Selective Covalent Binding of Acrylonitrile to Cys 186 in Rat Liver

Donald E. Nerland,* Jian Cai, and Frederick W. Benz. Department of Pharmacology & Toxicology, University of Louisville Medical School,. Louisville, Ke...
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Chem. Res. Toxicol. 2003, 16, 583-589

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Selective Covalent Binding of Acrylonitrile to Cys 186 in Rat Liver Carbonic Anhydrase III In Vivo Donald E. Nerland,* Jian Cai, and Frederick W. Benz Department of Pharmacology & Toxicology, University of Louisville Medical School, Louisville, Kentucky 40292 Received December 31, 2002

Covalent binding of reactive chemical species to tissue proteins is a common, but poorly understood, mechanism of toxicity. Identification of the proteins and the specific amino acid residues within the proteins that are chemically modified will aid our understanding of the toxification/detoxification mechanisms involved in covalent binding. Acrylonitrile (AN) is a commercial vinyl monomer that is acutely toxic and readily binds to tissue proteins. Total covalent binding of AN to tissue proteins is highly correlated with acute toxicity. Twodimensional PAGE and autoradiography were used to locate proteins in male rat liver cytosol that are radiolabeled following administration of [2,3-14C]AN in vivo. Four intensely labeled spots were prominent in the autoradiogram and formed an apparent “charge-train” at approximately 30 kDa. Tryptic peptide mapping by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS was used to identify all of the spots as carbonic anhydrase III (CAIII). HPLC of the tryptic digests combined with MALDI-TOF MS was used to localize the radiolabel to tryptic fragment T22 containing amino acids 171-187. This tryptic fragment contains two Cys residues (Cys181 and Cys186) in the rat CAIII sequence. Electrospray ionization ion-trap MS was used to sequence the peptide and establish that only Cys186 was labeled. Thus, although AN is considered to be highly reactive, our data indicate that it does not react indiscriminately with rat CAIII but rather is selective for one out of five Cys residues. Rat liver CAIII has previously been shown to protect cells against oxidative stress. Our data suggest that CAIII is also capable of scavenging reactive xenobiotics and may help prevent covalent binding to more critical macromolecules.

Introduction AN1 is a vinyl monomer used extensively in the manufacture of acrylic fibers, plastics, synthetic rubber, and acrylamide. Over 8.8 billion pounds of AN are produced yearly worldwide and U.S. production was more than 3 billion pounds in 1999 (1, 2). Workers may be exposed to AN in an industrial setting during production and polymerization, while other people may be exposed during transportation of AN or through cigarette smoking or industrial pollution. AN is acutely toxic (3). Despite a considerable amount of research, the mechanism responsible for the acute toxicity of AN has not been established. Three potential mechanisms have been suggested (4). AN is oxidatively metabolized to cyanide by the mixed function oxidase system (5-7). Although cyanide has been implicated in the acute toxicity of AN (8, 9), it has been argued that cyanide alone cannot account for the acute toxicity of AN (4). Similarly, AN produces a profound depletion of GSH in all tissues examined (10) but GSH depletion alone is not acutely toxic (4). * To whom correspondence should be addressed. Tel: (502)852-5560. Fax: (502)852-7868. E-mail: [email protected]. 1 Abbreviations: AN, acrylonitrile; CA, carbonic anhydrase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; ESI, electrospray ionization; IAM, iodoacetamide; IPG, immobilized pH gradient; GST, glutathione S-transferase; MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; MS/MS, tandem mass spectrometry; PVDF, poly(vinylidene difluoride); rCAIII, rat liver CA III; rGSTM, rat GST µ-family.

Third, AN can covalently bind to tissue proteins (4). However, unlike many of the more extensively studied toxicants, AN does not require metabolic activation to covalently bind to proteins. AN is an R,β-unsaturated nitrile that undergoes Michael-like addition reactions and can cyanoethylate cellular nucleophiles (11). AN is a soft electrophile that reacts preferentially with soft nucleophiles such as thiols. Despite its reactivity, we have found that the in vivo interactions can also be highly specific. We observed that the in vivo covalent modification of the rGSTs was limited to the µ-class of transferases (12) and was highly specific for rGSTM1. The specificity for rGSTM1 over rGSTM2 could be explained by a single amino acid difference that enhanced the reactivity of the target Cys residue. In the course of the GST studies mentioned above, we observed in the autoradiogram an unidentified but highly labeled protein that migrated on SDS-PAGE at approximately 28 kDa, i.e., slightly above the 27 kDa band characterized as rGSTs. In the current investigation, we have utilized two-dimensional (2D) gel electrophoresis combined with MALDI-TOF and ESI MS/MS to characterize this protein as CAIII. In addition, we show that of the five Cys residues in CAIII, only Cys186 reacts with AN in vivo.

Experimental Procedures Chemicals. AN (>99% pure), containing 35-45 ppm hydroquinone monomethyl ether as a polymerization inhibitor, was

10.1021/tx0256883 CCC: $25.00 © 2003 American Chemical Society Published on Web 04/08/2003

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purchased from Aldrich Chemical Co. (Milwaukee, WI). [2,314C]AN (>99% radiochemical purity) with specific activities from 5.5 to 24.2 Ci/mol was obtained from Sigma Chemical Co. (St. Louis, MO). Radiochemical purity was checked by HPLC prior to its use. Buffer reagents and protease inhibitors were purchased from Sigma Chemical Co. SDS-PAGE reagents and protein molecular weight markers were obtained from Bio-Rad Laboratories (Hercules, CA). IPG strips and IPG buffers used for isoelectric focusing were purchased from Amersham-Pharmacia (Piscataway, NJ). Animals and Tissue Treatments. Male Sprague-Dawley rats, weighing 250-350 gm, were obtained from Harlan (Indianapolis, IN). All animals were maintained on a 12 h lightdark cycle at 22 ( 2 °C. The rats were acclimated for at least 1 week prior to use and were allowed Purina Rat Chow (Ralston Purina Co., St. Louis, MO) and water ad libitum. Rats were injected subcutaneously with 115 mg/kg (2.2 mmol/kg) of [2,314C]AN (0.5-2.0 mCi/kg) or the distilled water vehicle. Two hours following the injection, the rats were sacrificed and the livers were perfused in situ and excised. The liver was minced into small pieces using scissors and homogenized using a Dounce homogenizer in 3 vol of isotonic KCl containing 10 mM TrisHCl buffer (pH 7.4) and protease inhibitors (Pepstatin A, 0.7 µg/mL; Leupeptin, 0.5 µg/mL; and EDTA, 0.3 mg/mL). The cytosolic fraction was isolated by centrifuging the homogenate at 109 000g for 60 min. Electrophoresis, Blotting, and Detection. The first dimension of the 2D PAGE was performed using 13 cm, pH 3-10, Immobiline DryStrips. The protein samples were dissolved in the rehydration buffer (8 M urea, 2% (w/v) CHAPS, and 2% IPG 3-10 L buffer), and the strips were rehydrated according to the manufacturer’s directions. The initial voltage of 200 V was ramped to 500 V over 2 h, from 500 to 3500 V over 3 h, and then held at 3500 V until a total of 76 kV hr was reached. Prior to the second-dimension run, the strips were equilibrated for 15 min in 50 mM Tris, pH 8.8, 8.0 M urea, 30% glycerol, 0.3% SDS plus DTT (2%), followed by a 15 min equilibration in the same buffer plus IAM (2%). The second-dimension separation was carried out in a vertical electrophoresis apparatus using 15% gels. The proteins were detected by staining with 0.5% Coomassie Brilliant Blue R250 in 30% methanol containing 10% acetic acid. Alternatively, the proteins were blotted onto PVDF (ImmobilonP, Millipore) membranes using the method of Szewczyk and Kozloff (13) and the radiolabeled proteins were detected by autoradiography using Kodak BioMax MS (Rochester, NY) film with a Kodak BioMax LE intensifying screen. In-Gel Digestion. Radiolabeled spots were excised from Coomassie-stained gels and digested in situ using trypsin. Briefly, the gel bands were cut into 1 mm cubes, destained with 50 mM NH4HCO3/acetonitrile (1:1, v/v), reduced with DTT, alkylated with IAM, and digested overnight at 37 °C using sequencing grade modified trypsin (Promega, Madison, WI). HPLC of Tryptic Fragments. The tryptic fragments were separated by HPLC using a Vydac C18 reversed-phase column (2 mm × 250 mm, 5 µm, 300 Å) at a flow rate of 200 µL/min. A step gradient elution was performed using 0.1% TFA in water: acetonitrile (95:5) as buffer A and 0.1% TFA in water:acetonitrile (20:80) as buffer B. The initial conditions were 2% B, then increased to 20% B over 7 min, and followed by a gradient to 60% B over 90 min. The column effluent was monitored at 214 nm, and fractions were collected at 1 min intervals. MALDI-TOF MS. The protein digest (1 µL) was mixed with 1 µL of R-cyano-4-hydroxy-trans-cinnamic acid (10 mg/mL in 0.1% TFA:acetonitrile, 1:1,v/v). One microliter of the mixture was deposited onto a fast evaporation nitrocellulose matrix surface, dried at room temperature, washed twice with 1.5 µL of 5% formic acid, and analyzed with a TofSpec 2E (Micromass) MALDI-TOF mass spectrometer using the reflectron mode. The mass axis was calibrated with a calibration file of peaks from trypsin autolysis and adjusted with trypsin peak (m/z 2211.10) as the mass lock. Database searches were performed using MS-

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Figure 1. Autoradiogram of a 2D electrophoretic gel prepared from the liver of a male Sprague-Dawley rat that was treated with a dose of 115 mg/kg of [2,314C]AN. Spots labeled 1-4 indicate the position of proteins identified as rCAIII. Spots labeled A-C indicate the position of previously identified proteins GSTM2 (A), GSTM1 (B), and the R- and β-chains of hemoglobin (C). Fit (http://prospector.ucsf.edu) by comparing the monoisotopic peaks of the peptides with the theoretical molecular weight of peptides produced by digestion of the proteins in the SWISSPROT/TrEMBL database (http://us.expasy.org). MS/MS Analysis and De Novo Sequencing. MS/MS sequencing was performed using a Finnigan LCQ Duo ion trap mass spectrometer (Finnigan Corp., San Jose, CA) equipped with an ESI source. High-purity helium was introduced as the collision buffer gas, and nitrogen was used as the sample nebulization gas. Peptides were introduced by infusion through a fused-silica capillary into the ionization source at 2.5 µL/min, using a syringe pump. The ESI source was operated at a voltage of 4500 V, and the interface capillary heater was set to 120 °C. All spectra were obtained in positive ion mode and recorded at unit mass resolution. Database searches were performed using the programs MS-Seq (http://prospector.ucsf.edu) to interrogate the SWISS-PROT/TrEMBL database.

Results Separation of Protein Adducts by 2D PAGE and Protein Identification with MALDI-TOF MS. Previous experiments performed in our laboratory demonstrated that some of the major covalent adducts in the liver of rats treated with [2,3-14C]AN are localized in two protein bands on SDS-PAGE of the cytosolic fraction at approximately 27 and 28 kDa (12). The 27 kDa adduct was isolated from the cytosol using affinity chromatography and identified as rGSTM1 using MALDI-TOF MS. The protein(s) corresponding to the 28 kDa band did not bind to the affinity resin and was not identified. To characterize this protein, 2D PAGE was used to separate the proteins in liver cytosol prepared from control and rats treated with [2,3-14C]AN. Autoradiography showed that the 27-30 kDa region of the gel contained four highly radiolabeled spots that formed a “charge train” along the pH axis (Figure 1). To identify these four spots and the site(s) of AN incorporation, corresponding spots from the livers of control and [2,3-14C]AN-treated rats were reduced with DTT and the Cys residues were alkylated with IAM. Following trypsin digestion and mass analysis using MALDI-TOF MS, 15 peptide fragments were detected and could be assigned to peptides derived from rCAIII based on the amino acid sequences available from the SWISS-PROT data bank (P14141). These fragments had

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Table 1. Masses of CAIII Tryptic Peptides Isolated from Control and AN-Treated Rats calcdc peptidea T10 T14 T8 T3 T15 T18 T13 T17-T18 T5 T22*e T22* T22 T12 T2 T26 T23

errord (Da)

residues

sequenceb

mass (Da)

control

AN-treated

80-88 126-134 67-75 24-35 135-147 153-164 113-125 151-164 39-56 171-187 171-187 171-187 91-112 3-23 226-250 188-211

GGPLSGPYR YNTFGEALK VVFDDTFDR GDNQSPIELHTK QPDGIAVVGIFLK GEFQILLDALDK YAAELHLVHWNPK EKGEFQILLDALDK HDPSLQPWSVSYDPGSAK EAPFNHFDPSJLFPABR EAPFNHFDPSBLFPAJR EAPFNHFDPSBLFPABR QFHLHWGSSDDHGSEHTVDGVK EWGYASHNGPEHWHELYPIAK SLFASAENEPPVPLVGNWRPPQPIK DYWTYHGSFTTPPBEEBIVWLLLK

902.46 1041.52 1112.51 1337.66 1355.78 1360.72 1576.81 1617.86 1969.92 2059.90 2059.90 2063.90 2474.10 2520.13 2742.45 3014.40

-0.01 0.01 -0.03 -0.03 -0.00 -0.02 -0.04 -0.03 -0.02 ndf nd -0.03 0.05 nd 0.09 nd

0.03 -0.02 0.00 0.00 0.02 0.01 -0.01 -0.01 0.01 -0.01 -0.01 -0.01 0.02 0.01 0.00 0.06

a Tryptic digestion fragments from CAIII. b Letters B and J in the sequence are the Cys residues alkylated by IAM (B) or AN (J). Masses are the calculated monoisotopic masses for the sequences in the table. d Errors are the difference between the calculated monoisotope masses and the masses detected in the samples. e An asterisk indicates that the peptide contains an AN adduct. f Not detected (nd).

c

one of the two Cys residues is labeled by AN. In addition, because not all of the protein could be accounted for using MALDI-TOF MS, we could not exclude the possibility that other nucleophilic sites were modified by AN. To clarify this ambiguity, HPLC analyses of the tryptic digests were performed. Parenthetically, four additional radioactive spots at slightly lower mass and between spots 3 and 4 (Figure 1) were also analyzed. Three of the spots also matched rCAIII but with lower sequence coverage, while the fourth spot remains unidentified. The three lower molecular mass spots matching rCAIII probably reflect some proteolysis occurring during liver homogenization and sample preparation.

Figure 2. Segment of the MALDI-TOF mass spectrum of a tryptic digest of rCAIII prepared from control (A) and ANtreated male rats (B).

mass errors ranging from -0.04 to 0.09 Da and covered almost 80% of the amino acid sequence (Table 1). All four spots produced the same match to rCAIII. The major difference in the MALDI-TOF spectra of the rCAIII tryptic fragments from control and AN-treated rats was that in treated rats an additional signal at m/z 2060.90 (T22*) was observed (Table 1 and Figure 2, only the m/z region 2050-2075 is illustrated). This new peptide had a mass 4 Da less than the corresponding control peptide T22. This fragment contains two Cys residues at sequence positions 181 and 186. Cyanoethylation of one of these Cys residues by AN would yield an ion 4 Da less than predicted for dicarboxamidomethylation of Cys181 and 186 by IAM. Unfortunately, from the MALDI-TOF spectra, we are unable to determine which

HPLC Separation of Tryptic Peptides Derived from the 28 kDa Radioactive Band following SDSPAGE. To determine if AN was covalently bound to additional sites on rCAIII, the cytosolic fraction of the livers from control and [2,3-14C]AN-treated rats was separated using 12% SDS-PAGE as described previously (12). The protein bands at 28 kDa were excised, reduced with DTT, alkylated with IAM, and digested with trypsin. The tryptic digests were separated by HPLC using a C18reversed phase column. The chromatograms of the two digests were nearly identical except that an additional peak at 44.7 min was present in the chromatogram from the AN-treated rat and the height of the peak at 41 min was decreased (Figure 3). Liquid scintillation spectrometry of the eluate showed that 90% of the total radioactivity added to the column eluted as a single fraction between 44 and 45 min, while the remaining 10% was present in the void volume. An aliquot of the radioactive peptide fraction was concentrated and analyzed by MALDI-TOF MS (Figure 3, inset). The major monoisotopic ion in the chromatogram at m/z 2061.12 was consistent with peptide T22*. The ion at m/z 1031.61 is due to the doubly charged ion [M + 2H] 2+ of the same fragment, and the ion at m/z 2744.71 results from carryover from the peak eluting at 43.5 min. These data indicate that AN reacts with rCAIII in vivo essentially exclusively on either Cys181 or Cys186 or is distributed equally between them. To resolve this ambiguity, the peptides were sequenced using MS/MS.

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Figure 3. C18-HPLC of tryptic fragments of CAIII prepared from a control rat and a rat treated with [2,314C]AN. The chromatogram labeled A is from the AN-treated rat, while chromatogram B is from the control rat. The arrow indicates the position of the additional peak that is present in the chromatogram from the AN-treated rat that contained 90% of the radioactivity applied to the column. The insert illustrates the MALDI-TOF spectrum of the radioactive peak containing the AN-adducted peptide.

MS/MS with Chemical-Induced Dissociation of rCAIII Peptides T22 and T22*. Portions of the tryptic digests of the 28 kDa protein bands from control and ANtreated animals that were used in the HPLC analysis (Figure 3) were infused into a Finnigan LCQ Duo ion trap mass spectrometer for sequence analysis. For the control sample, the doubly charged ion of peptide T22 at m/z 1033.5 was selected for chemical-induced dissociation. The product ion spectrum for this peptide is illustrated in Figure 4A. A search of the SWISS-PROT/TrEMBL database using these product ions verified the peptide as being derived from rCAIII. Similarly, for the ANtreated sample, the doubly charged ion of peptide T22* at m/z 1031.5 was selected for sequencing. The product ion spectrum for this peptide is illustrated in Figure 4B. A comparison of the y4+ and y5+ ions, which contain Cys186 but not Cys181, in the two spectra shows that these two ions from the treated sample are 4 Da less than those in the control sample. Conversely, the b12+ and b13+ ions, which contain Cys181 but not Cys186, are at the same m/z values in both samples. These data indicate unambiguously that rCAIII is alkylated exclusively on Cys186 by AN in vivo. Considerably less intense signals from other relevant ions, not illustrated in Figure 4, confirmed this conclusion. Specifically, in the control sample, the mass difference of the ion pair b10+ (m/z 1142.6) to b11+ (m/z 1302.6) and the ion pair b15+ (m/z 1730.8) to b16+ (m/z 1890.8) of 160 Da confirmed that both Cys181 and Cys186 were modified by carboxamidomethyl groups. In the spectrum from the AN-treated rat, the mass difference of the ion pair b10+ (m/z 1142.6) to b11+ (m/z 1302.6) of 160 Da

matches that observed in the control sample but the mass difference of the ion pair b15+ (m/z 1730.8) to b16+ (m/z 1886.8) of 156 Da can only occur if AN specifically alkylated on Cys186.

Discussion In this paper, we demonstrated that after subcutaneous administration to rats, one of the major protein adducts formed with AN is rCAIII. This high reactivity was determined to be due to the essentially exclusive reactivity of one of the five Cys residues in the protein, namely, Cys186. Recently, Koen and Hanzlik have noted that in the case of bromobenzene reactive metabolites and presumably other reactive chemical species such as AN, the alkylation of cellular proteins is purely a chemical process that conforms to a second-order kinetic expression where the rate of adduct formation ) k2 [nucleophile][electrophile] (14). Thus, for the soft electrophile AN, the amount of a protein adduct formed over time reflects both the concentration of the nucleophilic site(s) within the protein and the inherent reactivity of each site. Additionally, they suggested that covalent binding to abundant and apparently nonessential proteins could be considered a detoxification process that prevents the electrophile from binding to critical protein targets. It would appear that CAIII could fulfill such a role. The true physiological function of CAIII is currently unknown but some of its properties suggest it may have functions other than simply catalyzing the hydration of carbon dioxide. The specific activity of CAIII is only 1%

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Figure 4. MS/MS with chemical-induced dissociation of rCAIII peptides T22 and T22*. Panel A is the spectrum obtained from the control rat, and panel B is the spectrum from the AN-treated rat.

that of CAII, and it is insensitive to inhibition by concentrations of sulfonamides that readily inhibit the other isozymes (15-17). CAIII is highly abundant in several tissues. Specifically, it comprises ≈5% of the soluble protein of male rat liver (18), ≈ 8% of some skeletal muscles (19), ≈15% of mature soleus muscle (20), and up to 25% of adipocytes (21, 22). The level of CAIII in the liver of male rats is regulated by androgens, being up to 30 times greater than female rats (23, 24). However, it declines to nearly undetectable levels by 24 months of age (23, 25). In the liver, CAIII is not uniformly distributed throughout the lobule. The highest concentrations are present in the oxidizing environment of the perivenous hepatocytes with lesser amounts present in the periportal hepatocytes (26). A similar zonation pattern is observed for several isoforms of cytochrome P450 (27, 28). Cytochrome P450 is responsible for the metabolic activation of several chemicals to reactive intermediates that covalently bind to liver proteins (29). The hepatotoxic agents, acetaminophen (30) and bromobenzene (14), require metabolic activation by cytochrome P450 to electrophilic species prior to covalently binding to proteins and are reported to form covalent adducts with CAIII. However, the site of covalent attachment of either agent to CAIII was not reported. It would appear that coexpression of cytochrome P450 and a reactive protein in the same hepatocyte population increases the prob-

ability of covalent binding of reactive metabolites to a scavenger protein, perhaps CAIII (31, 32). CAIII has also been proposed to have a cytoprotective action against oxidative stress. For example, NIH/3T3 cells transfected to overexpress CAIII demonstrated reduced steady state levels of reactive oxygen species and an increased proliferation rate and were protected against H2O2-induced apoptosis as compared to controls (33). In addition, during oxidative stress, CAIII has been shown to undergo reversible S-thiolation reactions with GSH (18, 34-36). Although both Cys181 and Cys186 in rCAIII can be glutathiolated (37), Cys186 has been suggested to be more reactive (38). This reversible glutathiolation has been proposed to offer protection to the reactive Cys residues in proteins from more damaging irreversible oxidative reactions (32). This raises the question as to whether the tissue proteins with reactive thiols that participate in glutathiolation during oxidative stress are the same thiols that would be susceptible to alkylation by AN and other soft nucleophiles. As we show in this paper, this is certainly the case for Cys186 in rCAIII. From the relative heights of the signals in Figure 2B, it is clear that over 50% of rCAIII has participated in scavenging the acute toxin AN. If CAIII plays a significant role as an intracellular antioxidant as has been suggested (31, 32), AN exposure would certainly impair this function. The fact that AN also causes profound depletion of GSH in all tissues,

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References

Figure 5. Space-filling model of diglutathiolated rCAIII (PDB: 1FLJ). The two GSH residues have been removed for clarity. The surface location of both Cys181 and Cys186 is evident. The proximity of Cys186 to Lys211 could account for its higher reactivity. The model was drawn with Rasmol.

especially liver (10), would further exacerbate the redox imbalance. The factor(s) that promotes the selective reactivity of Cys186 over the other four Cys residues in rCAIII is not intuitively obvious. Two factors known to influence the reactivity of protein sulfhydryls are the degree of solvent exposure and the pKa of the thiol group (39). The crystal structure of diglutathiolated rCAIII reveals that both Cys181 and Cys186 are surface-exposed and thus would appear to be equally available for reaction with AN (Figure 5) (37). The normal pKa value of a sulfhydryl group is generally considered to be about 8.5, but this can vary greatly (39). Low pKa thiols are generally more reactive because it is the thiolate anion, rather than the sulfhydryl group, that participates in the rate-limiting step of thiol-disulfide exchange and alkylation (39). Lys211 is close to Cys186 in space. In the native protein, the Lys side chain should be free to rotate closer to the thiol group of Cys186 and this positive charge would act to lower the pKa of Cys186 and enhance its reactivity. In conclusion, although AN is considered to be highly reactive, our data indicate that it does not react indiscriminately with rat CAIII but rather is selective for one out of five Cys residues. rCAIII has previously been shown to protect cells against oxidative stress. Our data suggest that CAIII is also capable of scavenging reactive xenobiotics and may help prevent covalent binding to more critical macromolecules.

Acknowledgment. This research was supported by National Institute of Environmental Health Science Grants ES06141 to F.W.B. The Biomolecular Mass Spectrometry Laboratory of the University of Louisville is supported in part by NIH Grant 1 S10 RR11368-01 A1, the State of Kentucky Physical Facilities Trust Fund, the University of Louisville School of Medicine, and the University of Louisville Research Foundation.

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