins have no apparent effect in the assay and are not themselves readily oxidizable by metal-catalyzed systems. 34 Aldehydes introduced through the adduction of lipid peroxidation breakdown products (e.g., 4hydroxynonenal, MDA, acrolein) can react with DNPH and thus can be detected by this method. These adducts are somewhat unstable and the extent to which they actually contribute to protein-associated carbonyl levels in tissue extracts remains to be established. Reagents required for running the Western blot immunoassay are all available from commercial sources and the only special equipment employed is an SDS-PAGE/electroblotting apparatus, available in most biochemistry laboratories. Due to the nature of Western blotting, the assay is only semiquantitative. This drawback can be overcome by running sizeexclusion HPLC instead of S D S - P A G E if there is enough sample available. 35 Alternatively, the quantitation of total DNPH-derivatized protein carbonyls can be achieved by use of an ELISA, as described by Buss et aL 36 The actual procedure for running the Western blot immunoassay for protein carbonyls is very straightforward and there are few "tricks" required to ensure its success. Detailed procedures have been described in previous p u b l i c a t i o n s . 31'34'35'37 Directions and some useful pointers follow. Derivitization Reagents DNPH/TFA stock solution: 20 mM DNPH in 20% (v/v) trifluoroacetic acid (TFA). Dissolve the DNPH in 100% TFA and then dilute with 32 L. J. Yan, R. L. Levine, and R. S. Sohal, Proc. Natl. Acad. Sci. U.S.A. 94, 11168 (1997). 33 L. J. Yan and R. S. Sohal, Proc. Natl. Acad. Sci. U.S.A. 95, 12896 (1998). 34 y_j. Lee and E. Shacter, Arch. Biochem. Biophys. 321, 175 (1995). 35 R. L. Levine, J. Williams, E. R. Stadtman, and E. Shacter, Methods Enzymol. 233, 346 (1994). 36n . BUSS,T. P. Chan, K. B. Sluis, N. M. Domigan, and C. C. Winterbourn, Free Radic. Biol. Med. 23, 361 (1997). 37 E. Shacter, J. A. Williams, E. R. Stadtman, and R. L. Levine, in "Free Radicals: A Practical Approach" (N. Punchard and F. Kelly, eds.). Oxford Univ. Press, Oxford, 1994.
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water. Note that most commercial sources of DNPH contain roughly 30% H20. For a 10-ml stock DNPH/TFA solution, dissolve 40 mg of dry DNPH (52 mg of the labeled "wet" powder) in 1 ml of 100% TFA and then dilute with 4 ml water. 20% TFA (v/v) (reagent control) 24% SDS in H20 (24 g/100 ml) 2 M Tris base containing 30% glycerol 2-mercaptoethanol (2-ME), undiluted stock (14.4 M)
Samples Any protein solution may be used, including purified proteins and cell and tissue extracts. Extracts containing SDS are most suitable as the derivitization reaction and gels contain SDS buffer.
Reaction with Dinitrophenylhydrazine Derivitization is carried out in a solution containing 6% SDS/5 mM DNPH/5% TFA. The concentration of protein may be as high as 10 mg/ ml; use a concentration that will be convenient for gel loading (e.g., to load 2-20/zl of sample per lane). Start the derivitization reaction by mixing 2 volumes of protein solution (e.g., 10 txl) with 1 volume of 24% SDS (e.g., 5 tzl). Then add 1 volume of DNPH/TFA stock solution (e.g., 5 txl). Incubate at room temperature for 5-30 min. Longer derivitization times lead to a nonspecific incorporation of DNP, which will interfere with correct interpretation of the results. Immediately following derivitization, neutralize the sample and prepare for final gel loading by adding 1/3 volume (e.g., 6.7 /~1) of Tris/glycerol +__2-ME. The sample should turn from yellow to orange on neutralization. To run reducing gels, add 1 volume of 2-ME to 4 volumes of the Tris/glycerol solution. In most cases, heating is not performed. Load and run the S D S - P A G E gels promptly using the conditions described by Laemmli. 38 The total time elapsed between initiation of the derivitization reaction and running the gels should not take more than about 45-60 min for 20 samples.
38 U. K. Laemmli, Nature London 227, 680 (1970).
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Electroblotting Western transfer is carried out according to standard procedures. 39,4° In our hands, the lowest nonspecific immunoassay backgrounds are achieved with PVDF paper (e.g., Immobilon-P from Millipore). Free DNPH present in the samples will not interfere with immunodetection and does not contribute a background. When nonreducing gels are run (i.e., no 2-ME is added to the samples), treat the gels with 2-ME prior to transfer. This reduction of the proteins facilitates immunodetection of DNP groups in proteins containing large numbers of disulfide bonds (e.g., albumin)? 1 After electrophoresis, soak the gel in 1% 2-ME in Tris/glycine transfer buffer for 5 rain at room temperature. Then, rinse the gel in transfer buffer for 5 min to remove excess 2-ME. Set up the transfer. 39 H. Towbin, T. Staehelin, and J. Gordon, Proc. Natl. Acad. Sci. U.S.A. 76, 4350 (1979). 4oj. M. Gershoni and G. E. Palade, Anal. Biochem. 131, 1 (1983). 4a R. Radi, K. M. Bush, T. P. Cosgrove, and B. A. Freeman, Arch. Biochem. Biophys. 286, 117 (1991). 4z D. A. Davis, K. Dorsey, P. T. Wingfield, S. J. Stahl, J. Kaufman, H. M. Fales, and R. L. Levine, Biochemistry 35, 2482 (1996). 43 j. A. Thomas, Y.-C. Chai, and C.-H. Jung, Methods Enzymol. 233, 385 (1994). 44 K. L. Maier, A. G. Lenz, I. Beck-speier, and U. Costabel, Methods Enzymol. 251, 455 (1995). 45 R. L. Levine, L. Mosoni, B. S. Berlett, and E. R. Stadtman, Proc. Natl. Acad. Sci. U.S.A. 93, 15036 (1996). 46 M. A. Smith, L. M. Sayre, V. E. Anderson, P. L. R. Harris, M. F. Beal, N. Kowall, and G. Perry, J. Histochem. Cytochem. 46, 731 (1998). 47 L. J. Yan and R. S. Sohal, Anal. Biochem. 265, 176 (1998). 48 C. Leewenburgh, J. E. Rasmussen, F. F. Hsu, D. M. Mueller, S. Pennathur, and J. W. Heinecke, J. Biol. Chem. 272, 3520 (1998). 49 M. A. Smith, P. L. R. Harris, L. M. Sayre, J. S. Beckman, and G. Perry, J. Neurosci. 17, 2653 (1997). 5o y. Z. Ye, M. Strong, Z, Q. Huang, and J. S. Beckman, Methods Enzymol. 269, 201 (1996). 51 H. Ischiropoulos, L. Zhu, J. Chen, M. Tsai, J. C. Martin, C. D. Smith, and J. S. Beckman, Arch. Biochem. Biophys. 298, 431 (1992). 52j. p. Eiserich, C. E. Cross, A. D. Jones, B. Halliwell, and A. Van der Vliet, J. Biol. Chem. 271, 19199 (1996). 53 M. K. Shigenaga, H. H. Lee, B. C. Blount, E. T. Shigeno, H. Yip, and B. N. Ames, Proc. Natl. Acad. Sci. U.S.A. 94, 3211 (1997). 54 M. E. Rosenfeld, J. C. Khoo, E. Miller, S. Parthasarathy, W. Palinski, and J. L. Witztum, J. Clin. Invest. 87, 90 (1991). 55 j. G. Kim, F. Sabbagh, N. Santanam, J. N. Wilcox, R. M. Medford, and S. Parthasarathy, Free Radic. Biol. Med. 23, 251 (1997). ~6K. J. A. Davies and M. E. Delsignore, J. Biol. Chem. 262, 9908 (1987). 57 R. E. Pacifici and K. J. A. Davies, Methods Enzymol. 186, 485 (1990).
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Immunoassay Reagents TBS: 20 mM Tris/500 mM NaC1 Tween TBS: TBS containing 0.05% (v/v) Tween 20 Tween TBSA: Tween TBS containing 1% bovine serum albumin (BSA) Primary antibody solution: We employ a monoclonal anti-DNP antibody (a mouse IgE) marketed by Sigma (St. Louis, MO). Use at a 1:1000 dilution in Tween/TBSA. Other anti-DNP antibodies are also available commercially. These need to be tested to find the best dilution for obtaining high specificy with low backgrounds. Secondary antibody solution: biotinylated rat antimouse IgE from Southern Biotechnology Assoc. (Birmingham, AL). Use at at a 1 : 4000 dilution in Tween TBSA.
Immunoassay Procedure Block the blot for 30 rain using either 3% BSA in TBS or 5% milk. Incubation with the primary antibody is generally overnight at 4 ° or for at least 3 hr at room temperature. After treating with the anti-DNP antibody, wash the blot well with Tween/TBS. Incubate with the secondary antibody for 1 hr at room temperature. Final detection is carried out using a biotinavidin-peroxidase-conjugated system from Vector Laboratories and a chemiluminescence reagent (e.g., from NEN/Dupont or Amersham). Note that cell and tissue extracts often contain biotin-binding proteins that will react with biotin-avidin reagents. These proteins are detected by running the immunoassay in the absence of the primary (anti-DNP) antibody and can serve as convenient internal markers. If their presence interferes with the carbonyl assay, use a secondary antibody that is conjugated directly to peroxidase.
Staining of Total Protein Bands After completion of the immunoassay, protein bands can be visualized by washing the blots extensively (>1 day with several buffer changes) in TBS and then staining with Amido black (0.1% in 40% methanol/10% acetic acid). Destain with 40% methanol/10% acetic acid. By staining the same blot as used for the immunoassay, bands detected by staining and by immunoassay can be lined up accurately. There is no need to run replicate gels in order to visualize protein bands.
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Controls Two negative controls should be run when new samples are tested. Control for the Anti-DNP Reaction. This is accomplished by "derivatizing" with TFA without DNPH and performing the remainder of the protocol as usual. Samples treated in this manner should show no bands in the immunoassay. If any protein bands are seen, determine by process of elimination whether they derive from reactivity with the primary antibody, secondary antibody, or the biotin-avidin detection system. Control for Specificity of the Secondary Antibody. In our experience, most problems with "nonspecific" background derive from problems with the secondary antibody. It is a good idea to test this periodically by running the immunoassay without the primary antibody step. In addition, positive and negative control samples should be run in all gels. Any oxidatively modified protein and its native (or sodium borohydride-reduced) counterpart can be employed. We routinely use fibrinogen that has been oxidized by exposure either to iron/ascorbate or to ionizing radiation. 27 Protocols for preparing oxidatively modified proteins can be found in the literature. 5'27'31 Use a positive control of known carbonyl content (i.e., nmol/mg protein) to obtain an estimate of the extent of oxidation of positive protein bands. Use the negative control to gauge how long to do the chemiluminescence exposure (i.e., this band should be negative at all exposure times).
[39] D N A D a m a g e I n d u c e d b y U l t r a v i o l e t a n d V i s i b l e L i g h t a n d Its W a v e l e n g t h D e p e n d e n c e
By
CHRISTOPHER
KIELBASSA and
BERND
EPE
Introduction DNA damage induced by solar radiation in mammalian cells consists largely of two types of modification: pyrimidine dimers and oxidative modifications. 1 Pyrimidine dimers, which can be subdivided into cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts, are the characteristic and most abundant modifications after direct excitation of DNA, although they can also be formed indirectly by energy transfer from other excited 1 j, Cadet, M. Berger, T. Douki, B. Morin, S. Raoul, J.-L. R a v a n a t , a n d S. Spinelli, Biol. Chem. 378~ 1275 (1997).
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Copyright © 2000 by Academic Press All rights of reproduction in any form reserved. 0076-6879/00 $30.00
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Protein Oxidative Damage
By EMILY SHACTER Introduction Proteins can undergo numerous covalent changes on exposure to oxidants. 1'2 Some of these changes result from direct attack of a free radical on the protein molecule whereas others are incurred indirectly such as through covalent attachment of oxidation by-products. One mode of direct oxidative attack on a protein derives from site-specific metal-catalyzed oxidation (MCO) in which the reduced form of a protein-bound transition metal (e.g., Fe 2+, Cu +) reduces H 2 0 2 to form a reactive intermediate (e.g., O H . , ferryl radical) in the immediate proximity of amino acid side chains. 3'4 Radical-mediated oxidation induces the formation of amino acyl carbonyl groups (aldehydes and ketones) in amino acids, especially in Lys, Arg, Pro, Thr. 4'5 A metal-catalyzed attack on His and Tyr residues leads to the formation of oxo-His 6 and dityrosine, 7 respectively. The sulfhydryl moiety of Cys is highly prone to oxidative attack by several mechanisms, leading to the formation of disulfide bonds and thiyl radicals. 8'9 Similarly, numerous oxidative pathways lead to the oxidation of Met residues, leading to the formation of methionine sulfoxide. TMVal, Leu, and Tyr residues are oxidized to hydroxy and hydroperoxy derivatives on exposure to y-irradiation in O2.U Reaction of myeloperoxidase-derived HOC1 with Tyr, Trp, Lys, and Met residues leads to the formation of chlorotyrosine, chloramines, aldehydes, and methionine sulfoxide, a2-15 Peroxynitrite, generated from the
t R. T. Dean, S. Fu, R. Stocker, and M. J. Davies, Biochem. J. 324, 1 (1997). 2 B. S. Berlett and E. R. Stadtman, J. Biol. Chem. 272, 20313 (1997). 3 A. Samuni, J. Aronovitch, D. Godinger, M. Chevion, and G. Czapski, Eur. J. Biochem. 137, 119 (1983). n E. R. Stadtman, Free Radic. Biol. Med. 9, 315 (1990). 5 A. Amici, R. L. Levine, L. Tsai, and E. R. Stadtman, J. Biol. Chem. 264, 3341 (1989). 6 S. A. Lewisch and R. L. Levine, Anal Biochem. 231, 440 (1995). 7 C. Giulivi and K. J. A. Davies, Methods Enzymol. 233, 363 (1994). 8 M-L. Hu, Methods Enzymol. 233, 380 (1994). 9 B. Kalyanaraman, Biochem. Soc. Syrup. 61, 55 (1995). t0 W. Vogt, Free Radic. Biol. Med. 18, 93 (1995). u S. L. Fu and R. T. Dean, Biochem. J. 324, 41 (1997). 12 L. J. Hazell, J. J. M. van den Berg, and R. Stocker, Biochem. J. 302, 297 (1994). 13 C.-Y. Yang, Z.-W. Gu, H.-X. Yang, M. Yang, A, M. Gotto, and C. V. Smith, Free Radic. Biol. Med. 23, 82 (1997).
METHODS IN ENZYMOLOGY,VOL. 319
Copyright © 2000 by AcademicPress All rights of reproductionin any form reserved. 0076-6879/00 $30.00
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interaction of nitric oxide and superoxide, causes nitrosylation of tyrosine residues and oxidative modification of other amino acid residues, including Cys, Trp, Met, and Phe, 16 but does not induce protein carbonyls (R. L. Levine, personal communication). Indirect modification of protein amino acyl side chains occurs through adduct formation. For example, lipid peroxidation breakdown products such as hydroxynonenal (HNE) and malondialdehyde (MDA) bind covalently to Lys, His, and Cys residues, leading to the addition of aldehyde moieties to the protein. 17,18Glutathiolation of Cys residues 19 and addition of p-hydroxyphenylacetaldehyde to Lys residues 2° also occur under conditions of oxidative stress. Carboxymethyllysine represents a form of protein modification generated by oxidation products of sugars and lipids. 2'21 A summary of well-characterized protein amino acyl oxidative modifications is provided in Table I, and a list of assays commonly employed to measure these modifications is given in Table II. Note that ozone can induce a similar spectrum of protein oxidative modifications as MCO systems and has a particularly strong ability to induce protein carbonyl groups. 22'23 Singlet oxygen generated by exposure to UV light or by photochemical methods has a greater tendency to attack Trp, His, Tyr, Met, and Cys residues and a lesser capacity to induce carbonyls. 24-26 This article focuses on the detection of carbonyl groups as markers of oxidative protein modification. Protein carbonyls become elevated under various conditions of oxidative stress and are generated readily under experimental conditions in vitro, such as through exposure to iron/ascorbate, 5
14 S. L. Hazen and J. W. Heinecke, J. Clin. Invest. 99, 2075 (1997). 15 A, J. Kettle, FEBS Lett. 379, 103 (1996). 16 H. Ischiropoulos and A. B. A1-Mehdi, FEBS Lett. 364, 279 (1995). 17 K. Uchida and E. R. Stadtman, Methods Enzymol. 233, 371 (1994). 18 j. R. Requena, M. X. Fu, M. U. Ahmed, A. J. Jenkins, T. J. Lyons, J. W. Baynes, and S. R. Thorpe, Biochem. J. 322, 317 (1997). 19 C.-K. Lii, Y.-C. Chai, W. Zhao, J. A. Thomas, and S. Hendrich, Arch. Biochem. Biophys. 308, 231 (1994). 20 S. L. Hazen, J. P. Gaut, F. F. Hsu, J. R. Crowley, A. d'Avignon, and J. W. Heinecke, J. Biol. Chem. 27, 16990 (1997). 21 T. P. Degenhardt, S. R. Thorpe, and J. W. Baynes, Cell. Mol. Biol. 44, 1139 (1998). 22 C. E. Cross, A. Z. Reznik, L. Packer, P. A. Davis, Y. J. Suzuki, and B. Halliwell, Free Radic. Res. Commun. 15, 347 (1992). 23 B. S. Berlett, R. L. Levine, and E. R. Stadtman, J. Biol. Chem. 271, 4177 (1996). 24 D. Balasubramanian, X. Du, and J. S. Zigler, Photochem. PhotobioL 52, 761 (1990). z5 H. R. Shen, J. D. Spikes, P. Kopecekova, and J. Kopecek, J. Photochem. PhotobioL B 34, 203 (1996). 26 M. L. Hu and A. L. Tappel, Photochem. Photobiol. 56, 357 (1992).
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TABLE I EXAMPLE OF OXIDATIVE PROTEIN MODIFICATIONS
Modification Disulfides, mixed disulfides (e.g., glutathiolation) Methionine sulfoxide Carbonyls oxo-Histidine Dityrosine Chlorotyrosine Nitrotyrosine Tryptophanyl modifications [e.g., (N-formyl)kynurenine] Hydro(pero)xy derivatives Chloramines, deamination Lipid peroxidation adducts (MDA, HNE, acrolein) Glycoxidation adducts Amino acid oxidation adducts Cross-links, aggregates, fragments
Amino acids modified Cys Met All (especially Lys, Arg, Pro, Thr) His Tyr Tyr Wyr Trp
Oxidizing sources" All, ONOO (with GSSG) All, ONOOAll y-ray, MCO, I o 2 y-ray, MCO, 10 2 HOC1 ONOOy-ray, IO2, MCO, ozone
Val, Leu, Tyr, Trp Lys Lys, Cys, His
y-ray HOC1 y-ray, MCO (not HOC1)
Lys Lys, Cys, His several
Glucose HOCI All
a MCO, metal-catalyzed oxidation; all = y-ray, MCO, HOC1, 102,
ozone.
ionizing radiation, 27 singlet oxygen, 2s and o z o n e ) 2 Their chemical stability m a k e s t h e m suitable targets for laboratory m e a s u r e m e n t . Early techniques for the detection of protein carbonyls were based either on i n c o r p o r a t i o n of tritium t h r o u g h reduction with tritiated b o r o h y d r i d e 29 or on incorporation of a stable dinitrophenyl ( D N P ) a d d u c t t h r o u g h reaction of the carbonyls with 2,4-dinitrophenylhydrazine ( D N P H ) . 3° Because D N P absorbs light at 370 nm, the c a r b o n y l groups can be m e a s u r e d spectrophotometrically. While reliable and quantitative, these two techniques suffer f r o m three significant technical drawbacks: (a) T h e y require the use of a substantial a m o u n t of purified protein ( ~ 1 - 2 mg); (b) the derivitizing reagents have to be r e m o v e d prior to analysis in o r d e r to reduce b a c k g r o u n d levels sufficiently to allow detection of p r o t e i n - b o u n d label (either H 3 or D N P ) ; and (c) they are unable to differentiate oxidized f r o m nonoxidized proteins in cell or tissue extracts. 27E. Shacter, J. A. Williams, and R, L. Levine, Free Radic. Biol. Med. 18, 815 (1995). 28j. A. Silvester, G. S. Timmins, and M. J. Davies, Arch. Biochem. Biophys. 3511,249 (1998). 29A. Lenz, U. Costabel, S. Shaltiel, and R. L. Levine, Anal, Biochem. 177~419 (1989). 30R. L. Levine, D. Garland, C. N. Oliver, A. Amici, I. Climent, A. Lenz, B. Ahn, S. Shaltiel, and E. R. Stadtman, Methods Enzymol. 186, 464 (1990).
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TABLE II METHODSFORDETECTIONOF OXIDATIVEPROTEINMODIFICATIONS a Modification Disulfides Thiyl radicals Glutathiolation Methionine sulfoxide Carbonyls
2-oxo-His Dityrosine Chlorotyrosine Nitrotyrosine Tryptophanyl Hydroperoxides Lipid peroxidation adducts Amino acid oxidation adducts Cross-links, aggregates, fragments
Methods of detection
References b
SDS-gel electrophoresis (_+2-ME); DTNB Electron spin resonance spectroscopy RP-HPLC/mass spectroscopy; IEF; S35-Cys/Chx/SDS-PAGE CNBr cleavage/amino acid analysis DNPH/Western blot/ELISA/ immunocytochemistry/HPLC/ A370; reduction with Naborotritide Amino acid analysis Fluorescence; proteolysis or hydrolysis/HPLC Hydrolysis/nitroso-napthol/HPLC; HBr hydrolysis-GC/MS Immunoassay; hydrolysis/HLPC; HPLC/electrochemical detection Fluoroscence spectroscopy; amino acid analysis (alk. hydrolysis) KI/Ig/NaBH4/hydrolysis/OPAHPLC NaBH4/hydrolysis/OPA-HPLC; hydrolysis-GC/MS; DNPH; immunoassays Reduction/hydrolysis/NMR SDS-gel electrophoresis; HPLC
41 9 19,42,43 44,45 This article and 30,35,36,46 29,47 6 7,48 14,15 49-53
11 17,18,54,55 20 12,56,57
"DTNB, dithiobisnitrobenzoate; IEF, isoelectric focusing; DNPHI, dinitrophenylhydrazine; CNBr, cyanogen bromide; Chx, cyclohexamide; OPA, o-pthaldialdehyde. b References for some methods currently in use. Other methods are also available.
W e s t e r n Blot I m m u n o a s s a y for P r o t e i n C a r b o n y l s Several years ago, we described a new m e t h o d for detection of p r o t e i n carbonyls. 31 T h e m e t h o d takes advantage of the fact that D N P groups are i m m u n o g e n i c and that a n t i - D N P antibodies are available commercially. By derivatizing proteins with D N P H in an s o d i u m dodecyl sulfate (SDS) buffer, oxidized protein can be detected by p e r f o r m i n g S D S - p o l y a c r y l a m i d e gel electrophoresis ( S D S - P A G E ) , electroblotting to nitrocellulose (Western blotting), and assaying with a n t i - D N P antibodies (immunoassay). 3aE. Shacter, J. A. Williams, M. Eim, and R. L. Levine, Free Radic. Biol. Med. 17, 429 (1994).
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This method has significant advantages in that it requires very little protein (as little as 50 ng of a 50-kDa protein oxidized to the extent of 0.3-0.5 mol carbonyl/mol protein) and it identifies which proteins in a complex mixture are oxidized and which are not. 31 The sensitivity of the method for detecting minor oxidized proteins in a cell can be enhanced greatly by preparing subcellular fractions prior to derivatization and performance of the Western blot immunoassay. 32'33 The carbohydrate groups in glycoproteins have no apparent effect in the assay and are not themselves readily oxidizable by metal-catalyzed systems. 34 Aldehydes introduced through the adduction of lipid peroxidation breakdown products (e.g., 4hydroxynonenal, MDA, acrolein) can react with DNPH and thus can be detected by this method. These adducts are somewhat unstable and the extent to which they actually contribute to protein-associated carbonyl levels in tissue extracts remains to be established. Reagents required for running the Western blot immunoassay are all available from commercial sources and the only special equipment employed is an SDS-PAGE/electroblotting apparatus, available in most biochemistry laboratories. Due to the nature of Western blotting, the assay is only semiquantitative. This drawback can be overcome by running sizeexclusion HPLC instead of S D S - P A G E if there is enough sample available. 35 Alternatively, the quantitation of total DNPH-derivatized protein carbonyls can be achieved by use of an ELISA, as described by Buss et aL 36 The actual procedure for running the Western blot immunoassay for protein carbonyls is very straightforward and there are few "tricks" required to ensure its success. Detailed procedures have been described in previous p u b l i c a t i o n s . 31'34'35'37 Directions and some useful pointers follow. Derivitization Reagents DNPH/TFA stock solution: 20 mM DNPH in 20% (v/v) trifluoroacetic acid (TFA). Dissolve the DNPH in 100% TFA and then dilute with 32 L. J. Yan, R. L. Levine, and R. S. Sohal, Proc. Natl. Acad. Sci. U.S.A. 94, 11168 (1997). 33 L. J. Yan and R. S. Sohal, Proc. Natl. Acad. Sci. U.S.A. 95, 12896 (1998). 34 y_j. Lee and E. Shacter, Arch. Biochem. Biophys. 321, 175 (1995). 35 R. L. Levine, J. Williams, E. R. Stadtman, and E. Shacter, Methods Enzymol. 233, 346 (1994). 36n . BUSS,T. P. Chan, K. B. Sluis, N. M. Domigan, and C. C. Winterbourn, Free Radic. Biol. Med. 23, 361 (1997). 37 E. Shacter, J. A. Williams, E. R. Stadtman, and R. L. Levine, in "Free Radicals: A Practical Approach" (N. Punchard and F. Kelly, eds.). Oxford Univ. Press, Oxford, 1994.
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water. Note that most commercial sources of DNPH contain roughly 30% H20. For a 10-ml stock DNPH/TFA solution, dissolve 40 mg of dry DNPH (52 mg of the labeled "wet" powder) in 1 ml of 100% TFA and then dilute with 4 ml water. 20% TFA (v/v) (reagent control) 24% SDS in H20 (24 g/100 ml) 2 M Tris base containing 30% glycerol 2-mercaptoethanol (2-ME), undiluted stock (14.4 M)
Samples Any protein solution may be used, including purified proteins and cell and tissue extracts. Extracts containing SDS are most suitable as the derivitization reaction and gels contain SDS buffer.
Reaction with Dinitrophenylhydrazine Derivitization is carried out in a solution containing 6% SDS/5 mM DNPH/5% TFA. The concentration of protein may be as high as 10 mg/ ml; use a concentration that will be convenient for gel loading (e.g., to load 2-20/zl of sample per lane). Start the derivitization reaction by mixing 2 volumes of protein solution (e.g., 10 txl) with 1 volume of 24% SDS (e.g., 5 tzl). Then add 1 volume of DNPH/TFA stock solution (e.g., 5 txl). Incubate at room temperature for 5-30 min. Longer derivitization times lead to a nonspecific incorporation of DNP, which will interfere with correct interpretation of the results. Immediately following derivitization, neutralize the sample and prepare for final gel loading by adding 1/3 volume (e.g., 6.7 /~1) of Tris/glycerol +__2-ME. The sample should turn from yellow to orange on neutralization. To run reducing gels, add 1 volume of 2-ME to 4 volumes of the Tris/glycerol solution. In most cases, heating is not performed. Load and run the S D S - P A G E gels promptly using the conditions described by Laemmli. 38 The total time elapsed between initiation of the derivitization reaction and running the gels should not take more than about 45-60 min for 20 samples.
38 U. K. Laemmli, Nature London 227, 680 (1970).
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Electroblotting Western transfer is carried out according to standard procedures. 39,4° In our hands, the lowest nonspecific immunoassay backgrounds are achieved with PVDF paper (e.g., Immobilon-P from Millipore). Free DNPH present in the samples will not interfere with immunodetection and does not contribute a background. When nonreducing gels are run (i.e., no 2-ME is added to the samples), treat the gels with 2-ME prior to transfer. This reduction of the proteins facilitates immunodetection of DNP groups in proteins containing large numbers of disulfide bonds (e.g., albumin)? 1 After electrophoresis, soak the gel in 1% 2-ME in Tris/glycine transfer buffer for 5 rain at room temperature. Then, rinse the gel in transfer buffer for 5 min to remove excess 2-ME. Set up the transfer. 39 H. Towbin, T. Staehelin, and J. Gordon, Proc. Natl. Acad. Sci. U.S.A. 76, 4350 (1979). 4oj. M. Gershoni and G. E. Palade, Anal. Biochem. 131, 1 (1983). 4a R. Radi, K. M. Bush, T. P. Cosgrove, and B. A. Freeman, Arch. Biochem. Biophys. 286, 117 (1991). 4z D. A. Davis, K. Dorsey, P. T. Wingfield, S. J. Stahl, J. Kaufman, H. M. Fales, and R. L. Levine, Biochemistry 35, 2482 (1996). 43 j. A. Thomas, Y.-C. Chai, and C.-H. Jung, Methods Enzymol. 233, 385 (1994). 44 K. L. Maier, A. G. Lenz, I. Beck-speier, and U. Costabel, Methods Enzymol. 251, 455 (1995). 45 R. L. Levine, L. Mosoni, B. S. Berlett, and E. R. Stadtman, Proc. Natl. Acad. Sci. U.S.A. 93, 15036 (1996). 46 M. A. Smith, L. M. Sayre, V. E. Anderson, P. L. R. Harris, M. F. Beal, N. Kowall, and G. Perry, J. Histochem. Cytochem. 46, 731 (1998). 47 L. J. Yan and R. S. Sohal, Anal. Biochem. 265, 176 (1998). 48 C. Leewenburgh, J. E. Rasmussen, F. F. Hsu, D. M. Mueller, S. Pennathur, and J. W. Heinecke, J. Biol. Chem. 272, 3520 (1998). 49 M. A. Smith, P. L. R. Harris, L. M. Sayre, J. S. Beckman, and G. Perry, J. Neurosci. 17, 2653 (1997). 5o y. Z. Ye, M. Strong, Z, Q. Huang, and J. S. Beckman, Methods Enzymol. 269, 201 (1996). 51 H. Ischiropoulos, L. Zhu, J. Chen, M. Tsai, J. C. Martin, C. D. Smith, and J. S. Beckman, Arch. Biochem. Biophys. 298, 431 (1992). 52j. p. Eiserich, C. E. Cross, A. D. Jones, B. Halliwell, and A. Van der Vliet, J. Biol. Chem. 271, 19199 (1996). 53 M. K. Shigenaga, H. H. Lee, B. C. Blount, E. T. Shigeno, H. Yip, and B. N. Ames, Proc. Natl. Acad. Sci. U.S.A. 94, 3211 (1997). 54 M. E. Rosenfeld, J. C. Khoo, E. Miller, S. Parthasarathy, W. Palinski, and J. L. Witztum, J. Clin. Invest. 87, 90 (1991). 55 j. G. Kim, F. Sabbagh, N. Santanam, J. N. Wilcox, R. M. Medford, and S. Parthasarathy, Free Radic. Biol. Med. 23, 251 (1997). ~6K. J. A. Davies and M. E. Delsignore, J. Biol. Chem. 262, 9908 (1987). 57 R. E. Pacifici and K. J. A. Davies, Methods Enzymol. 186, 485 (1990).
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Immunoassay Reagents TBS: 20 mM Tris/500 mM NaC1 Tween TBS: TBS containing 0.05% (v/v) Tween 20 Tween TBSA: Tween TBS containing 1% bovine serum albumin (BSA) Primary antibody solution: We employ a monoclonal anti-DNP antibody (a mouse IgE) marketed by Sigma (St. Louis, MO). Use at a 1:1000 dilution in Tween/TBSA. Other anti-DNP antibodies are also available commercially. These need to be tested to find the best dilution for obtaining high specificy with low backgrounds. Secondary antibody solution: biotinylated rat antimouse IgE from Southern Biotechnology Assoc. (Birmingham, AL). Use at at a 1 : 4000 dilution in Tween TBSA.
Immunoassay Procedure Block the blot for 30 rain using either 3% BSA in TBS or 5% milk. Incubation with the primary antibody is generally overnight at 4 ° or for at least 3 hr at room temperature. After treating with the anti-DNP antibody, wash the blot well with Tween/TBS. Incubate with the secondary antibody for 1 hr at room temperature. Final detection is carried out using a biotinavidin-peroxidase-conjugated system from Vector Laboratories and a chemiluminescence reagent (e.g., from NEN/Dupont or Amersham). Note that cell and tissue extracts often contain biotin-binding proteins that will react with biotin-avidin reagents. These proteins are detected by running the immunoassay in the absence of the primary (anti-DNP) antibody and can serve as convenient internal markers. If their presence interferes with the carbonyl assay, use a secondary antibody that is conjugated directly to peroxidase.
Staining of Total Protein Bands After completion of the immunoassay, protein bands can be visualized by washing the blots extensively (>1 day with several buffer changes) in TBS and then staining with Amido black (0.1% in 40% methanol/10% acetic acid). Destain with 40% methanol/10% acetic acid. By staining the same blot as used for the immunoassay, bands detected by staining and by immunoassay can be lined up accurately. There is no need to run replicate gels in order to visualize protein bands.
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Controls Two negative controls should be run when new samples are tested. Control for the Anti-DNP Reaction. This is accomplished by "derivatizing" with TFA without DNPH and performing the remainder of the protocol as usual. Samples treated in this manner should show no bands in the immunoassay. If any protein bands are seen, determine by process of elimination whether they derive from reactivity with the primary antibody, secondary antibody, or the biotin-avidin detection system. Control for Specificity of the Secondary Antibody. In our experience, most problems with "nonspecific" background derive from problems with the secondary antibody. It is a good idea to test this periodically by running the immunoassay without the primary antibody step. In addition, positive and negative control samples should be run in all gels. Any oxidatively modified protein and its native (or sodium borohydride-reduced) counterpart can be employed. We routinely use fibrinogen that has been oxidized by exposure either to iron/ascorbate or to ionizing radiation. 27 Protocols for preparing oxidatively modified proteins can be found in the literature. 5'27'31 Use a positive control of known carbonyl content (i.e., nmol/mg protein) to obtain an estimate of the extent of oxidation of positive protein bands. Use the negative control to gauge how long to do the chemiluminescence exposure (i.e., this band should be negative at all exposure times).
[39] D N A D a m a g e I n d u c e d b y U l t r a v i o l e t a n d V i s i b l e L i g h t a n d Its W a v e l e n g t h D e p e n d e n c e
By
CHRISTOPHER
KIELBASSA and
BERND
EPE
Introduction DNA damage induced by solar radiation in mammalian cells consists largely of two types of modification: pyrimidine dimers and oxidative modifications. 1 Pyrimidine dimers, which can be subdivided into cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts, are the characteristic and most abundant modifications after direct excitation of DNA, although they can also be formed indirectly by energy transfer from other excited 1 j, Cadet, M. Berger, T. Douki, B. Morin, S. Raoul, J.-L. R a v a n a t , a n d S. Spinelli, Biol. Chem. 378~ 1275 (1997).
METHODS IN ENZYMOLOGY, VOL. 319
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