Histone carbonylation in vivo and in vitro
Histone carbonylation in vivo and in vitro
Histone carbonylation in vivo and in vitro
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Biochem. J. (2000) 351, 769–777 (Pr<strong>in</strong>ted <strong>in</strong> Great Brita<strong>in</strong>) 769<strong>Histone</strong> <strong>carbonylation</strong> <strong>in</strong> <strong>vivo</strong> <strong>and</strong> <strong>in</strong> <strong>vitro</strong>Georg T. WONDRAK*, Daniel CERVANTES-LAUREAN†, Ela<strong>in</strong>e L. JACOBSON†‡§ <strong>and</strong> Myron K. JACOBSON*‡§*College of Pharmacy, University of Kentucky, Lex<strong>in</strong>gton, KY 40506-0286, U.S.A., †Department of Cl<strong>in</strong>ical Sciences, University of Kentucky, Lex<strong>in</strong>gton, KY 40506-0286,U.S.A., ‡Lucille P. Markey Cancer Center, University of Kentucky, Lex<strong>in</strong>gton, KY 40506-0286, U.S.A., <strong>and</strong> §Advanced Science <strong>and</strong> Technology Commercialization Center,University of Kentucky, Lex<strong>in</strong>gton, KY 40506-0286, U.S.A.Non-enzymic damage to nuclear prote<strong>in</strong>s has potentially severeconsequences for the ma<strong>in</strong>tenance of genomic <strong>in</strong>tegrity. Introductionof carbonyl groups <strong>in</strong>to histones <strong>in</strong> io <strong>and</strong> <strong>in</strong> itrowas assessed by Western blot immunoassay <strong>and</strong> reductive<strong>in</strong>corporation of tritium from radiolabelled NaBH (sodiumborohydride). <strong>Histone</strong> H1 extracted from bov<strong>in</strong>e thymus, liver<strong>and</strong> spleen was found to conta<strong>in</strong> significantly elevated amountsof prote<strong>in</strong>-bound carbonyl groups as compared with core histones.The carbonyl content of nuclear prote<strong>in</strong>s of rat pheochromocytomacells (PC12 cells) was not greatly <strong>in</strong>creasedfollow<strong>in</strong>g oxidative stress <strong>in</strong>duced by H O , but was significantly<strong>in</strong>creased follow<strong>in</strong>g alkylat<strong>in</strong>g stress <strong>in</strong>duced by N-methyl-N-nitro-N-nitrosoguanid<strong>in</strong>e or by comb<strong>in</strong>ed oxidative <strong>and</strong> alkylat<strong>in</strong>gstress. Free ADP-ribose, a reduc<strong>in</strong>g sugar generated <strong>in</strong> thenucleus <strong>in</strong> proportion to DNA str<strong>and</strong> breaks, was shown to bea potent histone H1 carbonylat<strong>in</strong>g agent <strong>in</strong> isolated PC12 cellnuclei. Studies of the mechanism of histone H1 modification byADP-ribose <strong>in</strong>dicate that <strong>carbonylation</strong> <strong>in</strong>volves formation of astable acyclic ketoam<strong>in</strong>e. Our results demonstrate preferentialhistone H1 <strong>carbonylation</strong> <strong>in</strong> io, with potentially importantconsequences for chromat<strong>in</strong> structure <strong>and</strong> function.Key words: ADP-ribose, glycation, ketoam<strong>in</strong>e, alkylat<strong>in</strong>g stress,PC12.INTRODUCTIONNon-enzymic damage to prote<strong>in</strong>s contributes to biochemicalchanges characteristic of biological ag<strong>in</strong>g <strong>and</strong> many pathologicalconditions, <strong>in</strong>clud<strong>in</strong>g diabetes, atherosclerosis <strong>and</strong> Alzheimer’sdisease [1–3]. A number of recent studies have focused on nuclearprote<strong>in</strong>s as targets for non-enzymic modifications, which couldhave important implications for the ma<strong>in</strong>tenance of genomic<strong>in</strong>tegrity. For example, age-dependent <strong>in</strong>creases <strong>in</strong> deamidationof histone H1o occur <strong>in</strong> io [4] <strong>and</strong> exposure of histone H1 toioniz<strong>in</strong>g radiation generates prote<strong>in</strong>-bound hydroperoxides thatcan give rise to free radicals, which damage DNA [5].Prote<strong>in</strong> <strong>carbonylation</strong> represents non-enzymic damage thatcan result by a number of different pathways. Prote<strong>in</strong>-boundcarbonyl groups are generated by direct oxidation of am<strong>in</strong>o acidresidues, such as lys<strong>in</strong>e <strong>and</strong> arg<strong>in</strong><strong>in</strong>e, by reaction with aldehydes,such as 4-hydroxy-2-nonenal <strong>and</strong> malondialdehyde, produced bylipid peroxidation, <strong>and</strong> as a result of reaction with reduc<strong>in</strong>gsugars or their oxidation products [3,6,7]. The reaction withreduc<strong>in</strong>g sugars can generate prote<strong>in</strong> carbonyls as ketoam<strong>in</strong>esderived from early glycation reactions <strong>and</strong> by more complexglycoxidation reactions that lead to the generation of fluorescentadvanced glycation end products (AGEs) <strong>and</strong> prote<strong>in</strong> cross-l<strong>in</strong>ks[3,8,9]. The presence of nuclear prote<strong>in</strong> carbonyls has beendetected <strong>in</strong> io [10], but specific prote<strong>in</strong> targets have not yetbeen identified. The detection of a nuclear proteasome thatdegrades histones oxidized <strong>in</strong> itro [11] also suggests that nuclearprote<strong>in</strong> <strong>carbonylation</strong> probably has physiological relevance. Thepossibility that specific nuclear prote<strong>in</strong>s may be targets forprote<strong>in</strong> <strong>carbonylation</strong> by glycation is supported by reports ofaccumulation of AGEs on liver histones of streptozotoc<strong>in</strong><strong>in</strong>duceddiabetic rats [12] <strong>and</strong> glycation of high mobility group(HMG) prote<strong>in</strong>s <strong>in</strong> calf thymus [13].To further elucidate the biological significance of <strong>in</strong>tranuclearprote<strong>in</strong> <strong>carbonylation</strong>, we studied histone <strong>carbonylation</strong> <strong>in</strong> io<strong>and</strong> <strong>in</strong> itro. In the present study we report the identification ofhistone H1 as a preferential <strong>carbonylation</strong> target <strong>in</strong> io. Wedemonstrate the accumulation of carbonylated histones <strong>in</strong> ratpheochromocytoma cells (PC12 cells) follow<strong>in</strong>g alkylat<strong>in</strong>g stress.F<strong>in</strong>ally, we describe one possible mechanism of histone <strong>carbonylation</strong>us<strong>in</strong>g PC12 nuclei <strong>and</strong> isolated histone H1 as glycationtargets.MATERIALS AND METHODSChemicalsNaBH (radiolabelled sodium borohydride) was obta<strong>in</strong>ed fromNEN Life Science Products (Boston, MA, U.S.A.). All otherchemicals were purchased from Sigma Chemical Co. Calf tissues(thymus, liver <strong>and</strong> spleen), frozen <strong>in</strong> liquid nitrogen immediatelyafter collection, were from Pel-Freez Biologicals (Rogers, AR,U.S.A.).Preparation of AGE-BSAAGE-BSA was prepared as described by Takata et al. [14].Briefly, 1.6 g of BSA was dissolved with 3 g of D-glucose <strong>in</strong> 10 mlof 0.5 M sodium phosphate buffer (pH 7.4), conta<strong>in</strong><strong>in</strong>g 0.05%NaN . The solution was filter-sterilized through a 0.45 µm filter<strong>and</strong> <strong>in</strong>cubated <strong>in</strong> the dark for 90 days at 37 C. Follow<strong>in</strong>gdialysis aga<strong>in</strong>st water, the sample was freeze-dried.Large-scale isolation of histone H1 from calf thymusAll operations were carried out at 4 C. Chromat<strong>in</strong> was isolatedfrom calf thymus by extraction with 0.14 M NaCl0.05 MNa S O (pH 5.0), as described by Johns [15]. After repeated extraction with 5% (wv) HClO <strong>and</strong> centrifugation at 1500 gfor 30 m<strong>in</strong>, histone H1 was precipitated by addition of trichloroaceticacid (TCA) to a f<strong>in</strong>al concentration of 20% (wv).Abbreviations used: AGEs, advanced glycation end products; DNPH, 2,4-d<strong>in</strong>itrophenylhydraz<strong>in</strong>e; DTPA, diethylenetriam<strong>in</strong>epentaacetic acid; HMG,high mobility group; MALDI–FT-MS, matrix-assisted laser-desorption ionization–Fourier-transformed-MS; MNNG, N-methyl-N-nitro-N-nitrosoguanid<strong>in</strong>e;PC12 cells, rat pheochromocytoma cells; RP, reverse-phased; TCA, trichloroacetic acid.1 To whom correspondence should be addressed (e-mail mjacob1pop.uky.edu). 2000 Biochemical Society
770 G. T. Wondrak <strong>and</strong> othersThe histone H1 precipitate was collected by centrifugation at12000 g for 30 m<strong>in</strong> <strong>and</strong> resuspended <strong>in</strong> water. After dialysis(molecular-mass cut-off, 12000–14000 Da) aga<strong>in</strong>st water for48 h the sample was freeze-dried <strong>and</strong> stored at 4 C. SDSPAGE(12% polyacrylamide) was used to analyse the purity of thepreparation.Isolation of histone H1, core histones <strong>and</strong> total histones from calftissuesFrozen bov<strong>in</strong>e tissue (3 g) was ground on ice us<strong>in</strong>g a mortar <strong>and</strong>a pestle. First, <strong>in</strong>tact nuclei were prepared accord<strong>in</strong>g to a st<strong>and</strong>ardprocedure [16]. The ground tissue was homogenized <strong>in</strong> 20 ml ofice-cold 10 mM TrisHCl (pH 7.4), 10 mM NaCl, 3 mM MgCl , 0.5% Nonidet P40 <strong>and</strong> 1 mM PMSF (solution A) us<strong>in</strong>g aDounce homogenizer. Cell breakage was monitored by microscopyafter sta<strong>in</strong><strong>in</strong>g the sample with 0.5% Trypan Blue. Analiquot (5 ml) of the cell lysate was layered on to a 5 ml cushionof solution B [30% (wv) sucrose <strong>in</strong> solution A] <strong>and</strong> nuclei werepelleted by centrifugation at 2000 g for 5 m<strong>in</strong> at 4 C. For thepreparation of histone H1, the nuclear pellet was first extractedtwo times with three volumes of 5% (wv) HClO for 30 m<strong>in</strong> onice with occasional vortex-mix<strong>in</strong>g. HClO -<strong>in</strong>soluble material wassubsequently removed by centrifugation at 15000 g for 15 m<strong>in</strong>,<strong>and</strong> soluble prote<strong>in</strong>s were precipitated by add<strong>in</strong>g ice-cold TCA toa f<strong>in</strong>al concentration of 20% (wv). After 10 m<strong>in</strong> on ice, theprecipitated prote<strong>in</strong> was recovered by centrifugation at 15000 gfor 15 m<strong>in</strong> <strong>and</strong> the pellet was washed with diethyl ether. Theprote<strong>in</strong> was dissolved <strong>in</strong> water <strong>and</strong> was further purified bymicrodialysis aga<strong>in</strong>st water at 4 C. To prepare core histones, theHClO -<strong>in</strong>soluble material was extracted two times with threevolumes of 0.2 M H SO for 10 m<strong>in</strong> on ice, <strong>and</strong> further processed as <strong>in</strong>dicated above. For preparation of total histones, the nuclearpellet was directly extracted with 0.2 M H SO . For comparative purposes, histone H1 was also prepared byextract<strong>in</strong>g the nuclear pellet with 0.5 M NaCl, <strong>and</strong> core histoneswere prepared by subsequent extraction of the residual nuclearpellet with 2 M NaCl, as described previously [17]. Additionally,total histones were extracted with one volume of 12% (wv) SDSafter suspend<strong>in</strong>g the nuclear pellet <strong>in</strong> a stabiliz<strong>in</strong>g buffer conta<strong>in</strong><strong>in</strong>g5 mM TrisHCl (pH 7.4), 2.5 mM KCl <strong>and</strong> 5 mM MgCl (TKM buffer), <strong>and</strong> <strong>in</strong>cubat<strong>in</strong>g with 0.1 unitµl DNase I <strong>and</strong>25 µgml RNase A for 5 m<strong>in</strong> at 37 C.Pretreatment <strong>and</strong> isolation of <strong>in</strong>tact nuclei from PC12 cellsPC12 cells were grown, <strong>in</strong> Dulbecco’s modified Eagle’s mediumconta<strong>in</strong><strong>in</strong>g 5% (vv) fetal calf serum <strong>and</strong> 10% (vv) horseserum, to high density <strong>in</strong> T-75 flasks. After treatment with H O or N-methyl-N-nitro-N-nitrosoguanid<strong>in</strong>e (MNNG) as <strong>in</strong>dicated<strong>in</strong> Figure 3, the cells were removed from the flasks by tryps<strong>in</strong>treatment, <strong>and</strong> collected by centrifugation. Intact nuclei wereprepared accord<strong>in</strong>g to a st<strong>and</strong>ard procedure [16]. The nuclearpellet was suspended <strong>in</strong> TKM buffer <strong>and</strong> kept on ice.Incubation of <strong>in</strong>tact nuclei with ADP-ribose or D-glucose, <strong>and</strong>extraction of total histonesIntact nuclei (20 µl) were resuspended <strong>in</strong> 200 µl of TKM bufferconta<strong>in</strong><strong>in</strong>g 2 mM ADP-ribose or 2 mM D-glucose, <strong>and</strong> <strong>in</strong>cubatedfor 2 h at 37 C. After centrifugation, the nuclear pellet waswashed with TKM buffer <strong>and</strong> extracted with 300 µl of 0.2 MH SO for 10 m<strong>in</strong> on ice. After centrifugation at 15000 g for 15 m<strong>in</strong>, 75 µl of ice-cold 100% (wv) TCA was added to thesupernatant. After 10 m<strong>in</strong> on ice, the precipitated prote<strong>in</strong> wasrecovered by centrifugation at 15000 g for 15 m<strong>in</strong> <strong>and</strong> the pelletwas washed with diethyl ether. The histones were re-dissolved<strong>in</strong> water <strong>and</strong> immediately <strong>in</strong>cubated with 2,4-d<strong>in</strong>itrophenylhydraz<strong>in</strong>e(DNPH).Oxidative modification of histone H1Oxidative modification of histone H1 for <strong>carbonylation</strong> analysiswas performed accord<strong>in</strong>g to Chao et al. with slight modifications[18]. <strong>Histone</strong> H1 (1 mgml) was <strong>in</strong>cubated <strong>in</strong> physiological PBSconta<strong>in</strong><strong>in</strong>g 100 µM FeSO , 25 mM ascorbate <strong>and</strong> 25 mM H O at 37 C for 4 h The reagents were removed by microdialysis(MEGA System Microdialyzer; molecular-mass cut-off, 12000–14000 Da; Health Products, Rockford, IL, U.S.A.) <strong>and</strong> analiquot of the prote<strong>in</strong> solution was used for further analysis.Glycation of histone H1 or BSA by ADP-ribose at physiological pHIncubations of 1.0 ml conta<strong>in</strong>ed 50 mM potassium phosphate(pH 7.4), 1.0 mM ADP-ribose, 1.5 mg of histone H1 or BSA,<strong>and</strong> 0.015% NaN . Incubations were carried out at 37 C <strong>in</strong> thedark. Incubations at pH 9.0 were performed as reported previously[19]. When present, transition metal ion chelators EDTA,diethylenetriam<strong>in</strong>epentaacetic acid (DTPA), <strong>and</strong> other reagentswere added from concentrated solutions <strong>in</strong> phosphate buffer.Oxygen was excluded from the reaction mixtures by bubbl<strong>in</strong>gwith argon gas for 5 m<strong>in</strong> <strong>and</strong> then seal<strong>in</strong>g reaction vessels undera stream of argon. Reaction aliquots of 100 µl were dialysedaga<strong>in</strong>st distilled water us<strong>in</strong>g a microdialysis system.Immunodetection of prote<strong>in</strong>-bound carbonyl groupsDerivatization of prote<strong>in</strong>-bound carbonyl groups with DNPH<strong>and</strong> immunodetection were performed accord<strong>in</strong>g to the procedureof Shacter et al. [20]. Briefly, 5 µl of12% SDS was added to a5 µl aliquot of sample conta<strong>in</strong><strong>in</strong>g 5 µg of prote<strong>in</strong>. After additionof 10 µl of 10 mM DNPH <strong>in</strong> 10% (vv) trifluoroacetic acid, themixture was <strong>in</strong>cubated for 20 m<strong>in</strong> at room temperature. Thereaction mixture was neutralized <strong>and</strong> prepared for SDSPAGEby add<strong>in</strong>g 7.5 µl of 2 M Tris base conta<strong>in</strong><strong>in</strong>g 30% (vv) glycerol.Prote<strong>in</strong>s were separated by SDSPAGE <strong>and</strong> transferred on to anitrocellulose membrane us<strong>in</strong>g a semi-dry system. A second gelconta<strong>in</strong><strong>in</strong>g duplicate samples was run <strong>and</strong> sta<strong>in</strong>ed with CoomassieBlue. Additionally, equal transfer was checked by reversibleprote<strong>in</strong> sta<strong>in</strong><strong>in</strong>g of the nitrocellulose membrane with Ponceau S.The anti-DNP antibody (V401) was supplied by DAKO (Carp<strong>in</strong>teria,CA, U.S.A.) <strong>and</strong> used at 1:1000 dilution. The secondaryantibody was goat anti-rabbit conjugated with horseradishperoxidase (Jackson Immunoresearch Laboratories, Bar Harbor,ME, U.S.A.) used at 1:20000 dilution. Immunoblots werevisualized us<strong>in</strong>g ECL detection reagents supplied by Amersham,with exposure times between 5 s <strong>and</strong> 3 m<strong>in</strong>.Immunodetection of histone H1<strong>Histone</strong> H1 was identified on the same nitrocellulose membranespreviously used for immunodetection of prote<strong>in</strong>-bound carbonylgroups. Anti-(histone H1) serum from sheep was supplied byMa<strong>in</strong>e Biotechnology Services (Portl<strong>and</strong>, ME, U.S.A.), <strong>and</strong> wasused at 1:200 dilution. The secondary antibody was donkey antisheepIgG conjugated with horseradish peroxidase from Sigma,<strong>and</strong> was used at 1:5000 dilution. Immunoblots were visualizedus<strong>in</strong>g ECL detection reagents. 2000 Biochemical Society
<strong>Histone</strong> H1 <strong>carbonylation</strong> <strong>in</strong> <strong>vivo</strong> <strong>and</strong> <strong>in</strong> <strong>vitro</strong>771Reductive tritium <strong>in</strong>corporation <strong>in</strong>to glycated <strong>and</strong> oxidizedprote<strong>in</strong>sProte<strong>in</strong>-bound carbonyl groups were reduced <strong>and</strong> quantified bythe method of Lenz et al. [21]. All determ<strong>in</strong>ations were performed<strong>in</strong> triplicate. Briefly, an aliquot conta<strong>in</strong><strong>in</strong>g 200–600 µg of prote<strong>in</strong>was dissolved <strong>in</strong> 50 µl of water followed by addition of 6 µl of1 M TrisHCl (pH 8.5)10 mM EDTA <strong>and</strong> 14 µL of NaBH <strong>in</strong> 0.1 M NaOH (specific activity, 100 mCimmol). After <strong>in</strong>cubationat 37 C for 30 m<strong>in</strong>, 330 µl of water was added, the sample wascooled on ice, <strong>and</strong> 100 µl of ice-cold 100% TCA was added.After 10 m<strong>in</strong> on ice, the sample was centrifuged at 11000 g for15 m<strong>in</strong> <strong>and</strong> the precipitate was washed with ice-cold 20% TCA<strong>and</strong> diethyl ether. The pellet was re-dissolved <strong>in</strong> 10–20 µlof12%SDS <strong>and</strong> diluted with water to a f<strong>in</strong>al concentration of less than1% SDS. After centrifugation at 11000 g for 15 m<strong>in</strong>, prote<strong>in</strong> wasquantified us<strong>in</strong>g the bic<strong>in</strong>chon<strong>in</strong>ic acid-reaction kit from Pierce(Rockford, IL, U.S.A.). Sc<strong>in</strong>tillation cocktail (5 ml of EcoLumefrom ICN; Costa Mesa, CA, U.S.A.) was added <strong>and</strong> radioactivitywas determ<strong>in</strong>ed by liquid sc<strong>in</strong>tillation count<strong>in</strong>g on a Beckman LS6500 counter.Preparation <strong>and</strong> derivatization of N α -acetyl-N ε -(1-deoxy-ADPribulos-1-yl)-L-lys<strong>in</strong>eA 100 µl reaction mixture conta<strong>in</strong><strong>in</strong>g 50 mM potassium phosphate(pH 7.4), 10 mM ADP-ribose <strong>and</strong> 1.0 M N α -acetyl-L-lys<strong>in</strong>e,was <strong>in</strong>cubated for 24 h at room temperature, <strong>and</strong> samples wereanalysed periodically by reversed-phase (RP)-HPLC. The reducedketoam<strong>in</strong>e was prepared by addition of 6 µl of 1 M Tris(pH 8.5)10 mM EDTA <strong>and</strong> 14 µl of 200 mM NaBH <strong>in</strong> 0.1 MNaOH to 50 µl of the above reaction mixture. After <strong>in</strong>cubationat 37 C for 30 m<strong>in</strong>, the mixture was acidified with HCl, mixed,<strong>and</strong> neutralized with NaOH. The oxime derivative of theketoam<strong>in</strong>e was prepared by <strong>in</strong>cubation of the ketoam<strong>in</strong>e with1 M hydroxylam<strong>in</strong>e for 1 h at 37 C. All reaction products werepurified by preparative RP-HPLC for analysis by NMR <strong>and</strong> MS.RP-HPLC used a µBondapak C column (3.9 mm300 mm)from Waters (Milford, MA, U.S.A.) with a gradient elution.Runn<strong>in</strong>g solution A was 50 mM ammonium formate (pH 5.0)<strong>and</strong> runn<strong>in</strong>g solution B was 100% methanol. The concentrationof methanol was 0% for 15 m<strong>in</strong>, 0–5% from 15–20 m<strong>in</strong>, <strong>and</strong> 5%from 20–40 m<strong>in</strong>. The eluate was monitored at 254 nm. MS wasperformed us<strong>in</strong>g IonSpec HiRes MALDI–Fourier-transformed-MS (MALDI–FT-MS). Spectra were recorded with high resolution<strong>in</strong> negative ion mode after dissolv<strong>in</strong>g samples <strong>in</strong> H Omethanol (1:1, vv). The matrix used was 2,5-dihydroxybenzoicacid.RESULTSMetal-catalysed oxidation <strong>and</strong> glycation of histone H1 results <strong>in</strong>the <strong>in</strong>troduction of prote<strong>in</strong> carbonyl groupsIt is known that oxidation <strong>and</strong> glycation lead to the formationof carbonyl groups on target prote<strong>in</strong>s [3,8,9]. Prote<strong>in</strong>s modified bycarbonyl groups due to oxidative modifications can be detectedby immunoassay follow<strong>in</strong>g derivatization with the carbonylspecificreagent DNPH [20]. We exam<strong>in</strong>ed the feasibility of us<strong>in</strong>gthis approach to detect prote<strong>in</strong> damage on histone H1 aftermetal-catalysed oxidation or glycation by ADP-ribose. ADPriboseis a reduc<strong>in</strong>g sugar generated <strong>in</strong> the nucleus <strong>in</strong> proportionto DNA str<strong>and</strong> breaks follow<strong>in</strong>g genotoxic stress [22–24]. Figure1 shows SDSPAGE analysis of histone H1 sta<strong>in</strong>ed by CoomassieBlue (Figure 1A) <strong>and</strong> immunosta<strong>in</strong>ed for prote<strong>in</strong>-bound DNPepitopes (Figure 1B), follow<strong>in</strong>g various treatments. BSA <strong>in</strong>cubatedfor 90 days with 1.66 M D-glucose (AGE-BSA) served asa reference st<strong>and</strong>ard for the <strong>in</strong>troduction of carbonyl groups byglycation [14]. The AGE-BSA readily displayed carbonyl-specificepitopes on both monomeric <strong>and</strong> cross-l<strong>in</strong>ked forms of theprote<strong>in</strong> (lane 2). As little as 10 ng of AGE-BSA was readilydetectable by carbonyl immunosta<strong>in</strong><strong>in</strong>g. As reference st<strong>and</strong>ardsfor the direct <strong>in</strong>troduction of carbonyl groups by oxidativemechanisms, histone H1 was <strong>in</strong>cubated with H O alone or <strong>in</strong> thepresence of ascorbate <strong>and</strong> FeSO to establish conditions forthe Fenton reaction. Incubation with 25 mM H O alone atpH 7.4 did not result <strong>in</strong> detectable carbonyl immunosta<strong>in</strong><strong>in</strong>g(lane 4), but <strong>in</strong>clusion of ascorbate <strong>and</strong> FeSO along with H O resulted <strong>in</strong> strong immunosta<strong>in</strong><strong>in</strong>g <strong>in</strong>dicat<strong>in</strong>g extensive prote<strong>in</strong><strong>carbonylation</strong> <strong>and</strong> prote<strong>in</strong> degradation (lane 5). Strong carbonylimmunosta<strong>in</strong><strong>in</strong>g of both the monomer <strong>and</strong> cross-l<strong>in</strong>ked forms ofhistone H1 (lane 6) was detected. This f<strong>in</strong>d<strong>in</strong>g is consistent withour previous observations that <strong>in</strong>cubation of histone H1 <strong>in</strong> thepresence of ADP-ribose at pH 9.0 results <strong>in</strong> extensive prote<strong>in</strong>cross-l<strong>in</strong>k<strong>in</strong>g [19]. The immunosta<strong>in</strong><strong>in</strong>g of this sample was dependentupon reaction with DNPH s<strong>in</strong>ce immunosta<strong>in</strong><strong>in</strong>g wascompletely elim<strong>in</strong>ated when DPNH was omitted (lane 7). Incubationof histone H1 for 7 days with 1 mM ADP-ribose at pH7.4 resulted <strong>in</strong> strong carbonyl immunosta<strong>in</strong><strong>in</strong>g (lane 9). Detectableimmunosta<strong>in</strong><strong>in</strong>g of the histone H1 monomer occurredafter <strong>in</strong>cubation at pH 7.4 for only 2 h (lane 8). Interest<strong>in</strong>gly,upon prolonged exposure of the film, untreated histone H1 (lane3) displayed detectable carbonyl immunosta<strong>in</strong><strong>in</strong>g, whereas equalamounts of untreated BSA (lane 1) or core histones did not(results not shown). This suggested that histone H1 may have asignificant background level of <strong>carbonylation</strong> <strong>in</strong> io.The results of Figure 1 <strong>in</strong>dicated that carbonyl-specificimmunosta<strong>in</strong><strong>in</strong>g would be useful for the characterization ofdamage to histone H1 by oxidation <strong>and</strong>or glycation. Wequantified the amount of prote<strong>in</strong>-bound carbonyls after oxidationor glycation with ADP-ribose by reductive tritium <strong>in</strong>corporation,from NaBH , <strong>in</strong>to the target prote<strong>in</strong> accord<strong>in</strong>g to a st<strong>and</strong>ardprocedure [21]. The results are shown <strong>in</strong> Table 1. Prote<strong>in</strong>s not<strong>in</strong>cubated or <strong>in</strong>cubated <strong>in</strong> the absence of D-glucose or ADPribose<strong>in</strong>corporated very low levels of radiolabel. However,untreated histone H1 displayed nearly 3-fold higher levels of<strong>in</strong>corporation than untreated BSA (nmol carbonylmg prote<strong>in</strong>)aga<strong>in</strong> <strong>in</strong>dicat<strong>in</strong>g the presence of a small but significant amount ofprote<strong>in</strong>-bound carbonyl groups on histone H1 <strong>in</strong> io. Prereductionof the histone H1 sample with NaBH reduced the<strong>in</strong>corporation of radiolabel on histone H1 by 4-fold <strong>in</strong>dicat<strong>in</strong>gthat <strong>in</strong>corporation of radiolabel was not due to non-specific<strong>in</strong>corporation (results not shown). Aga<strong>in</strong>, AGE-BSA served asreference st<strong>and</strong>ard <strong>and</strong> nearly 100 nmol of carbonyl groups permg of prote<strong>in</strong> were detected. Consistent with the results observedby carbonyl-immunosta<strong>in</strong><strong>in</strong>g (Figure 1) <strong>in</strong>cubation of histoneH1 with H O alone resulted <strong>in</strong> barely-detectable formation ofcarbonyl groups, while Fenton reaction conditions resulted <strong>in</strong>the formation of more than 80 nmol of carbonyl groups per mgof histone H1. Incubation of histone H1 with ADP-ribose at pH7.4 for 1 or 7 days resulted <strong>in</strong> the formation of 25.1 <strong>and</strong>38.7 nmol of carbonyl groups per mg of prote<strong>in</strong> respectively. Theamount of modification by ADP-ribose <strong>in</strong> 1 day at pH 7.4 wasapproximately 50% of that observed follow<strong>in</strong>g <strong>in</strong>cubation for1 day at alkal<strong>in</strong>e pH, which is known to accelerate the reaction[19]. On a molar basis, <strong>in</strong>cubation with ADP-ribose produced1 mol of carbonyl group per mol of histone H1 prote<strong>in</strong>, even atpH 7.4. The quantification reported <strong>in</strong> Table 1 is <strong>in</strong> goodagreement with the <strong>in</strong>tensity of the carbonyl immunosta<strong>in</strong><strong>in</strong>gshown <strong>in</strong> Figure 1, <strong>in</strong>dicat<strong>in</strong>g that the immunosta<strong>in</strong><strong>in</strong>g reflectsthe relative levels of prote<strong>in</strong> modification by carbonyl groups. 2000 Biochemical Society
772 G. T. Wondrak <strong>and</strong> othersFigure 1Carbonylation of BSA <strong>and</strong> histone H1: detection by carbonyl immunosta<strong>in</strong><strong>in</strong>gProte<strong>in</strong> samples [4 µg <strong>in</strong>(A) <strong>and</strong> 1 µg <strong>in</strong>(B)] were analysed by SDS/PAGE (12% polyacrylamide) with Coomassie sta<strong>in</strong><strong>in</strong>g (A) or after DNPH-derivatization <strong>and</strong> transfer on to nitrocellulose withcarbonyl immunosta<strong>in</strong><strong>in</strong>g (B). Lanes 1 <strong>and</strong> 2 conta<strong>in</strong>ed BSA <strong>in</strong>cubated for 90 days at pH 7.4 without sugar (lane 1) or <strong>in</strong> the presence of 1.66 M D-glucose (lane 2). Lanes 3–9 conta<strong>in</strong>ed histoneH1 <strong>in</strong>cubated for 1 day at pH 7.4 with no additions (lane 3), for 2 h at pH 7.4 with 25 mM H 2 O 2 (lane 4), or for 4 h at pH 7.4 with 25 mM H 2 O 2 , 25 mM ascorbate <strong>and</strong> 100 µM FeSO 4 (lane5). Lanes 6–9 conta<strong>in</strong>ed 1 mM ADP-ribose <strong>in</strong>cubated for 1 day at pH 9.0 (lane 6), as <strong>in</strong> lane 6 except that DNPH was omitted from the procedure (lane 7), at pH 7.4 for 2 h (lane 8) or at pH7.4 for 7 days (lane 9).Table 1 3H-<strong>in</strong>corporation <strong>in</strong>to BSA <strong>and</strong> histone H1 after reaction with various agents: quantification of prote<strong>in</strong>-bound carbonylsProte<strong>in</strong> samples (200–600 µg) were reacted with NaBT 4 <strong>and</strong> tritium <strong>in</strong>corporation was quantified as described <strong>in</strong> the Materials <strong>and</strong> methods section. Data are given as the meansS.D.(n 3). The metal-catalysed oxidation (MCO) system conta<strong>in</strong>ed 25 mM H 2 O 2 , 25 mM ascorbate <strong>and</strong> 100 µM FeSO 4 .Prote<strong>in</strong> bound carbonylsProte<strong>in</strong>Compound(concentration) Reaction time pH3 H-<strong>in</strong>corporation(c.p.m./µg of prote<strong>in</strong>)(nmol carbonyl/mg ofprote<strong>in</strong>)(mol ofcarbonyl/mol ofprote<strong>in</strong>)BSA None – – 10012 1.50.2 0.100.01BSA None 7 days 7.4 11110 1.70.2 0.110.01BSA Glucose (1.66 M) 90 days 7.4 66101040 10015.8 6.811.08H1 None – – 2547 3.90.1 0.080.00H1 None 7 days 7.4 25931 3.90.5 0.080.01H1 ADP-ribose (1 mM) 1 days 7.4 1910130 29.02.0 0.610.04H1 ADP-ribose (1 mM) 7 days 7.4 2810120 42.61.8 0.890.04H1 ADP-ribose (1 mM) 1 days 9.0 3410180 51.72.7 1.090.06H1 H 2 O 2 (25 mM) 2 h 7.4 30855 4.70.8 0.100.02H1 MCO 4 h 7.4 5600510 84.97.8 1.780.16Background levels of prote<strong>in</strong>-bound carbonyl groups on histoneH1 <strong>and</strong> other nuclear prote<strong>in</strong>s <strong>in</strong> mammalian tissuesOur detection of significant <strong>carbonylation</strong> of untreated histoneH1 led us to further <strong>in</strong>vestigate the carbonyl background levelson histone extracted from bov<strong>in</strong>e tissue. Extracted histones wereanalysed by SDSPAGE followed by Coomassie Blue sta<strong>in</strong><strong>in</strong>g<strong>and</strong> carbonyl immunosta<strong>in</strong><strong>in</strong>g (Figure 2). Carbonyl immunosta<strong>in</strong><strong>in</strong>gdemonstrated that the histone H1-fractions extractedfrom bov<strong>in</strong>e thymus, liver <strong>and</strong> spleen conta<strong>in</strong>ed significantlyelevated amounts of prote<strong>in</strong>-bound carbonyl groups comparedto the core histone-fractions (Figure 2B). This difference wasmost pronounced <strong>in</strong> the thymus, but was also apparent <strong>in</strong> theliver <strong>and</strong> the spleen. This pattern of histone <strong>carbonylation</strong> wasseen with several different batches of bov<strong>in</strong>e tissue. Consistentwith the patterns observed <strong>in</strong> the immunoblots, reductive tritium<strong>in</strong>corporation <strong>in</strong>to the histone H1-fraction from thymus was7-fold greater than the <strong>in</strong>corporation <strong>in</strong>to core histones(3.90 nmolmg of histone H1 compared with 0.56 nmolmg ofcore histone). As a control for artifactual <strong>in</strong>troduction of carbonylgroups dur<strong>in</strong>g preparation of histone H1, samples conta<strong>in</strong><strong>in</strong>gtotal histones were also exam<strong>in</strong>ed. In these samples only histoneH1 showed appreciable carbonyl immunosta<strong>in</strong><strong>in</strong>g. Interest<strong>in</strong>gly,<strong>in</strong> histone H1-fractions, two additional prote<strong>in</strong> b<strong>and</strong>s migrat<strong>in</strong>gwith apparent molecular-masses slightly below 29 kDa showedstrong carbonyl immunosta<strong>in</strong><strong>in</strong>g (as <strong>in</strong>dicated by the arrowheads<strong>in</strong> Figures 2A <strong>and</strong> 2B). Based on apparent molecular size <strong>and</strong>extraction characteristics, these prote<strong>in</strong>s are most probablyHMG12, members of the basic HMG nuclear prote<strong>in</strong>s [13].These prote<strong>in</strong>s are typically contam<strong>in</strong>ants <strong>in</strong> histone H1 preparationsobta<strong>in</strong>ed by perchloric acid- or NaCl-extraction ofnuclei [13]. To further exclude the artifactual <strong>in</strong>troduction ordestruction of prote<strong>in</strong> carbonyl groups dur<strong>in</strong>g histone preparation<strong>and</strong> analysis, several control experiments were performed(Figures 2C <strong>and</strong> 2D). Alternative methods of histone preparation,<strong>in</strong>volv<strong>in</strong>g extraction of total histone <strong>in</strong>to 12% SDS or sequentialsalt extraction of histone H1 <strong>and</strong> core histones, gave the sameresults for carbonyl immunosta<strong>in</strong><strong>in</strong>g as shown <strong>in</strong> Figure 2 (results 2000 Biochemical Society
<strong>Histone</strong> H1 <strong>carbonylation</strong> <strong>in</strong> <strong>vivo</strong> <strong>and</strong> <strong>in</strong> <strong>vitro</strong>773Figure 3stress<strong>Histone</strong> <strong>carbonylation</strong> <strong>in</strong> PC12 cells under oxidative <strong>and</strong> alkylat<strong>in</strong>gFigure 2Increased levels of <strong>carbonylation</strong> on histone H1 <strong>in</strong> bov<strong>in</strong>e tissuesTotal histone (H t ), histone H1 (H1), <strong>and</strong> core histones (H c ) were prepared from fresh nuclei frombov<strong>in</strong>e tissues (thymus, liver <strong>and</strong> spleen) as described <strong>in</strong> the Materials <strong>and</strong> methods section.Prote<strong>in</strong> samples [5 µg <strong>in</strong> (A) <strong>and</strong> 2 µg <strong>in</strong> (B)] were analysed by SDS/PAGE (15%polyacrylamide) with Coomassie sta<strong>in</strong><strong>in</strong>g (A) or after DNPH-derivatization <strong>and</strong> transfer on tonitrocellulose with carbonyl immunosta<strong>in</strong><strong>in</strong>g (B). As a control (C: Ponceau sta<strong>in</strong>; D: carbonylimmunosta<strong>in</strong><strong>in</strong>g), histone H1 fractions were processed as above, but <strong>in</strong> the presence (lane 2)or absence of DNPH (lane 1), <strong>and</strong> after pre-reduction with NaBH 4 (lane 3). The arrowheads<strong>in</strong>dicate the position of other nuclear prote<strong>in</strong>s with high carbonyl immunosta<strong>in</strong><strong>in</strong>g, presumablyHMG1/2.PC12 cells grown to high density <strong>in</strong> T-75 flasks were sham-treated (A–C, lane 1) or exposedto H 2 O 2 (2 mM) added to the medium for 1 h (A–C, lane 2). Another group of cells wasexposed to H 2 O 2 (2 mM), added to the medium for 1 h <strong>and</strong> MNNG (20 µg/ml) was added forthe last 20 m<strong>in</strong> (A–C, lane 3). In another set of experiments, PC12 cells were sham-treated(D–F, lane 1) or exposed to MNNG (20 µg/ml), added to the medium for 1 h (D–F, lane 2).After preparation of the nuclei, total histones were extracted with 0.2 M H 2 SO 4 . Prote<strong>in</strong> samples[8 µg <strong>in</strong>(A) <strong>and</strong> (D); 2 µg <strong>in</strong>(B), (C), (E) <strong>and</strong> (F)] were analysed by SDS/PAGE (15%polyacrylamide) with Coomassie sta<strong>in</strong><strong>in</strong>g (A <strong>and</strong> D) or after DNPH-derivatization transferred onto nitrocellulose with carbonyl immunosta<strong>in</strong><strong>in</strong>g (C <strong>and</strong> F) <strong>and</strong> subsequent immunosta<strong>in</strong><strong>in</strong>g forhistone H1 (B <strong>and</strong> E). As a control, a duplicate sample as <strong>in</strong> (A–C, lane 3) was processedwithout DNPH (A–C, lane 4).not shown). Omission of DNPH dur<strong>in</strong>g the derivatization step ofthe histone H1 fraction (Figure 2D, lane 1) or pre-reductionof this fraction with NaBH (Figure 2D, lane 3) elim<strong>in</strong>ated thecarbonyl immunosta<strong>in</strong><strong>in</strong>g displayed by the complete reactionmixture (Figure 2D, lane 2).Stress-<strong>in</strong>duced histone <strong>carbonylation</strong> <strong>in</strong> cultured mammalian cellsPC12 cells, a widely used model for the study of oxidativedamage <strong>in</strong> mammalian neuronal-like cells [25], were used to<strong>in</strong>vestigate histone <strong>carbonylation</strong> follow<strong>in</strong>g oxidative <strong>and</strong> alkylat<strong>in</strong>gstress (Figure 3). Exposure to H O (2 mM) was chosen as an oxidative stress [11], <strong>and</strong> exposure to the alkylat<strong>in</strong>g agentMNNG (20 µgml), known to <strong>in</strong>duce DNA str<strong>and</strong> breaks <strong>and</strong>rapid ADP-ribose polymer synthesis <strong>and</strong> turnover [22], waschosen as an alkylat<strong>in</strong>g stress. Follow<strong>in</strong>g treatment, nuclei wereisolated, histones were extracted <strong>and</strong> analysed by SDSPAGE,followed by Coomassie Blue sta<strong>in</strong><strong>in</strong>g, histone H1 immunosta<strong>in</strong><strong>in</strong>g<strong>and</strong> carbonyl immunosta<strong>in</strong><strong>in</strong>g. Samples from controlcells showed background carbonyl immunosta<strong>in</strong><strong>in</strong>g that wasespecially pronounced <strong>in</strong> the HMG12-region of the gel (Figure3C, lane 1 <strong>and</strong> Figure 3F, lane 1). Treatment with H O <strong>in</strong>duced Figure 4nucleiCarbonylation of histones by ADP-ribose <strong>in</strong> <strong>in</strong>tact mammalianIntact nuclei from PC12 cells (50 µl) were <strong>in</strong>cubated with 2 mM D-glucose or 2 mM ADPriboseat pH 7.4 <strong>in</strong> TKM buffer at 37 C for 2 h Total histones were extracted <strong>and</strong> equal aliquots(8 µg) were analysed by SDS/PAGE (15% polyacrylamide) with subsequent Coomassie sta<strong>in</strong><strong>in</strong>g(A). Another aliquot (2 µg) was reacted with DNPH <strong>and</strong>, follow<strong>in</strong>g SDS/PAGE, was transferredon to a nitrocellulose membrane <strong>and</strong> analysed by carbonyl immunosta<strong>in</strong><strong>in</strong>g (C) <strong>and</strong> subsequentimmunosta<strong>in</strong><strong>in</strong>g for histone H1 (B). Lane 1 shows an <strong>in</strong>cubation without added sugar, lane 2shows addition of D-glucose <strong>and</strong> lane 3 shows addition of ADP-ribose. The migration positionsof histone H1 (H1) <strong>and</strong> core histones (Hcore) are <strong>in</strong>dicated on the right.only a modest <strong>in</strong>crease of <strong>carbonylation</strong> of the upper histone H1b<strong>and</strong> (Figure 3C, lane 2), but a considerable decrease of<strong>carbonylation</strong> <strong>in</strong> the HMG12 region of the gel, most probably<strong>in</strong>dicat<strong>in</strong>g a rapid turnover of this material <strong>in</strong> response to 2000 Biochemical Society
774 G. T. Wondrak <strong>and</strong> othersFigure 5 Comparative glycation of histone H1 by different sugars <strong>and</strong>effects of antioxidant conditions <strong>and</strong> reagents on prote<strong>in</strong> cross-l<strong>in</strong>k<strong>in</strong>g <strong>and</strong><strong>carbonylation</strong>(A <strong>and</strong> B) Reaction mixtures conta<strong>in</strong><strong>in</strong>g histone H1 <strong>and</strong> 1 mM of different sugars were<strong>in</strong>cubated at pH 7.4. At day 7, reaction aliquots were analysed by SDS/PAGE (12%polyacrylamide) with subsequent silver sta<strong>in</strong><strong>in</strong>g (A) or carbonyl immunosta<strong>in</strong><strong>in</strong>g (B). Lane 1shows a control reaction with no added sugar <strong>and</strong> the sugars present <strong>in</strong> the other lanes wereD-glucose (lane 2), D-ribose (lane 3), ADP-glucose (lane 4), <strong>and</strong> ADP-ribose (lane 5). (C <strong>and</strong>D) Reaction mixtures conta<strong>in</strong><strong>in</strong>g histone H1, 1.0 mM ADP-ribose, <strong>and</strong> other additions were<strong>in</strong>cubated at pH 7.4. At day 7, aliquots were analysed by SDS/PAGE (12% polyacrylamide) withsubsequent silver sta<strong>in</strong><strong>in</strong>g (C) <strong>and</strong> carbonyl immunosta<strong>in</strong><strong>in</strong>g (D). Lane 1 shows a reactionwithout added ADP-ribose. Lanes 2–7 show reactions with ADP-ribose <strong>and</strong> no other additions(lane 2), addition of 5 mM DTPA under argon (lane 3), 5 mM am<strong>in</strong>oguanid<strong>in</strong>e (lane 4), 5 mMN α -acetyl-L-cyste<strong>in</strong>e (lane 5), 5 mM mannitol (lane 6), or 100 units/ml of catalase (lane 7).M <strong>and</strong> D refer to the migration position of histone H1 monomer <strong>and</strong> dimer respectively.oxidative stress. Treatment with MNNG resulted <strong>in</strong> less prote<strong>in</strong><strong>in</strong> the histone H1 b<strong>and</strong> (Figures 3D <strong>and</strong> 3E, lanes 2), but<strong>in</strong>creased carbonyl immunosta<strong>in</strong><strong>in</strong>g of histone H1 (Figure 3F,lane 2). Remarkably, Coomassie sta<strong>in</strong> (Figure 3D, lane 2) <strong>and</strong>carbonyl immunosta<strong>in</strong><strong>in</strong>g (Figure 3F, lane 2) revealed newprote<strong>in</strong> b<strong>and</strong>s migrat<strong>in</strong>g at slightly less than 68 kDa, a regionwhere histone H1 dimers would appear (Figure 1, lane 9).However, the histone H1 immunosta<strong>in</strong><strong>in</strong>g could not confirm theidentity of this heavily carbonylated prote<strong>in</strong> as histone H1-dimer(Figure 3E, lane 2). When H O was adm<strong>in</strong>istered <strong>in</strong> comb<strong>in</strong>ationwith MNNG, strong carbonyl immunosta<strong>in</strong><strong>in</strong>g of histone H1<strong>and</strong> core histones was observed (Figure 3C, lane 3). Controlsdemonstrat<strong>in</strong>g the requirement for DNPH-derivatization forimmunosta<strong>in</strong><strong>in</strong>g (Figure 3C, lane 4) <strong>and</strong> rul<strong>in</strong>g out non-specificprote<strong>in</strong> b<strong>in</strong>d<strong>in</strong>g dur<strong>in</strong>g immunosta<strong>in</strong><strong>in</strong>g (Figure 3C, lane 5) arealso shown.<strong>Histone</strong>s <strong>in</strong> isolated mammalian nuclei are rapidly carbonylatedby ADP-riboseThe modest effect of H O treatment, the strong effects of MNNG treatment, <strong>and</strong> the synergistic effect of oxidative <strong>and</strong>alkylat<strong>in</strong>g stress on nuclear prote<strong>in</strong> <strong>carbonylation</strong> <strong>in</strong> PC12 cellsFigure 6 Reaction of N α -acetyl-L-lys<strong>in</strong>e with ADP-ribose at pH 7.4A reaction conta<strong>in</strong><strong>in</strong>g 1 M N α -acetyl-L-lys<strong>in</strong>e was <strong>in</strong>cubated with 10 mM ADP-ribose <strong>in</strong> 50 mMpotassium phosphate buffer (pH 7.4). A reaction aliquot was analysed by RP-HPLC, with UVdetection at 254 nm at zero time (A) <strong>and</strong> after 24 h (B). At 24 h the reaction mixture wassubjected to reduction by NaBH 4 (C). The isolated reaction product P1 was <strong>in</strong>cubated withhydroxylam<strong>in</strong>e before RP-HPLC analysis (D). The positions of ADP-ribose (ADPR) <strong>and</strong> reactionproducts P1–P4 are <strong>in</strong>dicated.suggested that accumulation of histone carbonyl groups <strong>in</strong>volvesmechanisms <strong>in</strong> addition to direct oxidative <strong>in</strong>troduction ofcarbonyl groups <strong>in</strong>to nuclear prote<strong>in</strong>s. S<strong>in</strong>ce MNNG-<strong>in</strong>ducedDNA damage is known to result <strong>in</strong> the <strong>in</strong>tranuclear generationof the reduc<strong>in</strong>g sugar ADP-ribose [22,23], the possibility that thehistone <strong>carbonylation</strong> was derived from glycation <strong>and</strong>orglycoxidation reactions <strong>in</strong>itiated by ADP-ribose was exam<strong>in</strong>ed.Freshly prepared nuclei from PC12 cells were <strong>in</strong>cubated for 2 h<strong>in</strong> a nuclear stabilization buffer at pH 7.4 conta<strong>in</strong><strong>in</strong>g 2 mMADP-ribose or 2 mM D-glucose. Extracted prote<strong>in</strong>s were analysedby SDSPAGE followed by Coomassie Blue sta<strong>in</strong><strong>in</strong>g,histone H1 immunosta<strong>in</strong><strong>in</strong>g <strong>and</strong> carbonyl immunoblott<strong>in</strong>g (Figure4). The presence of ADP-ribose resulted <strong>in</strong> strong carbonylspecificimmunosta<strong>in</strong><strong>in</strong>g (Figure 4C, lane 3), while the presenceof D-glucose resulted <strong>in</strong> barely-detectable immunosta<strong>in</strong><strong>in</strong>g (Figure4C, lane 2) <strong>and</strong> histones from a control <strong>in</strong>cubation withoutadded sugars did not show detectable immunosta<strong>in</strong><strong>in</strong>g (Figure4C, lane 1) at the chosen exposure time of the film. When ADP- 2000 Biochemical Society
<strong>Histone</strong> H1 <strong>carbonylation</strong> <strong>in</strong> <strong>vivo</strong> <strong>and</strong> <strong>in</strong> <strong>vitro</strong>775Scheme 1The ADP-ribose derived ketoam<strong>in</strong>e <strong>and</strong> its epimeric reduction productsN α -acetyl-L-lys<strong>in</strong>e was reacted with ADP-ribose at pH 7.4, as described <strong>in</strong> the Materials <strong>and</strong> methods section. Treatment of the ketoam<strong>in</strong>e (product 1) with NaBH 4 yielded two epimers (products2 <strong>and</strong> 3).ribose was used, the majority of carbonyl immunoreactivity waspresent <strong>in</strong> the histone H1 region, especially <strong>in</strong> the upper b<strong>and</strong> asalready observed <strong>in</strong> <strong>in</strong>tact PC12 cells exposed to H O <strong>and</strong>MNNG (Figure 3). Immunoreactivity also appeared <strong>in</strong> theregion of the core histones with the exception of histone H4(lowest b<strong>and</strong> of core histones).Carbonylation of histone H1 by ADP-ribose is more potent thanby other sugars <strong>and</strong> does not <strong>in</strong>volve oxidative pathwaysS<strong>in</strong>ce ADP-ribose was much more potent than glucose <strong>in</strong> caus<strong>in</strong>ghistone <strong>carbonylation</strong> <strong>in</strong> isolated nuclei, its <strong>carbonylation</strong> reactivitywas compared with other reduc<strong>in</strong>g sugars <strong>in</strong> experiments<strong>in</strong> which sugars were <strong>in</strong>cubated at pH 7.4 with histone H1 for anextended period (1 week) due to the low <strong>carbonylation</strong> activity ofmost of the sugars tested. As seen <strong>in</strong> Figure 5 ADP-ribose causedextensive <strong>carbonylation</strong> (Figure 5B, lane 5), D-ribose caused alesser amount (Figure 5B, lane 3), <strong>and</strong> neither D-glucose (Figure5B, lane 2) nor ADP-glucose (Figure 5B, lane 4) caused significant<strong>carbonylation</strong>, even at the extended <strong>in</strong>cubation times used <strong>in</strong> thisexperiment. The amount of <strong>carbonylation</strong> caused by ADP-ribose(0.82 molemole of histone H1) <strong>and</strong> D-ribose (0.06 molemole ofhistone H1), determ<strong>in</strong>ed by <strong>in</strong>corporation of NaBH , correlatedwith the relative amounts of immunosta<strong>in</strong><strong>in</strong>g caused by thesesugars.To determ<strong>in</strong>e if the <strong>carbonylation</strong> of histone H1 by ADPribose<strong>in</strong>volved oxidative reaction pathways, several compoundsknown to <strong>in</strong>terfere with the glycoxidation of prote<strong>in</strong>s by D-glucose [26] were exam<strong>in</strong>ed for their effects on prote<strong>in</strong> <strong>carbonylation</strong>by ADP-ribose (Figures 5C <strong>and</strong> 5D). Differentreagents <strong>in</strong>clud<strong>in</strong>g the transition metal ion chelator DTPA <strong>in</strong> theabsence of oxygen (lane 3), the nucleophilic antioxidant N α -acetyl-L-cyste<strong>in</strong>e (lane 5), the hydroxyl radical scavenger mannitol(lane 6), <strong>and</strong> the presence of catalase for the removal oftraces of hydrogen peroxide (lane 7) did not <strong>in</strong>terfere withhistone <strong>carbonylation</strong> by ADP-ribose. Remarkably, only the dicarbonylscavenger <strong>and</strong> <strong>in</strong>hibitor of advanced glycation, am<strong>in</strong>oguanid<strong>in</strong>e[27], showed a slight <strong>in</strong>hibitory effect on <strong>carbonylation</strong>(Figure 5D, lane 4). In contrast, prote<strong>in</strong> cross-l<strong>in</strong>k<strong>in</strong>g was fullyblocked. These data strongly suggest that histone H1 <strong>carbonylation</strong>by ADP-ribose at pH 7.4 is not a result of glycoxidation,but rather early glycation.Structure elucidation of the carbonyl compound from earlyglycation by ADP-riboseA model system designed to allow structural elucidation wasdeveloped to further characterize the nature of the carbonylderivatives that rapidly accumulate on histone H1 dur<strong>in</strong>g reactionwith ADP-ribose at pH 7.4. N α -acetyl-L-lys<strong>in</strong>e, which imitatespeptide-bound lys<strong>in</strong>e as the most likely glycation target onhistone H1, was reacted with ADP-ribose at pH 7.4. Reactionproducts were characterized by RP-HPLC as shown <strong>in</strong> Figure 6.Incubation for 24 h resulted <strong>in</strong> conversion of more than 90% ofthe ADP-ribose <strong>in</strong>to material termed P1, which migrated primarilyas a s<strong>in</strong>gle peak at approximately 29 m<strong>in</strong> (Figures 6A<strong>and</strong> 6B). Although stable under the conditions of <strong>in</strong>cubation <strong>and</strong>HPLC, P1 was partially degraded upon lyophilization <strong>and</strong> it wastherefore derivatized for further analysis. Upon treatment withNaBH , P1 was quantitatively transformed <strong>in</strong>to a mixture of twoputative reduction products, termed P2 <strong>and</strong> P3, which migratedat approximately 26 <strong>and</strong> 27 m<strong>in</strong> respectively (Figure 6C). 2000 Biochemical Society
776 G. T. Wondrak <strong>and</strong> othersTreatment of P1 with hydroxylam<strong>in</strong>e yielded material (P4) thatmigrated as a s<strong>in</strong>gle peak at approximately 32 m<strong>in</strong> (Figure 6D).MALDI–FT-MS of P2 <strong>and</strong> P3 material revealed a s<strong>in</strong>gle [MH]−of 730.190 Da, compatible with P2 <strong>and</strong> P3 represent<strong>in</strong>g a mixtureof 2-epimeric N α -acetyl-N ε -(1-deoxy-ADP-pentit-1-yl)-Llys<strong>in</strong>e(C H N O P , calculated [MH]− 730.195 Da). Analysisof P4 revealed a s<strong>in</strong>gle [M-H]− of 743.200 Da, compatiblewith the oxime of a ketoam<strong>in</strong>e (C H N O P , calculated[MH]− 743.180 Da) derived from ADP-ribose <strong>and</strong> N α -acetyl-L-lys<strong>in</strong>e. The proposed oxime structure for P4 was furthervalidated by C-NMR analysis that revealed a C-signal (δ;153.5 ppm) <strong>in</strong> the expected range of an oxime carbon (145–165 ppm) [28]. In addition to this signal, the expected signals forthe five aden<strong>in</strong>e carbon atoms (δ; 121.5 ppm, 142.4 ppm, 151.7ppm, 154.5 ppm <strong>and</strong> 157.2 ppm) were observed (spectrum notshown). These chromatographic <strong>and</strong> spectroscopic data were <strong>in</strong>complete agreement with our previous analysis of the <strong>in</strong>itialketoam<strong>in</strong>e product formed from the reaction of n-butylam<strong>in</strong>ewith ADP-ribose, where detailed H-NMR analysis establishedthe carbonyl group at position 2 [29]. Taken together, thechromatographic <strong>and</strong> spectroscopic data <strong>in</strong>dicate that the earlyglycation product (P1) is most probably the ketoam<strong>in</strong>e N α -acetyl-N ε -(1-deoxy-ADP-ribulos-1-yl)-L-lys<strong>in</strong>e that is convertedto epimeric reduction products follow<strong>in</strong>g treatment withNaBH (Scheme 1). The ADP-ribose-derived ketoam<strong>in</strong>e onhistone H1 would be expected to react with DNPH because, <strong>in</strong>contrast to ketoam<strong>in</strong>es orig<strong>in</strong>at<strong>in</strong>g from glycation by hexoses orother pentoses, the 5-phosphate substitution of the D-riboseprevents stabilization by a closed r<strong>in</strong>g hemiketal [8]. Prior studieshave shown that DNPH readily forms hydrazones with theketoam<strong>in</strong>es derived from glyceraldehyde s<strong>in</strong>ce this sugar is tooshort to cyclize [30]. Consistent with this suggestion, am<strong>in</strong>oguanid<strong>in</strong>e,which effectively blocked advanced glycation, asassessed by prote<strong>in</strong> crossl<strong>in</strong>k<strong>in</strong>g (Figures 5C <strong>and</strong> 5D), did notblock the formation of the prote<strong>in</strong>-bound carbonyl groups.Although am<strong>in</strong>oguanid<strong>in</strong>e forms a hydrazone with ketoam<strong>in</strong>es,the molar excess of DNPH dur<strong>in</strong>g the derivatization reactionshould displace most of the am<strong>in</strong>oguanid<strong>in</strong>e. It is possible thatthe slight reduction <strong>in</strong> immunosta<strong>in</strong><strong>in</strong>g of the histone H1monomer observed <strong>in</strong> reactions conta<strong>in</strong><strong>in</strong>g am<strong>in</strong>oguanid<strong>in</strong>e(Figure 5D, lane 4) may be the result of <strong>in</strong>complete displacementby DNPH.DISCUSSIONProte<strong>in</strong> <strong>carbonylation</strong> is elevated <strong>in</strong> pathological conditions <strong>and</strong>ag<strong>in</strong>g [1,3,7,31]. <strong>Histone</strong>s are important potential targets forprote<strong>in</strong> <strong>carbonylation</strong> due to their <strong>in</strong>volvement <strong>in</strong> chromat<strong>in</strong>structure <strong>and</strong> function. Prior studies have detected prote<strong>in</strong>boundcarbonyls <strong>in</strong> the nuclear fraction, although specific prote<strong>in</strong>acceptors were not identified [10]. The present study has demonstrated,by two different methods, that histone H1 is one ofthe nuclear prote<strong>in</strong>s modified by carbonyl groups <strong>in</strong> io. Theimmunological method for the detection of oxidized prote<strong>in</strong>s,based on the derivatization of prote<strong>in</strong>-bound carbonyl groupswith DNPH <strong>and</strong> subsequent detection of the DNP-epitopes byimmunoblott<strong>in</strong>g [32], has led to the identification of a number ofprote<strong>in</strong>s that accumulate oxidative damage dur<strong>in</strong>g the ag<strong>in</strong>gprocess [33]. The successful application of this method to detecthistone carbonyl groups should facilitate the study of histone<strong>carbonylation</strong> under different physiological conditions.The higher level of prote<strong>in</strong> <strong>carbonylation</strong> <strong>in</strong> histone H1 relativeto core histones is consistent with the higher accessibility of thisl<strong>in</strong>ker histone as compared to core histones [34]. The carbonylcontent of the histone H1 fraction from calf thymus (approximately4 nmol of carbonylmg prote<strong>in</strong>) as quantified by <strong>in</strong>corporationof radiolabel (Table 1) is similar to the reportedlevels of total prote<strong>in</strong>-bound carbonyl groups <strong>in</strong> aged hum<strong>and</strong>ermal fibroblasts <strong>in</strong> tissue culture [35]. It will be <strong>in</strong>terest<strong>in</strong>g toexam<strong>in</strong>e the possibility that histone <strong>carbonylation</strong> <strong>in</strong>creases withtissue age, as has been demonstrated for carbonyl content oftotal prote<strong>in</strong> <strong>in</strong> different tissues [31]. The half-life of histoneswith<strong>in</strong> non-proliferat<strong>in</strong>g mouse cells ranges between four <strong>and</strong>five months, potentially allow<strong>in</strong>g the accumulation of nonenzymichistone damage with ag<strong>in</strong>g [36].Our demonstration that histone <strong>carbonylation</strong> is <strong>in</strong>creasedfollow<strong>in</strong>g stress <strong>in</strong> PC12 cells must be <strong>in</strong>terpreted <strong>in</strong> light ofevidence that the nucleus conta<strong>in</strong>s a proteasome that preferentiallydegrades histones oxidized <strong>in</strong> itro [11]. Thus thecontent of prote<strong>in</strong> carbonyls obta<strong>in</strong>ed from cells or tissuesprobably reflects the relative rates of formation <strong>and</strong> degradationof prote<strong>in</strong>s conta<strong>in</strong><strong>in</strong>g carbonyl groups. In itro, H1 exhibited thehighest rate of proteasomal degradation among histone fractionsfollow<strong>in</strong>g oxidative damage by H O [37]. Our detection of onlyslight <strong>in</strong>creases <strong>in</strong> the accumulation of carbonylated H1 <strong>in</strong> PC12cells follow<strong>in</strong>g H O treatment may only reflect efficient degradationof the oxidized H1 <strong>in</strong> these cells follow<strong>in</strong>g oxidative stress.This possibility is supported by the decreased content of carbonyls<strong>in</strong> other nuclear prote<strong>in</strong>s (the putative HMG12 samples) fromH O -treated PC12 cells (Figure 3). Studies of the nuclearproteasome have shown that more highly oxidized histonesconta<strong>in</strong><strong>in</strong>g cross-l<strong>in</strong>ked histone–DNA aggregates have decreasedproteolytic susceptibility [37]. Thus it is possible that the histone<strong>carbonylation</strong> detected <strong>in</strong> our studies follow<strong>in</strong>g alkylat<strong>in</strong>g stressor comb<strong>in</strong>ations of alkylat<strong>in</strong>g <strong>and</strong> oxidative stress may be due tothe accumulation of prote<strong>in</strong> damage that is not recognized, orpoorly recognized, by the proteasome.S<strong>in</strong>ce prote<strong>in</strong> <strong>carbonylation</strong> <strong>in</strong>volves a complex chemistry thatcan result <strong>in</strong> formation of carbonyls by multiple pathways, morestudies will be required to identify the source(s) of histonecarbonyl groups <strong>in</strong> io. One pathway that seems likely toaccount for histone <strong>carbonylation</strong> follow<strong>in</strong>g alkylat<strong>in</strong>g stress ishistone glycation. Monofunctional alkylat<strong>in</strong>g agents. likeMNNG, are potent <strong>in</strong>ducers of DNA str<strong>and</strong> cleavage, whichactivates the nuclear enzymes poly(ADP-ribose) polymerase <strong>and</strong>poly(ADP-ribose) glycohydrolase. The co-ord<strong>in</strong>ated activities ofthese two enzymes results <strong>in</strong> the conversion of a large fractionof the millimolar-range NAD pool with<strong>in</strong> m<strong>in</strong>utes to free ADPribose<strong>in</strong> close proximity to basic histones [22,23]. The possibilitythat histone <strong>carbonylation</strong> <strong>in</strong> io is the result at least <strong>in</strong> part ofglycation is supported by earlier studies by Hilz <strong>and</strong> co-workersthat described ADP-ribose conjugates of histone H1 <strong>in</strong> hepatomacells follow<strong>in</strong>g alkylat<strong>in</strong>g stress [38] with chemical stability similarto histone glycation conjugates [29].Although the <strong>in</strong>volvement of free ADP-ribose <strong>in</strong> histone<strong>carbonylation</strong> <strong>in</strong> io is hypothetical at this po<strong>in</strong>t, several resultsreported <strong>in</strong> the present study support that possibility: (1) ADPribosereadily results <strong>in</strong> the rapid <strong>in</strong>troduction of carbonyls <strong>in</strong>toH1 at physiological pH (Figure 1); (2) ADP-ribose is considerablymore potent than other sugars exam<strong>in</strong>ed <strong>in</strong> the <strong>in</strong>troduction ofcarbonyls <strong>in</strong>to H1 (Figure 5); (3) ADP-ribose causes <strong>carbonylation</strong>of histones <strong>in</strong> nuclei isolated from PC12 cells (Figure 4); (4)H1 was the dom<strong>in</strong>ant histone target for <strong>carbonylation</strong> by ADPribose<strong>in</strong> nuclei, a pattern rem<strong>in</strong>iscent of that <strong>in</strong> tissues <strong>and</strong> cells(Figures 2 <strong>and</strong> 3). As to the nature of the carbonyl groupsgenerated from ADP-ribose, the <strong>carbonylation</strong> of histone H1by ADP-ribose <strong>in</strong> itro dur<strong>in</strong>g early glycation via oxygen<strong>in</strong>dependentpathways (Figure 5) <strong>and</strong> the rapid conversion ofADP-ribose <strong>and</strong> N α -acetyl-L-lys<strong>in</strong>e at physiological pH <strong>in</strong>to aketoam<strong>in</strong>e product (Figure 6 <strong>and</strong> Scheme 1) supports the 2000 Biochemical Society
<strong>Histone</strong> H1 <strong>carbonylation</strong> <strong>in</strong> <strong>vivo</strong> <strong>and</strong> <strong>in</strong> <strong>vitro</strong>777conclusion that an acyclic ketoam<strong>in</strong>e is the primary source ofprote<strong>in</strong>-bound carbonyl groups derived from ADP-ribose. Theketoam<strong>in</strong>e or its stabilized reduction product may be a usefulbiomarker for assess<strong>in</strong>g the relevance of ADP-ribose <strong>in</strong>duced<strong>carbonylation</strong> <strong>in</strong> io.Enzymic post-translational modifications of histones that<strong>in</strong>clude methylation, acetylation, ubiquit<strong>in</strong>ation, poly(ADPribosyl)ation<strong>and</strong> phosphorylation are <strong>in</strong>volved <strong>in</strong> the regulationof multiple chromat<strong>in</strong> functions [39]. Thus it is plausible thathistone <strong>carbonylation</strong> may alter chromat<strong>in</strong> function. It has beenreported that nucleosomal histone prote<strong>in</strong> protects DNA fromiron-mediated damage [40]. Nuclear polyam<strong>in</strong>es like sperm<strong>in</strong>eare effective antioxidants protect<strong>in</strong>g DNA from attack by reactiveoxygen species, <strong>and</strong> a sperm<strong>in</strong>e-carbonyl adduct has been identifiedas a result of this function [41]. In an analogous manner,preferential histone <strong>carbonylation</strong> could serve to protect thechemical <strong>in</strong>tegrity of DNA. On the other h<strong>and</strong>, oxidative DNAdamage has been demonstrated as a result of the decay of histoneH1-bound hydroperoxides <strong>in</strong> itro, suggest<strong>in</strong>g a deleterious effectof oxidized histones on genomic <strong>in</strong>tegrity [5]. The methodology<strong>and</strong> results described <strong>in</strong> the present study should prove useful <strong>in</strong><strong>in</strong>vestigat<strong>in</strong>g the functional significance of <strong>in</strong>tranuclear prote<strong>in</strong><strong>carbonylation</strong>.This research was supported by grants from the National Institutes of Health(CA43894 <strong>and</strong> NS38496) <strong>and</strong> Niadyne Inc. (University of Kentucky, Lex<strong>in</strong>gton, KY,U.S.A.). M. K. J. <strong>and</strong> E. L. J. are co-founders of Niadyne Inc., whose sponsoredresearch is managed <strong>in</strong> accordance with the University of Kentucky conflict-of<strong>in</strong>terestpolicies. The assistance of Kristi Ballard with cell culture is gratefullyacknowledged. Mass spectral analyses were performed by the University of KentuckyMass Spectrometry Facility directed by Dr Jan Pyrek.REFERENCES1 Halliwell, B. <strong>and</strong> Gutteridge, J. M. C. (1989) Free radicals, age<strong>in</strong>g, <strong>and</strong> disease. InFree Radicals <strong>in</strong> Biology <strong>and</strong> Medic<strong>in</strong>e, pp. 279–313, Clarendon Press, Oxford2 Brownlee, M. (1995) Advanced prote<strong>in</strong> glycosylation <strong>in</strong> diabetes <strong>and</strong> ag<strong>in</strong>g. Annu.Rev. Med. 46, 223–2343 Berlett, B. S. <strong>and</strong> Stadtman, E. R. (1997) Prote<strong>in</strong> oxidation <strong>in</strong> ag<strong>in</strong>g, disease, <strong>and</strong>oxidative stress. J. Biol. Chem. 272, 20313–203164 L<strong>in</strong>dner, H., Sarg, B., Hoertnagl, B. <strong>and</strong> Helliger, W. (1998) The microheterogeneity ofthe mammalian H1(0) histone. Evidence for an age-dependent deamidation. J. Biol.Chem. 273, 13324–133305 Luxford, C., Mor<strong>in</strong>, B., Dean, R. T. <strong>and</strong> Davies, M. J. (1999) <strong>Histone</strong> H1- <strong>and</strong> otherprote<strong>in</strong>- <strong>and</strong> am<strong>in</strong>o acid-hydroperoxides can give rise to free radicals which oxidizeDNA. Biochem. J. 344, 125–1346 Stadtman, E. R. (1995) Role of oxidized am<strong>in</strong>o acids <strong>in</strong> prote<strong>in</strong> breakdown <strong>and</strong>stability. Methods Enzymol. 258, 379–3937 Dean, R. T., Fu, S., Stocker, R. <strong>and</strong> Davies, M. J. (1997) Biochemistry <strong>and</strong> pathologyof radical-mediated prote<strong>in</strong> oxidation. Biochem. J. 324, 1–188 Ligg<strong>in</strong>s, J. <strong>and</strong> Furth, A. J. (1997) Role of prote<strong>in</strong>-bound carbonyl groups <strong>in</strong> theformation of advanced glycation endproducts. Biochim. Biophys. Acta 1361, 123–1309 Fu, S., Fu, M. X., Baynes, J. W., Thorpe, S. R. <strong>and</strong> Dean, R. T. (1998) Presence ofdopa <strong>and</strong> am<strong>in</strong>o acid hydroperoxides <strong>in</strong> prote<strong>in</strong>s modified with advanced glycationend products (AGEs): am<strong>in</strong>o acid oxidation products as a possible source ofoxidative stress <strong>in</strong>duced by AGE prote<strong>in</strong>s. Biochem. J. 330, 233–23910 Pleshakova, O. V., Kutsyi, M. P., Sukharev, S. A., Sadovnikov, V. B. <strong>and</strong> Gaziev, A. I.(1998) Study of prote<strong>in</strong> carbonyls <strong>in</strong> subcellular fractions isolated from liver <strong>and</strong>spleen of old <strong>and</strong> gamma-irradiated rats. Mech. Age<strong>in</strong>g Dev. 103, 45–5511 Ullrich, O., Re<strong>in</strong>heckel, T., Sitte, N., Hass, R., Grune, T. <strong>and</strong> Davies, K. J. (1999)Poly-ADPribose polymerase activates nuclear proteasome to degrade oxidativelydamaged histones. Proc. Natl. Acad. Sci. U.S.A. 96, 6223–622812 Gugliucci, A. <strong>and</strong> Bendayan, M. (1995) <strong>Histone</strong>s from diabetic rats conta<strong>in</strong> <strong>in</strong>creasedlevels of advanced glycation end products. Biochem. Biophys. Res. Commun. 212,56–6213 Med<strong>in</strong>a, L. <strong>and</strong> Haltiwanger, R. S. (1998) Calf thymus high mobility group prote<strong>in</strong>sare nonenzymatically glycated but not significantly glycosylated. Glycobiology 8,191–19814 Takata, K., Horiuchi, S., Araki, N., Shiga, M., Saitoh, M. <strong>and</strong> Mor<strong>in</strong>o, Y. (1988)Endocytic uptake of nonenzymatically glycosylated prote<strong>in</strong>s is mediated by ascavenger receptor for aldehyde-modified prote<strong>in</strong>s. Biochemistry 263, 14819–1482515 Johns, E. W. (1964) Studies on histones. 7. Preparative methods for histone fractionsfrom calf thymus. Biochem. J. 92, 55–5916 Bonnieu, A., Rech, J., Jeanteur, P. <strong>and</strong> Fort, P. (1989) Requirements for c-fos mRNAdown regulation <strong>in</strong> growth stimulated mur<strong>in</strong>e cells. Oncogene 4, 881–88817 Ruiz-Carillo, A. <strong>and</strong> Jorcano, J. L. (1979) An octamer of core histones <strong>in</strong> solution:central role of the H3-H4 tetramer <strong>in</strong> the self-assembly. Biochemistry 18, 760–76818 Chao, C. C., Ma, Y. S. <strong>and</strong> Stadtman, E. R. (1997) Modification of prote<strong>in</strong> surfacehydrophobicity <strong>and</strong> methion<strong>in</strong>e oxidation by oxidative systems. Proc. Natl. Acad. Sci.U.S.A. 94, 2969–297419 Cervantes-Laurean, D., Jacobson, E. L. <strong>and</strong> Jacobson, M. K. (1996) Glycation <strong>and</strong>glycoxidation of histones by ADP-ribose. J. Biol. Chem. 271, 10461–1046920 Shacter, E., Williams, J. A., Lim, M. <strong>and</strong> Lev<strong>in</strong>e, R. L. (1994) Differential susceptibilityof plasma prote<strong>in</strong>s to oxidative modification: exam<strong>in</strong>ation by western blotimmunoassay. Free Radical Biol. Med. 17, 429–43721 Lenz, A. G., Costabel, U., Shaltiel, S. <strong>and</strong> Lev<strong>in</strong>e, R. L. (1989) Determ<strong>in</strong>ation ofcarbonyl groups <strong>in</strong> oxidatively modified prote<strong>in</strong>s by reduction with tritiated sodiumborohydride. Anal. Biochem. 177, 419–42522 Jacobson, M. K., Levi, V., Juarez-Sal<strong>in</strong>as, H., Barton, R. A. <strong>and</strong> Jacobson, E. L.(1980) Effect of carc<strong>in</strong>ogenic N-alkyl-N-nitroso-compounds on nicot<strong>in</strong>amide aden<strong>in</strong>ed<strong>in</strong>ucleotide metabolism. Cancer Res. 40, 1797–180223 Jacobson, E. L., Antol, K. M., Juarez-Sal<strong>in</strong>as, H. <strong>and</strong> Jacobson, M. K. (1983)Poly(ADP-ribose) metabolism <strong>in</strong> UV irradiated human fibroblasts. J. Biol. Chem. 258,102–10724 Jacobson, E. L., Cervantes-Laurean, D. <strong>and</strong> Jacobson, M. K. (1997) ADP-ribose <strong>in</strong>glycation <strong>and</strong> glycoxidation reactions. Adv. Exp. Med. Biol. 419, 371–37925 Shearman, M. S., Ragan, C. I. <strong>and</strong> Iversen, L. L. (1994) Inhibition of PC12 cell redoxactivity is a specific, early <strong>in</strong>dicator of the mechanism of beta-amyloid-mediated celldeath. Proc. Natl. Acad. Sci. U.S.A. 91, 1470–147526 Elgawish, A., Glomb, M., Friedl<strong>and</strong>er, M. <strong>and</strong> Monnier, V. M. (1996) Involvement ofhydrogen peroxide <strong>in</strong> collagen cross-l<strong>in</strong>k<strong>in</strong>g by high glucose <strong>in</strong> <strong>vitro</strong> <strong>and</strong> <strong>in</strong> <strong>vivo</strong>.J. Biol. Chem. 271, 12964–1297127 Edelste<strong>in</strong>, D. <strong>and</strong> Brownlee, M. (1992) Mechanistic studies of advanced glycosylationend product <strong>in</strong>hibition by am<strong>in</strong>oguanid<strong>in</strong>e. Diabetes 41, 26–2928 Silverste<strong>in</strong>, R. M., Bassler, G. C. <strong>and</strong> Morril, T. C. (1991) 13 C-NMR spectroscopy. InSpectrometric Identification of Organic Compounds, pp. 227–265, John Wiley &Sons, New York29 Cervantes-Laurean, D., M<strong>in</strong>ter, D. E., Jacobson, E. L. <strong>and</strong> Jacobson, M. K. (1993)Prote<strong>in</strong> glycation by ADP-ribose: studies of model conjugates. Biochemistry 32,1528–153430 Acharya, A. S. <strong>and</strong> Mann<strong>in</strong>g, J. M. (1980) Amadori rearrangement of glyceraldehydehemoglob<strong>in</strong>Schiff base adducts. A new procedure for the determ<strong>in</strong>ation of ketoam<strong>in</strong>eadducts <strong>in</strong> prote<strong>in</strong>s. J. Biol. Chem. 255, 7218–722431 Stadtman, E. R. (1992) Prote<strong>in</strong> oxidation <strong>and</strong> ag<strong>in</strong>g. Science 257, 1220–122432 Lev<strong>in</strong>e, R. L., Williams, J. A., Stadtman, E. R. <strong>and</strong> Shacter, E. (1994) Carbonyl assaysfor determ<strong>in</strong>ation of oxidatively modified prote<strong>in</strong>s. Methods Enzymol. 233, 346–35733 Yan, L. J., Lev<strong>in</strong>e, R. L. <strong>and</strong> Sohal, R. S. (1997) Oxidative damage dur<strong>in</strong>g ag<strong>in</strong>gtargets mitochondrial aconitase. Proc. Natl. Acad. Sci. U.S.A. 94, 11168–1117234 Wolffe, A. P. (1992) Chromat<strong>in</strong> structure. In Chromat<strong>in</strong> Structure <strong>and</strong> Function,pp. 4–67, Academic Press, London35 Oliver, C. N., Ahn, B.-W., Moerman, E. J., Goldste<strong>in</strong>, S. <strong>and</strong> Stadtman, E. R. (1987)Age-related changes <strong>in</strong> oxidized prote<strong>in</strong>s. J. Biol. Chem. 262, 5488–549136 Commerford, S. L., Carsten, A. L. <strong>and</strong> Cronkite, E. P. (1982) <strong>Histone</strong> turnover with<strong>in</strong>nonproliferat<strong>in</strong>g cells. Proc. Natl. Acad. Sci. U.S.A. 79, 1163–116537 Ullrich, O., Sitte, N., Sommerburg, O., S<strong>and</strong>ig, V., Davies, K. J. A. <strong>and</strong> Grune, T.(1999) Influence of DNA b<strong>in</strong>d<strong>in</strong>g on the degradation of oxidized histones by the 20Sproteasome. Arch. Biochem. Biophys. 362, 211–21638 Kreimeyer, A., Wielkens, P., Adamietz, P. <strong>and</strong> Hilz, H. (1984) DNA repair-associatedADP-ribosylation <strong>in</strong> <strong>vivo</strong>. Modification of histone H1 differs from that of the pr<strong>in</strong>cipalacceptor prote<strong>in</strong>s. J. Biol. Chem. 259, 890–89639 Strahl, B. D. <strong>and</strong> Allis, C. D. (2000) The language of covalent histone modifications.Nature (London) 403, 41–4540 Enright, H. U., Miller, W. J. <strong>and</strong> Hebbel, R. P. (1992) Nucleosomal histone prote<strong>in</strong>protects DNA from iron-mediated damage. Nucleic Acids Res. 20, 3341–334641 Chol Ha, H., Sirisoma, N. S., Kuppusamy, P., Zweier, J. L., Woster, P. M. <strong>and</strong> Casero,R. A. (1998) The natural polyam<strong>in</strong>e sperm<strong>in</strong>e functions directly as a free radicalscavenger. Proc. Natl. Acad. Sci. U.S.A. 95, 11140–11145Received 8 May 2000/26 June 2000; accepted 24 July 2000 2000 Biochemical Society