Reactivity with Tris(hydroxymethyl) - American Chemical Society

Sep 6, 2003 - In this study, rabbit antiserum was raised against acrolein-modified keyhole limpet hemocyanin in the expectation that it would allow im...
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Chem. Res. Toxicol. 2003, 16, 1196-1201

Communications Reactivity with Tris(hydroxymethyl)aminomethane Confounds Immunodetection of Acrolein-Adducted Proteins Philip C. Burcham,*,† Frank R. Fontaine,† Dennis R. Petersen,‡ and Simon M. Pyke§ Molecular Toxicology Research Group, Department of Clinical & Experimental Pharmacology, Department of Chemistry, The University of Adelaide, Adelaide, South Australia 5005, Australia, and Department of Pharmaceutical Sciences, University of Colorado Health Sciences Center, Denver, Colorado Received June 3, 2003

The toxic R,β-unsaturated aldehyde acrolein readily attacks proteins, generating adducts at cysteine, histidine, and lysine residues. In this study, rabbit antiserum was raised against acrolein-modified keyhole limpet hemocyanin in the expectation that it would allow immunodetection of adducted proteins in biological samples. Using slot-blot and enzyme-linked immunosorbent assays, the antiserum detected acrolein-modified protein with high sensitivity and specificity. Adduct immunodetection was strongly inhibited by acrolein-modified polylysine but not polyhistidine. Efforts to develop a Western blotting method for detecting adducted proteins in cell lysates were hampered by irreproducible outcomes, evidently due to adduct instability during SDS-PAGE. Indeed, adducts generated via brief exposure of a model protein to acrolein displayed pH- and concentration-dependent instability to tris(hydroxymethyl)aminomethane (Tris), a nucleophilic buffer used in protein electrophoresis. The effect was most striking when Tris solutions were buffered to pH 8.0 and higher. In contrast, adducts formed during extended exposure to acrolein (g60 min) were completely stable to Tris. The time dependence of susceptibility raised the possibility that Tris interfered with specific steps in lysine modification, which involves stepwise Michael addition of two molecules of acrolein to the same residue, followed by condensation and dehydration to form a heterocyclic adduct, N-(3-formyl-3,4-dehydropiperidino)lysine. We hypothesize that carbonyl-retaining Michael adducts may react with Tris by forming imines with the primary amine of the buffer. Consistent with this idea, triethanolamine, a tertiary amine buffer unable to form imines, had no effect on acrolein-adducted protein. These effects of Tris may explain difficulties in the detection of acrolein-adducted proteins during conventional Western blotting procedures.

Introduction ACR (2-propenal)1 is a highly toxic R,β-unsaturated aldehyde that contributes to many diverse pathological states. Its pronounced toxicity reflects the possession of two strongly electron deficient centers, ensuring that ACR readily alkylates DNA, RNA, and protein (1). The reaction with cysteine residues in proteins is especially favored (2). ACR also attacks the imidazole ring nitrogen of histidine and the -amino group of lysine (1, 2). Typically, these reactions proceed via Michael type * To whom correspondence should be addressed. Tel: 61-8-83035287. Fax: 61-8-8224-0685. E-mail: [email protected]. † Department of Clinical & Experimental Pharmacology, The University of Adelaide. ‡ Department of Pharmaceutical Sciences, University of Colorado Health Sciences Center. § Department of Chemistry, The University of Adelaide. 1Abbreviations: ACR, acrolein; ACR-BSA, acrolein-modified bovine serum albumin; BSA, bovine serum albumin; KLH, keyhole limpet hemocyanin; Tris, tris(hydroxymethyl)aminomethane.

addition to the C-3 of ACR, forming carbonyl-retaining derivatives (1). Consequently, extensive protein carbonylation accompanies ACR-mediated toxicity in cells (3). Uchida and associates discovered that during lysine modification, two molecules of ACR add to a given residue, forming a bis-Michael adduct (Scheme 1), which undergoes condensation and dehydration reactions to form a six-membered heterocycle, N-(3-formyl-3,4-dehydropiperidino)lysine (FDP-lysine) (4, 5). FDP-lysine is also a reactive carbonyl species, undergoing nucleophilic addition by glutathione at its β-carbon (6). Although the chemistry of protein modification by ACR is quite well-understood, the identity of proteins that sustain damage during cellular exposure to this compound is unknown. While several proteins are sensitive to ACR in vitro (e.g., R-1-proteinase, carbonic anhydrase, etc.) (7, 8), the in vivo relevance of such observations is unclear. The present work was conducted with a view to developing an immunochemical approach to detect ACRadducted proteins in cell extracts.

10.1021/tx0341106 CCC: $25.00 © 2003 American Chemical Society Published on Web 09/06/2003

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Chem. Res. Toxicol., Vol. 16, No. 10, 2003 1197 Scheme 1. Steps in the Adduction of Lysine by ACR

Experimental Procedures Materials. Male Swiss mice, 4-6 weeks of age, and adult male NZ White rabbits (approximately 1 kg) were obtained from the Waite Institute, Adelaide, South Australia. KLH and chemiluminescent substrate reagent (SuperSignal West Pico) were purchased from Pierce. Polylysine-HBr (MW-LALLS 19.6 kDa) and polyhistidine-HCl (MW-LALLS 16.1 kDa) were obtained from the Sigma Chemical Company. Synthesis of Hapten Carrier Protein Conjugates and Antisera Preparation. KLH (5 mg/mL) was reacted overnight with 10 mM ACR in 50 mM sodium phosphate buffer (pH 7) at 37 °C. Using a spectrophotometric assay for carbonyl content to estimate the extent of modification (9), each KLH molecule contained a minimum of 255 carbonyl adducts after the overnight reaction with ACR (assuming an average molecular mass of 4.5 × 105 Da for KLH). The solution was then diluted in PBS (50 mM, pH 7.4) and stored at - 20 °C until use. On the day of injection, thawed aliquots were diluted with 1 vol of complete or incomplete Freund’s adjuvant (for primary injection and subsequent boosts, respectively). A rabbit then received 10 subcutaneous injections at different sites (100 µg KLH per site), followed by eight booster injections at 3 week intervals. Ten days after the final boost, the rabbit was anaesthetized and 100 mL of whole blood was collected via cardiac puncture. Serum was then prepared and stored at -20 °C. Slot-Blot Methods. To prepare adducted protein for use as assay standard, ACR was added to a solution of BSA (2 mg/mL in 50 mM sodium phosphate buffer, pH 7.0) to give concentrations ranging from 0 to 500 µM. After 2 h at 37 °C, diluted aliquots containing 100 µg of BSA were transferred to individual wells of a Hofer 48 well slot-blot manifold containing a prewetted nitrocelluose membrane. Following sample application, the membranes were blocked for 1 h in PBS containing 5% nonfat milk powder. The membrane was then treated for 1 h with a 1/1000 dilution of ACR-KLH antiserum diluted in 5% milk/ PBS. It was then washed three times with PBS and once with TBS (25 mM, pH 7.4) before treatment with secondary antibody for 45 min (goat anti-rabbit, peroxidase-coupled, 1/10 000 dilution) in 5% milk/TBS. After the washing steps were repeated, the membrane was treated with chemiluminescence substrate and then exposed to film. ELISA For Crossreactivity of ACR/KLH Antisera. ELISA methods were based on those reported by Uchida and associates (5). ELISA antigens were prepared by treating BSA (2 mg/mL) with 2 mM concentrations of 10 diverse biogenic carbonyl compounds (37 °C, 2 h). Mixtures were then diluted to 50 µg/ mL in PBS, and 300 µL volumes (i.e., 15 µg BSA) were transferred to individual wells of round-bottomed 96 well plates (Nunc Nunclon). The plates were then incubated overnight at 4 °C before they were washed with PBS and stored at 4 °C. On the day of use, plates were rinsed four times with PBS containing Tween 20 (0.05% v/v), blocked with 1% BSA in PBS (200 µL/well) for 1 h, and then washed four times with PBSTween 20. Next, 100 µL volumes of antiserum dilutions (prepared in PBS containing 1% BSA and 0.05% Tween 20) over a range of 1/100 to 1/10 000 were added to each well for 90 min. The wells were then washed four times with PBS-Tween 20 before 100 µL of secondary antibody solution was incubated in each well for 60 min (goat anti-rabbit IgG conjugated to alkaline phosphatase, 1/5000 dilution in PBS containing 1% BSA and 0.1% Tween 20). The plates were then developed using pnitrophenyl phosphate reagent (Sigma-Fast) with absorbance measurements made at 405 nm using a plate reader.

Epitope Characterization Experiments. ACR-adducted polylysine and polyhistidine were prepared for use in antigen competition experiments as described by others (10). Briefly, aminoacyl polymers were dissolved in 50 mM sodium phosphate buffer (pH 7.0) to a concentration of 1 mg/mL. ACR was then added to a 1:1 molar ratio relative to respective monomer concentrations. Reactions were allowed to proceed for 4 h at 37 °C, and then, the mixtures were dialyzed overnight (Pierce SlideA-Lyser, 3.5 kDa cutoff) at 4 °C against 10 mM sodium phosphate buffer (pH 7.0). The resulting inhibitors were then stored at -20 °C. For epitope characterization experiments, antigen (ACRadducted BSA) was prepared by reacting ACR (100 µM) with 2 mg/mL BSA for 2 h at 37 °C in 50 mM sodium phosphate buffer (pH 7.0). After dilution 1:1 with SDS-PAGE loading buffer and heat denaturation, aliquots containing 200 µg of protein were loaded onto a 4-20% polyacrylamide gel (2D gel format). Following electrophoresis, protein was transferred to nitrocellulose before the membrane was blocked for 1 h in 5% milk/ PBS. The membrane was then placed in a Bio-Rad Protean II Multiscreen apparatus, and each lane was treated for 1 h with 1/1000 dilutions of ACR-KLH antiserum that had been preincubated with 0.001-100 µg/mL concentrations of either unmodified or modified polyhistidine or polylysine. After 60 min of incubation, membranes were processed as outlined above for the slot-blot immunoassay. Adduct Stability Experiments. ACR-modified assay substrate was prepared by treating BSA (10 mg/mL) with 1 mM ACR in 10 mM sodium phosphate (pH 7.0) for 20 min at 37 °C. Next, 100 µL aliquots were transferred to 0.6 mL tubes containing 100 µL of Tris-HCl to give final concentrations of 0, 10, 30, or 100 mM Tris at either pH 7.0, 8.0, or 9.0. After 60 min at 37 °C, the samples were diluted with SDS-PAGE sample buffer, and following heat denaturation, aliquots containing 20 µg of BSA were resolved via SDS-PAGE. The proteins were then transferred to nitrocellulose and assessed for adduct abundance using the immunochemical approach outlined above. In an additional experiment, ACR-adducted BSA was diluted with either (i) buffers to give final concentrations of 100 mM Tris-HCl or triethanolamine-HCl, both of which were adjusted to a final pH of either 7.0, 8.0, or 9.0, or (ii) NaOH, to give final concentrations ranging from 1 to 100 mM. In a related timecourse experiment, BSA (10 mg/mL) was treated with 1 mM ACR in 10 mM sodium phosphate (pH 7.0) at 37 °C. At various time points (15, 30, 60, 90, and 120 min), a 100 µL aliquot of ACR-modified BSA was subjected to a 60 min reaction with 100 mM Tris-HCl (pH 8.0) at 37 °C. Controls at each time point were treated with H2O only.

Results Selective Immunodetection of ACR-Protein Adducts. Via slot-blot analysis, ACR-adducted BSA was evaluated for adducts using the rabbit antiserum, with the results shown in Figure 1A. While it did not react with unmodified BSA (Figure 1A, band 1), the antiserum detected an increasing abundance of adducts in BSA that was exposed to 25-500 µM ACR for 2 h (bands 2-6). An ELISA was then used to assess the antiserum’s reactivity toward BSA that was modified by one of 10 carbonyl compounds of known endogenous origin (Figure 1B). The serum was highly selective for ACR-derived adducts and did not detect protein damage produced by closely related

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Figure 1. Characterization of anti-ACR/KLH serum. (A) Immunodetection of ACR adducts in BSA using a slot-blot method and rabbit polyclonal antiserum raised against ACR-KLH. Assay antigen was prepared by reacting BSA (2 mg/mL) in 50 mM sodium phosphate buffer (pH 7.0) for 2 h at 37 °C in the presence of ACR (0-500 µM). ACR concentrations used in each lane were as follows: lane 1, no ACR; lane 2, 25 µM; lane 3, 50 µM; lane 4, 100 µM; lane 5, 250 µM; and lane 6, 500 µM. (B) Rabbit antiserum against ACR-KLH detects adducts in BSA generated by ACR but not nine other carbonyl compounds of known endogenous origin. Each data point is expressed as a percentage of the response detected for ACR-modified BSA and represents the mean ( SE of three independent determinations. Assay substrate was prepared by reacting BSA (2 mg/mL) with 2 mM concentrations of each carbonyl compound for 2 h at 37 °C. To ensure that the various carbonyl compounds appreciably modified the BSA, a UV spectrophotometric assay was used to assess carbonylation prior to use in the preparation of ELISA plates (9). The degree of modification varied according to the reactivity of each compound: ACR and 4-hydroxynonenal introduced an additional approximately 12 carbonyl groups per BSA molecule; 2-pentenal, 2-hexenal, 2-heptenal, 2-octenal, and 2-nonenal introduced approximately four carbonyl groups per BSA; crotonaldehyde introduced approximately 2.5 carbonyl groups per BSA; and malondialdehyde and methylglyoxal generated approximately one carbonyl group per BSA molecule. (C) Inhibition of immunodetection of ACR-BSA by ACR-modified polylysine but not ACR-modified polyhistidine or unmodified polyamino acids. Inhibitors were prepared by reacting the respective polyamino acids with excess ACR as described in the Experimental Procedures. The respective treatments were as follows: lane 1, no inhibitor present; lane 2, 100 µg/mL poly-HIS; lane 3, 10 µg/mL poly-HIS; lane 4, 1 µg/mL poly-HIS; lane 5, 100 µg/mL ACR-modified polyHIS; lane 6, 10 µg/mL ACR-modified poly-HIS; lane 7, 1 µg/mL ACR-modified poly-HIS; lane 8, 100 µg/mL poly-LYS; lane 9, 10 µg/mL poly-LYS; lane 10, 1 µg/mL poly-LYS; lane 11, 10 µg/mL ACR-modified poly-LYS; lane 12, 1.0 µg/mL ACR-modified poly-LYS; lane 13, 0.1 µg/mL ACR-modified poly-LYS; lane 14, 0.01 µg/mL ACR-modified poly-LYS; and lane 15, 0.001 µg/mL ACR-modified poly-LYS.

aldehydes including malondialdehyde (which exists mainly as the enolate of β-hydroxy-ACR at physiological pH) and crotonaldehyde (a 2-alkenal possessing just one additional carbon group to ACR) (Figure 1B). We then sought to characterize the epitope involved in immunorecognition by anti-ACR/KLH serum (Figure 1C). This experiment exploited the ability of aldehydeadducted polyaminoacyl compounds to inhibit antigen recognition by antibodies raised against aldehyde-adducted proteins (10). Figure 1C shows that preincubating antiserum with up to 100 µg/mL of either unmodified polyhistidine (lanes 2-4), ACR-modified polyhistidine (lanes 5-7), or unmodified polylysine (lanes 8-10) had no effect on the immunorecognition of ACR-adducted BSA. In striking contrast, preincubation with 10, 1, or 0.1 µg/mL ACR-modified polylysine strongly attenuated antigen recognition (lanes 11-13), although the effect was lost at 0.01 and 0.001 µg/mL dilutions of the inhibitor (lanes 14 and 15).

Instability of ACR-Protein Adducts to Tris. Having established that the antiserum could detect ACRmodified lysine groups with high specificity, we sought to develop a Western blotting method to detect such damage in proteins from cells after exposure to ACR in vitro. In keeping with protocols used previously to detect aldehyde-adducted proteins, the lysis buffer used contained Tris (11). Unfortunately, these experiments were hampered by irreproducibility and poor stability of protein adducts upon freezing of cell lysates.2 At this point, we considered the possibility that Tris targets ACR adducts in a similar manner whereby it reacts with malondialdehyde adducts in frozen DNA samples (12). This seemed feasible since Michael adducts formed in the early stages of lysine adduction by ACR are saturated analogues of oxopropenal adducts formed during modification of the N2-amine of guanine by malondialdehyde. 2Burcham,

P. C., and Fontaine, F. R. Unpublished observation.

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Figure 2. (A) Time-dependent susceptibility of ACR adducts to Tris-HCl. Assay antigen was prepared by reacting BSA (10 mg/mL) for 15-120 min with 1 mM ACR in 10 mM sodium phosphate (pH 7.0) at 37 °C. At each time point, an aliquot of ACR-BSA was subjected to a 60 min treatment with 100 mM Tris-HCl (pH 8.0) at 37 °C. The control shown at each time point was treated with H2O: lane 1, 15 min, control; lane 2, 15 min + Tris; lane 3, 30 min, control; lane 4, 30 min + Tris; lane 5, 60 min, control; lane 6, 60 min + Tris; lane 7, 90 min, control; lane 8, 90 min + Tris; lane 9, 120 min, control; and lane 10, 120 min + Tris. (B) Concentration- and pH-dependent effect of Tris on “early” ACR adducts in BSA. ACR-BSA was treated for 60 min with Tris buffer at various concentrations and pH values: lane 1, control, no Tris; lane 2, 10 mM, pH 7.0; lane 3, 30 mM, pH 7.0; lane 4, 100 mM, pH 7.0; lane 5, 10 mM, pH 8.0; lane 6, 30 mM, pH 8.0; lane 7, 100 mM, pH 8.0; lane 8, 10 mM, pH 9.0; lane 9, 30 mM, pH 9.0; and lane 10, 100 mM, pH 9.0.

Because the latter reacts with Tris (12), we explored the possibility that ACR adducts are targets for this buffer. For this experiment, we modified BSA for 15-120 min with 1 mM ACR and then subjected it to a 60 min reaction in the presence of 100 mM Tris-HCl (pH 8.0). The high concentration of ACR used in the initial modification phase overcame the problem of adduct instability during SDS-PAGE that occurred at lower ACR concentrations.3 At the end of the second reaction, aliquots of reaction mixture were assessed for adducts via Western blotting. Figure 2A confirms that in the early stages of protein modification by ACR, lysine adducts were very susceptible to Tris, but that such susceptibility was lost upon extended modification. Therefore, adducts present at 15 (lane 2 vs lane 1) and 30 min (lane 3 vs lane 4) were largely abolished by Tris; yet, those predominating at 60, 90, or 120 min completely resisted the buffer (lanes 5-10). This ability of Tris to attack adducts generated during brief exposure of BSA to ACR is consistent with the expectation that Tris participates in adduct-trapping reactions at Michael adducts, forming imine adducts that are not recognized by the antiserum. The pH and concentration dependence of Tris’s reaction with ACR adducts was then assessed by reacting BSA with 1 mM ACR for 20 min and then subjecting it to a 60 min reaction at 37 °C with 0 (Figure 2B, lane 1), 10, 30, or 100 mM concentrations of Tris-HCl, buffered to a final pH of either 7.0 (lanes 2-4), 8.0 (lanes 5-7), or 9.0 (lanes 8-10). Figure 2B confirms that the effect of Tris on ACR adducts was both pH- and concentration-dependent, with the loss of adducts most prominent at 30 and 100 mM Tris at pH 8.0 (lanes 6 and 7) and pH 9.0 (lanes 9 and 10). Stability of Adducts to a Nonimine-Forming Buffer and NaOH. While these findings raise the possibility that Tris attacks “early” Michael adducts in 3Burcham,

P. C. Unpublished observation.

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Figure 3. Early ACR adducts are susceptible to Tris-HCl but not triethanolamine-HCl or NaOH. Assay substrate (ACRBSA) was prepared by reacting BSA (10 mg/mL) for 20 min with 2 mM ACR in 10 mM sodium phosphate (pH 7.0) at 37 °C. It was then treated for 60 min with either 100 mM Tris or triethanolamine buffer at different pH values (A) or 1-100 mM NaOH (B). The treatments for individual lanes were as follows: (A) lane 1, control, ACR-BSA only; lane 2, Tris, pH 7.0; lane 3, Tris, pH 8.0; lane 4, Tris, pH 9.0; lane 5, triethanolamine, pH 7.0; lane 6, triethanolamine, pH 8.0; and lane 7, triethanolamine, pH 9.0. (B) Lane 1, control ACR-BSA; lane 2, 1 mM NaOH; lane 3, 3 mM NaOH; lane 4, 10 mM NaOH; lane 5, 30 mM NaOH; and lane 6, 100 mM NaOH.

ACR-modified protein, an alternative explanation could be that the adducts are unstable in an alkaline medium, since the effect of Tris was most pronounced at pH 8.0 and pH 9.0 (Figure 2B). To address this, the effects of Tris on ACR adducts were compared to those of triethanolamine. A tertiary amine, triethanolamine, has strong buffering capacity over a comparable pH range to Tris but cannot participate in imine-forming reactions. The results in Figure 3A indicate that while Tris-HCl strongly diminished adduct intensity (lanes 2-4 cf. lane 1), triethanolamine-HCl actually caused a pH-dependent increase in the intensity of ACR adduction (lanes 5-7). This suggests that rather than abolishing adducts, a basic environment during the secondary incubation actually enhanced lysine adduction by residual ACR. The conclusion that ACR adducts are stable to bases was reinforced by the finding that they completely resisted 1-100 mM NaOH (Figure 3B).

Discussion The experimental approach used in this study, namely, the generation of antibodies against ACR-modified proteins, was based on methods used in other laboratories to detect aldehyde-adducted proteins (4, 5, 11). Using immunohistochemical methods, Uchida and co-workers have detected ACR-modified proteins in the affected tissues of several age-related health conditions, including Alzheimer’s disease (13), atherosclerosis (4), and diabetic renal disease (14). Such damage seems consistent with endogenous formation of ACR via oxidative fragmentation of unsaturated membrane lipids (15). However, despite the usefulness of these immunohistochemical approaches, few papers confirm the use of antibodies in Western blotting experiments to detect individual ACRadducted proteins. Tanaka et al. published immunoblots obtained using anti-ACR antibodies that reveal light modification of a few proteins in UV-damaged skin (16), while Nguyen et al. also showed light modification of several proteins in neutrophils after exposure to cigarette smoke extracts (17). Moreover, after extensively enriching the target via immunoprecipitation, Biswal et al. demonstrated modification of c-jun in ACR-treated cells

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via Western blotting (18). However, the small number of targets revealed in these studies seems inconsistent with the fact that ACR is a highly reactive electrophile that is expected to alkylate many cell proteins. Indeed, when we detected carbonylated proteins as hydrazinestabilized derivatives, we found that dozens of cell proteins were heavily modified during exposure of hepatocytes to the ACR precursor, allyl alcohol (3). Yet, reproducing these experiments using the anti-ACR/KLH serum prepared in the present study in a Western blot experiment revealed inconsistent modification in proteins from allyl alcohol-treated cells.2 Insights from the present work may explain such difficulties in the immunodetection of ACR-adducted proteins via Western blotting. Our findings indicate that the nucleophilic buffer constituent Tris exhibits time-, pH-, and concentration-dependent effects on the immunoreactivity of ACR-modified protein. In contrast, the nonimine-forming buffer triethanolamine did not diminish adduct abundance. These findings suggest the need for detailed chemical investigations to confirm formation of Tris-derivatized ACR-protein adducts but should also stimulate a search for alternatives to Tris-based electrophoresis systems. Our preliminary efforts in this area have proved unproductive since most alternative methods employ nucleophilic nitrogen compounds as obligatory buffer constituents; for example, we found that ACR adducts are also unstable to ammediol, a long-standing alternative to Tris (19).3 The present findings suggest that the polyclonal antiserum raised against ACR-modified KLH during these studies recognized more than one species at modified lysine residues (e.g., Figure 2A indicates the antiserum recognized early adducts that are altered by Tris, as well as stable adducts predominating at later time points). These observations are consistent with the properties of polyclonal antiserum, since it is likely to comprise clonal antibody subpopulations that are directed against different adducts formed by ACR at lysine residues (i.e., mono- and bis-Michael adducts in addition to FDP-lysine, Figure 1). Although previous studies have indicated that FDP-lysine is the dominant species forming upon extended modification of lysine side chains by ACR (4), our ongoing MS-based explorations of the kinetics of ACR modification of a model lysine-containing peptide indicate that acyclic mono- and bis-Michael adducts are highly prevalent in the early stages of the reactions, while FDPlysine is most abundant at later time points.4 The relevance of such findings to the biological setting remains to be clarified, but they raise the possibility that acyclic Michael adducts formed as reaction intermediates in FDP-lysine formation may contribute to the toxicological actions of ACR in living tissues, and especially during the rapid onset of toxicity accompanying acute, high-dose ACR exposure. Because adducts formed during the early stages of lysine modification appear to be reactive entities that survive SDS-PAGE conditions poorly, our findings raise the intriguing question as to the intracellular fate of early Michael adducts formed in cell proteins. As mentioned above, Uchida and associates have established that monoACR adducts at lysine can be targeted by a second ACR molecule to form cyclic species (15). However, the mono4Kaminskas, L. M., Burcham, P. C., and Pyke, S. M. Unpublished observations.

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Michael adduct also seems sufficiently reactive to participate in trapping reactions with neighboring biomolecules, in much the same way carbonyl-retaining ACR adducts in DNA cross-link with DNA and peptides (20).

Acknowledgment. We gratefully acknowledge the late Kerry Goodwin for her fine contributions to the preparation of rabbit antiserum and John Piper for helpful discussions. We are grateful for financial support received from the National Health and Medical Research Council of Australia (Project Grant 104878, P.C.B. and F.R.F.), the Faculty of Health Sciences Research Committee, and NIH/NIAAA 09300 (D.R.P.).

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Communications acrolein in human A549 lung adenocarcinoma cells due to thiol imbalance and covalent modifications. Chem. Res. Toxicol. 15, 180-186. (19) Bury, A. F. (1982) Evaluation of three sodium dodecyl sulphatepolyacrylamide gel electrophoresis buffer systems. J. Chromatogr. 213, 491-500.

Chem. Res. Toxicol., Vol. 16, No. 10, 2003 1201 (20) Kurtz, A. J., and Lloyd, R. S. (2003) 1,N2-deoxyguanosine adducts of acrolein, crotonaldehyde, and trans-4-hydroxynonenal crosslink to peptides via Schiff base linkage. J. Biol. Chem. 278, 5970-5976.

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