Stereochemical Configuration of 4-Hydroxy-2-nonenal-Cysteine Adducts and Their Stereoselective Formation in a Redox-regulated Protein*

4-Hydroxy-2-nonenal (HNE), a major racemic product of lipid peroxidation, preferentially reacts with cysteine residues to form a stable HNE-cysteine Michael addition adduct possessing three chiral centers. Here, to gain more insight into sulfhydryl modification by HNE, we characterized the stereochemical configuration of the HNE-cysteine adducts and investigated their stereoselective formation in redox-regulated proteins. To characterize the HNE-cysteine adducts by NMR, the authentic (R)-HNE- and (S)-HNE-cysteine adducts were prepared by incubating N-acetylcysteine with each HNE enantiomer, both of which provided two peaks in reversed-phase high performance liquid chromatography (HPLC). The NMR analysis revealed that each peak was a mixture of anomeric isomers. In addition, mutarotation at the anomeric center was also observed in the analysis of the nuclear Overhauser effect. To analyze these adducts in proteins, we adapted a pyridylamination-based approach, using 2-aminopyridine in the presence of sodium cyanoborohydride, which enabled analyzing the individual (R)-HNE- and (S)-HNE-cysteine adducts by reversed-phase HPLC following acid hydrolysis. Using the pyridylamination method along with mass spectrometry, we characterized the stereoselective formation of the HNE-cysteine adducts in human thioredoxin and found that HNE preferentially modifies Cys73 and, to the lesser extent, the active site Cys32. More interestingly, the (R)-HNE- and (S)-HNE-cysteine adducts were almost equally formed at Cys73, whereas Cys32 exhibited a remarkable preference for the adduct formation with (R)-HNE. Finally, the utility of the method for the determination of the HNE-cysteine adducts was confirmed by an in vitro study using HeLa cells. The present results not only offer structural insight into sulfhydryl modification by lipid peroxidation products but also provide a platform for the chemical analysis of protein S-associated aldehydes in vitro and in vivo.

Lipid peroxidation in tissue and in tissue fractions represents a degradative process, which is the consequence of the production and the propagation of free radical reactions primarily involving membrane polyunsaturated fatty acids and has been implicated in the pathogenesis of numerous diseases, including atherosclerosis, diabetes, cancer, and rheumatoid arthritis, as well as in drug-associated toxicity, post-ischemic reoxygenation injury, and aging (1). The peroxidative breakdown of polyunsaturated fatty acids has also been implicated in the pathogenesis of many types of liver injury and especially in the hepatic damage induced by several toxic substances. Lipid peroxidation leads to the formation of a broad array of different products with diverse and powerful biological activities. Among them is a variety of different aldehydes (2). The primary products of lipid peroxidation, lipid hydroperoxides, can undergo carbon-carbon bond cleavage via alkoxyl radicals in the presence of transition metals giving rise to the formation of short chain, unesterified aldehydes, or a second class of aldehydes still esterified to the parent lipid. These reactive aldehydic intermediates readily form covalent adducts with cellular macromolecules, including protein, leading to disruption of important cellular functions. The important agents that give rise to the modification of protein may be represented by ␣,␤unsaturated aldehydic intermediates, such as 2-alkenals, 4-hydroxy-2-alkenals, and 4-oxo-2-alkenals (3,4).
4-Hydroxy-2-nonenal (HNE), 2 among the reactive aldehydes, is a major product of lipid peroxidation and is believed to be largely responsible for the cytopathological effects observed during oxidative stress (2,5). HNE exerts these effects because of its facile reactivity with biological materials, particularly the sulfhydryl groups of proteins. The reaction of HNE with sulfhydryl groups leads to the formation of thioether adducts that further undergo cyclization to form cyclic hemiacetals (2). Although HNE also forms Michael adducts with the imidazole moiety of histidine residues and the ⑀-amino group of lysine residues (5), the formation of thiol-derived Michael adducts, stabilized as the cyclic hemiacetal, is considered to constitute the main reactivity of HNE, because of the nucleophilic potential of the sulfhydryl group compared with those of the imidazole and amine groups. However, because of the lack of specific and reliable methods for the determination of HNE-cysteine adducts, no study has so far quantitatively demonstrated their formation in proteins.
Because HNE generated in lipid peroxidation is a racemic mixture of 4R-and 4S-enantiomers (6), the HNE Michael adducts, possessing three chiral centers at C-2, C-4, and C-5 in the tetrahydrofuran moiety (Fig. 1A), are composed of at least eight isomers. In our previous study (7), we characterized the configurational isomers of an HNE-histidine adduct by NMR spectroscopy and by molecular orbital calculations, and we found that the configuration of the tetrahydrofuran ring could affect the electron delocalization features, which contribute to the stability of the adduct. Moreover, we raised monoclonal antibodies against (R)-HNE-and (S)-HNE-histidine adducts and observed differential cellular distributions of these adducts in vivo. Balogh et al. (8) recently characterized the stereochemical configurations of the HNEglutathione adduct by NMR experiments in combination with simulated annealing structure determinations. Despite these studies, however, the stereoselectivity of the HNE Michael addition adducts generated in proteins remains to be fully explored. In this study, to gain further structural insight into sulfhydryl modification by the lipid peroxidation product, we characterized the stereochemical configuration of the HNE-Nacetylcysteine adducts by NMR spectroscopy. In addition, we adapted a pyridylamination-based method for fluorescent labeling of the HNE-cysteine adducts, using 2-aminopyridine (2-AP) and sodium cyanoborohydride (NaCNBH 3 ), and successfully analyzed the individual (R)-HNE-and (S)-HNE-cysteine adducts by reversed-phase HPLC following acid hydrolysis. Furthermore, using the pyridylamination method along with mass spectrometry, we characterized the stereoselective formation of the HNE-cysteine adducts in human thioredoxin (Trx).

EXPERIMENTAL PROCEDURES
Materials-N-Acetyl-L-cysteine was obtained from Sigma. Human recombinant Trx was produced by a method described previously (9). Thiol determination of Trx using 5,5Ј-dithiobis-2-nitrobenzoic acid gave 4.5 sulfhydryl residues per Trx protein (mol/mol) (data not shown). Sequence grade-modified trypsin was obtained from Promega. 2-AP, bovine serum albumin (fatty acid-free), dithiothreitol, and iodoacetamide were obtained from Wako Pure Chemical Industries, Ltd. The stock solutions of HNE were prepared by the acid treatment (1 mM HCl) of HNE dimethylacetal, which was synthesized according to the procedure of De Montarby et al. (10). Enantioisomeric HNEs, (R)-HNE and (S)-HNE, were prepared by the enzymatic resolution of racemic HNE (11) and purified by chiral phase HPLC on a Chiral-Pak AS column (0.46 ϫ 25 cm) (Daicel Chemical Industries, Ltd., Osaka, Japan) eluted with hexane/2-propanol/trifluoroacetic acid (90:10:0.01, by volume), at a flow rate of 1.0 ml/min. The elution profiles were monitored by UV absorbance at 224 nm. The concentrations of racemic and enantioisomeric HNE stock solutions were determined by the measurement of UV absorbance at 224 nm (12).
HPLC Analysis of Pyridylaminated HNE-Cysteine Adducts-The HNE-N-acetylcysteine adducts (1 mM) were treated with 340 mM 2-AP and 90 mM NaCNBH 3 for 24 h at 37°C. The pyridylaminated HNE-N-acetylcysteine adducts were hydrolyzed in vacuo with 6 N HCl for 24 h at 110°C. The hydrolysates were then concentrated, dissolved in distilled water, and then analyzed by reverse phase HPLC on a Sun-niest C18 column (4.6 ϫ 250 mm inner diameter, Chroma-Nik, Japan). The samples were eluted with a gradient of water containing 0.1% trifluoroacetic acid (solvent A) and acetonitrile containing 0.1% trifluoroacetic acid (solvent B), (time ϭ 0 -40 min, 95 to 60% A; 40 -45 min, 60 to 0% A), at a flow rate of 0.8 ml/min. The elution profiles were monitored by absorbance at 230 nm and by fluorescence intensity (excitation, 315 nm; emission, 380 nm).
Pyridylamination of HNE-treated Trx-Trx (1 mg/ml) in 50 mM sodium phosphate buffer (pH 7.2) was incubated with 1 mM HNE for 5-30 min at 37°C. The mixture was then applied to a PD-10 column (Sephadex G-25), equilibrated in PBS, to separate protein-bound HNE from the free aldehyde. For determination of the HNE-cysteine adducts by the pyridylamination method, the reactions were terminated by incubating with 340 mM 2-AP and 16 mM NaCNBH 3 for 24 h at 37°C. After the incubation, they were extensively dialyzed against PBS to remove a large amount of the free probe. The pyridylaminated samples were hydrolyzed in vacuo with 6 N HCl for 24 h at 110°C. The hydrolysates were concentrated, dissolved in distilled water, and then analyzed by HPLC for the pyridylaminated HNE-cysteine adducts.
Modeling Analysis-The (R)-HNE, (S)-HNE, and HNE-cysteine Michael adduct molecules were generated and energyminimized using the Dundee PRODRG Server (13). The positions of the (R)-HNE, (S)-HNE, and HNE-cysteine Michael adduct were adjusted using Coot (14) and MolFeat (FiatLux Co., Japan) to avoid steric clash and to correct the direction of C-3 toward its target thiol group of Cys 32 . The surface electric potential was calculated from the coordinate of the human Trx structure (Protein Data Bank code 1ERW, reduced-form human Trx) using the program MolFeat.
Formation of HNE-Cysteine Adducts in HeLa Cells Exposed to HNE-HeLa cells were cultivated in Dulbecco's modified Eagle's medium supplemented with 5% heat-inactivated fetal bovine serum, 2 mM glutamine, and 100 units/ml penicillin/ streptomycin. The cells were cultured at 37°C in an incubator with 95% humidified atmosphere containing 5% CO 2 . The Trx activity was measured by the insulin reduction assay (15). To analyze the HNE-cysteine adducts, the HeLa cells were treated with 0 -50 M HNE for 30 min. The cells were then washed twice with PBS, lysed in lysis buffer (4% (w/v) CHAPS, 9 M urea, 40 mM Tris (base), and centrifuged at 10,000 rpm for 5 min at 4°C; the supernatant was pyridylaminated, and the HNE-cysteine adducts were determined by HPLC analysis following acid hydrolysis.
For determination of HNE-cysteine adducts using the pyridylamination method in combination with SDS-PAGE, total cell lysates from the HNE-treated HeLa cells were pyridylaminated and separated by SDS-PAGE. Protein bands from Coomassie-stained gels were then selected, excised manually, and treated in vacuo with 6 N HCl for 24 h at 110°C to hydrolyze proteins in gels. The hydrolysates were concentrated, dissolved in distilled water, and analyzed by HPLC for the pyridylaminated HNE-cysteine adducts.

RESULTS
Reactions of N-Acetylcysteine with Racemic and Enantioisomeric HNE-Because of the introduction of new chiral centers at C-2, C-4, and C-5 during the reaction, the HNE-cysteine adducts are composed of at least eight configurational isomers (Fig. 1A). To prepare individual (R)-HNE-and (S)-HNE-cysteine adducts, N-acetylcysteine was incubated with (R)and (S)-HNE enantiomers, respectively. As shown in Fig. 1B, the reversed-phase HPLC demonstrated that the reaction of N-acetylcysteine with (R)-HNE in sodium phosphate buffer (pH 7.2) for 24 h at 37°C mainly gave two products, R1 and R2, with relative amounts of 1:1. A similar HPLC profile was observed in the reaction of N-acetylcysteine with (S)-HNE, providing S1 and S2. The liquid chromatography-MS analysis of these peaks gave a pseudomolecular ion peak at m/z 320 (M ϩ H) ϩ (data not shown), which could be expected from the Michael addition-type HNE-cysteine adducts, suggesting that they all represent the products derived from the Michael addition of the sulfhydryl group to the C-3 of HNE.
Mutarotation at the Anomeric Center of HNE-Cysteine Adducts-The hemiacetal ring opens and reforms to give products with different configurations at the anomeric center. This equilibration occurs with all reducing saccharides and is accompanied by a change in optical rotation. This process of equilibrating the ratios of two anomers in carbohydrates is known as mutarotation. It can be assumed that the anomers in the four products (R1, R2, S1, and S2) also interconvert via an open chain form (reversible formation of internal hemiacetal linkage). We indeed observed the NOE cross-peaks (Fig. 4A) between the anomeric isomers (2R,4S,5S and 2S,4S,5S) in the NOESY analysis of S1. Similar cross-peaks were also observed in the NOESY analysis of three other products (R1, R2, and S2). Mutarotation of S1 in solution at equilibrium is shown in Fig. 4B.
Fluorescent Labeling of HNE-Cysteine Adducts-Because of the lack of a specific and reliable method for determination of the HNE-cysteine adducts, no study has so far quantitatively demonstrated their formation in the HNE-modified proteins. Hence, we attempted to establish a method for the detection of HNE-cysteine adducts. Our strategy for the fluorescent labeling of HNE-cysteine adducts in proteins is illustrated in Fig. 5. The method is based on the fact that HNE forms Michael addition adducts with specific amino acid residues possessing a cyclic hemiacetal ring, which upon reaction with 2-AP in the presence of NaCNBH 3 can be converted to the pyridylaminated derivatives. It was anticipated that the HNE-cysteine adducts after the pyridylamination were resistant to the process of acid hydrolysis. To test the validity of this procedure, we first attempted to detect the adducts in the hydrolyzed samples of authentic HNE-N-acetylcysteine adduct. As shown in Fig. 6, the pyridylamination of the HNE-cysteine adduct followed by acid hydrolysis gave two products in the reverse phase HPLC analysis. Based on the observations that the pyridylamination of the authentic (R)-HNE-and (S)-HNE-cysteine adducts gave main peaks at 27 and 28 min, respectively, these products were identified to be the pyridylaminated (R)-HNE-and (S)-HNE-cysteine adducts, respectively. However, we were unable to further separate the diastereomers of the individual (R)-HNE-and (S)-HNE-cysteine adducts (data not shown).
Stereoselective Formation of HNE-Cysteine Adducts in Human Trx-To investigate the formation of HNE-cysteine adducts in proteins, we utilized Trx, the major redox-regulated protein present in the mitochondria and cytosol (16). It has been shown that Trx is sensitive to HNE (17), and the redox residues (Cys 32 and Cys 35 ) in Trx represent primary targets for HNE modification (18). When Trx (1 mg/ml) was incubated with 0.5 mM HNE in 50 mM sodium phosphate buffer (pH 7.2) at 37°C, selective loss of cysteine was  observed (supplemental Fig. S2,  panel A). In addition, accompanied by the loss of cysteine residues, the enzyme activity of Trx linearly decreased to 30% of the initial value after 30 min (supplemental Fig. S2, panel B). To further examine the selective modification of the Trx cysteine residues, the HNE-treated Trx and cysteine-blocked Trx for 5 min were analyzed by MALDI-TOF MS. As shown in supplemental Fig. S3  (panel A), the analysis of the native Trx revealed a peak of m/z 11738. When Trx was incubated with 0.5 mM HNE in 50 mM sodium phosphate buffer (pH 7.2) for 5 min at 37°C, some adducted Trx subunits were observed (m/z 11,894), as well as the peak (m/z 12,050), corresponding to the addition of one to two molecules of HNE per Trx. However, the cysteineblocked Trx (m/z 12,023, corresponding to the alkylation of five cysteine residues in Trx) was resistant to the HNE adduction (supplemental Fig. S3, panel B). These results suggest that HNE exclusively binds to the active site cysteine residues of Trx and forms adduct, most probably the Michael addition-type HNE-cysteine adducts.
To characterize the formation of the HNE-cysteine adducts in Trx, the HNE-treated and untreated Trx were pyridylaminated, hydrolyzed, and analyzed by reverse phase HPLC. As shown in  (Fig. 7B). Such stereoselective formation of HNE-cysteine adducts was not observed in other sulfhydryl proteins, including gly-ceraldehyde-3-phosphate dehydrogenase. 3 These data and the observation that, upon reaction with racemic HNE, N-acetylcysteine generated almost equal amounts of the (R)-HNE-and (S)-HNE-cysteine adducts (Fig. 6) suggested the unique stereoselectivity of Trx cysteine residues toward HNE.

Identification of Target Cysteines in Human
Trx-Based on the preferential formation of the (R)-HNEcysteine adducts, we further characterized the stereochemistry of individual HNE-cysteine adducts generated in the HNE-modified Trx. Trx (1 mg/ml) in 50 mM sodium phosphate buffer (pH 7.2) was incubated with 1 mM HNE at 37°C. To identify a hypersensitive cysteine, the incubation time was fixed to 5 min. Trx after treatment with and without HNE was pyridylaminated, carbamidomethylated, and digested with trypsin. The resulting peptides were then separated by reverse phase HPLC as described under "Experimental Procedures." The peptide map demonstrated the appearance of one major fluorescent product (P-1) and several minor fluorescent products, including P-2, from the HNEtreated Trx (Fig. 8A), whereas the native Trx provided no fluorescent products (data not shown). These peptides were further purified and subjected to MALDI-TOF MS. P-1 and P-2 gave a peak of m/z 1382.4 (Fig. 8B) and 3234.0 Da, respectively (Fig. 8C). It was speculated that, relative to the calculated masses of the unmodified peptides, these mass values corresponded to the sequences Cys 73 -Lys 81 (M r of 1149.41) and Tyr 9 -Lys 36 (M r of 2944.35), respectively, i.e. an increased mass of 235 Da corresponds to the addition of pyridylaminated HNE per peptide for P-1 and an increased mass of 292 Da corresponds to the addition of pyridylaminated HNE (235 Da) and a carbamidomethyl group (57 Da) per peptide for P-2. The reason for the incomplete digestion at Lys 21 in P-2 remains unclear.
To confirm the HNE modification sites, the tryptic fragments P-1 and P-2 were further analyzed by MALDI TOF/TOF tandem mass spectrometer.    OCTOBER 16, 2009 • VOLUME 284 • NUMBER 42 to b8 were observed to increase by 235 Da, indicating that the HNE modification site in the sequence is on Cys 73 . The MS/MS spectrum of the [M ϩ H] ϩ at m/z 3234.0 from P-2 is shown in Fig. 9B. In the MS/MS analysis, the singly charged N-terminal fragment ions (b-series ions; b4 to b22) were observed. An increased mass of 57 Da was observed in the fragment ion y4. The fragment ions b24 and b25 were observed to increase by 235 Da, whereas the y-series fragment ions y5 to y23 were observed to increase by 292 Da, indicating the HNE modification site at Cys 32 and the carbamidomethylation site at Cys 35 . Thus, MS/MS analyses identified Cys 73 as the major and Cys 32 as the minor targets of HNE in Trx.

Stereoselective Formation of HNE-Cysteine Adducts
Stereochemistry of the HNE-Cysteine Adducts Generated at Cys 32 and Cys 73 -Finally, to characterize the stereochemistry of the HNE-cysteine adducts generated at Cys 32 and Cys 73 , we hydrolyzed the tryptic fragments P-1 and P-2 and analyzed the (R)-HNE-and (S)-HNE-cysteine adducts by reverse phase HPLC. As shown in Fig. 10, the (R)-HNE-and (S)-HNE-cysteine adducts were detected in the hydrolysates of both peptide fragments in the ratio of 4:3 (P-1) and 3:1 (P-2). Thus, the (R)-HNE-and (S)-HNE-cysteine adducts were almost equally formed at Cys 73 , whereas Cys 32 exhibited a remarkable preference for the adduct formation with (R)-HNE.
Modeling Analysis-To elucidate the mechanism of stereoselective formation of HNE-cysteine adducts in Trx, we per-formed a computational analysis. The conserved active site amino acids Trp 31 -Cys 32 -Gly 33 -Pro 34 -Cys 35 form L-shaped hydrophobic pocket, and the residues Val 59 and Met 74 also participate in the hydrophobic interactions with Trx substrates (19 -21). It can be expected that the hydrophobic HNE is trapped into this hydrophobic pocket. Cys 32 is exposed to the solvent; however, the sulfhydryl group of Cys 35 is buried behind Cys 32 (Fig. 11A). The side chains of Cys 62 and Cys 69 are also buried at opposite ends of the ␣3 helix (Fig. 11B) and are surrounded by extensive acidic residues (Glu 6 , Asp 60 , Asp 61 , Asp 64 , Glu 68 , and Glu 70 ). Thus, the steric hindrance and acidic environment may reduce their accessibility to HNE. On the other hand, because Cys 73 is completely exposed to the solvent, both HNE enantiomers may therefore be freely accessible to the cysteine residue (Fig. 11A). In addition, the neighboring Lys 72 , facilitating the deprotonation of the thiol group to form a nucleophilic thiolate anion, may further potentiate the reactivity of Cys 73 with HNE. These structural properties may be associated with the observation that the (R)-HNE-and (S)-HNEcysteine adducts were almost equally formed at Cys 73 (Fig. 10). Meanwhile, the partially exposed sulfur atom of Cys 32 is located at the bottom of the L-shaped hydrophobic active site pocket. The modeling analysis of the HNE-cysteine Michael adducts adjusted to avoid steric clash shows that they can fit into the Trx active site (Fig. 11C). (R)-HNE can bind to the hydrophobic pocket in the same orientation of the HNE-cysteine adducts, having a proper direction toward its target thiol group of Cys 32 (Fig. 11D), resulting in the predominant formation of the (R)-HNE-cysteine adduct. However, the configuration of the hydroxyl group in (S)-HNE disturbs the nucleophilic attack of Cys 32 at the C-3 carbon of (S)-HNE (Fig. 11E).
Stereoselective Formation of HNE-Cysteine Adducts in HeLa Cells Exposed to HNE-To demonstrate the utility of the method for the determination of the HNE-cysteine adducts, in vivo experiments using HeLa cells were carried out. When HeLa cells were exposed to HNE (50 M) for 30 min, the Trx activity decreased to 40% of the initial value after 30 min (Fig.  12A). We then sought to detect the HNE-cysteine adducts in the cells exposed to HNE. As shown in Fig. 12B 12C). In addition, using the pyridylamination method in combination with SDS-PAGE, we confirmed the presence of both (R)-HNE-and (S)-HNE-cysteine adducts in protein bands from Coomassie-stained SDS-polyacrylamide gels (Fig. 12D).

DISCUSSION
HNE, under physiological conditions, reacts most rapidly with sulfhydryl residues of proteins, result-   OCTOBER 16, 2009 • VOLUME 284 • NUMBER 42 ing in the formation of the HNE-cysteine Michael adducts (22). Because of the introduction of new chiral centers at C-2, C-4, and C-5 during the reaction, these adducts have been suggested to be composed of at least eight configurational isomers. We previously showed that the HNE-N-acetylcysteine Michael adduct was mainly detected as two peaks upon reverse phase HPLC analysis and suggested the multiplicity of primary products in the HNE/N-acetylcysteine reaction (23). However, this previous work on the HNE-cysteine adducts has focused on racemic HNE, and therefore, the stereoselectivity remains to be fully explored. Because stereochemically distinct substrates and products have different biological effects, it is important to define the stereochemistry of the HNE-cysteine adducts. In this study, to gain structural insight into sulfhydryl modification by HNE enantiomers, we characterized the configurational isomers of the HNE-N-acetylcysteine adducts by NMR spectroscopy and observed the product stereoselectivity upon reaction of N-acetylcysteine with HNE enantiomers. In addition, during the course of the NOESY analysis, we observed unusual intermolecular NOE cross-peaks between two anomeric isomers (Fig. 3), due probably to the rapid interconversion (mutarota-tion) between the isomers. This finding suggests that the adduct may be in the equilibrium between the ring-opened and ring-closed structures. This structural property may be characteristic of the 4hydroxy-2-alkenal-derived Michael adducts and important for understanding the biological impact on the formation of the adducts composed primarily of the four ring-opened and eight ring-closed structures.

Stereoselective Formation of HNE-Cysteine Adducts
To differentiate between various modes of carbonyl group formation, a method for the detection and quantification of protein carbonyl groups associated with the conjugation of protein sulfhydryl groups with lipid peroxidation products was previously developed (23). This method is based on the reduction of the adducts with NaB[ 3 H]H 4 to stable radioactive derivatives followed by cleavage of the thioether linkage upon treatment with Raney nickel. Although this procedure is not specific for the HNE-cysteine adducts, it can provide a means of determining the fraction of total free carbonyl groups introduced into proteins via reaction of ␣,␤-unsaturated aldehydes with protein sulfhydryl groups. In later studies, both HNE-histidine and HNE-lysine Michael adducts generated in peptides and proteins were analyzed by HPLC following o-phthaldehyde derivatization (24 -26). This method allowed quantitating the Michael addition-type HNE-histidine adducts and trace amounts of HNE-lysine adducts in Cu 2ϩ -oxidized low density lipoprotein (27). However, in terms of the HNE-cysteine adducts, no study has so far revealed that they are indeed formed in the HNE-modified proteins because of the lack of specific and reliable methods for the determination of the adducts. To analyze the HNE-cysteine adducts in protein, we adapted a reductive amination-based pyridylamination using 2-AP and NaCNBH 3 . This method was originally developed for detection of reducing sugars (28). Upon pyridylamination followed by acid hydrolysis of HNE-N-acetylcysteine, we detected two fluorescent products, which were identified to be the pyridylaminated (R)-HNEand (S)-HNE-cysteine adducts (Fig. 6). It is noteworthy that these 2-AP derivatives of the HNE-cysteine adducts were stable against the acid hydrolysis using 6 N HCl (110°C, 24 h). Thus, the mild derivatization conditions and derivatives resistant to acid hydrolysis permitted the reliable and accurate quantification of the HNE-cysteine adducts. The derivatives were fluorescent after acid hydrolysis and there-FIGURE 12. Detection of HNE-cysteine adducts in HeLa cells exposed to HNE. A, changes in Trx activity. HeLa cells were exposed to HNE (50 M) for 30 min. The Trx activity was measured by the insulin reduction assay. B and C, detection of the HNE-cysteine adducts in total cell lysates. HeLa cells were treated with 0 -50 M HNE for 30 min. The cells were then washed twice with PBS, lysed, and centrifuged at 10,000 rpm for 5 min at 4°C; the supernatant was pyridylaminated, and the HNE-cysteine adducts were determined by HPLC analysis following acid hydrolysis. D, detection of the HNE-cysteine adducts using the pyridylamination method in combination with SDS-PAGE. Total cell lysates from the HNE-treated HeLa cells were pyridylaminated and separated by SDS-PAGE. Protein bands (Fr. 1-3) from Coomassie-stained gels (left panel) were then selected, excised manually, and treated in vacuo with 6 N HCl for 24 h at 110°C to hydrolyze proteins in gels. The hydrolysates were concentrated, dissolved in distilled water, and analyzed by HPLC for the pyridylaminated HNE-cysteine adducts (right panel).