Polyclonal Antibodies to a Fluorescent 4-Hydroxy-2-nonenal (HNE

4-Hydroxy-2-nonenal (HNE)-Derived Lysine-Lysine. Cross-Link: Characterization and Application to. HNE-Treated Protein and in Vitro Oxidized Low-Densit...
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Chem. Res. Toxicol. 2000, 13, 406-413

Polyclonal Antibodies to a Fluorescent 4-Hydroxy-2-nonenal (HNE)-Derived Lysine-Lysine Cross-Link: Characterization and Application to HNE-Treated Protein and in Vitro Oxidized Low-Density Lipoprotein Guozhang Xu, Yahua Liu, and Lawrence M. Sayre* Department of Chemistry, Case Western Reserve University, Cleveland, Ohio 44106 Received December 6, 1999

Oxidative modification of low-density lipoprotein (LDL) is thought to play a key role in the etiology of atherosclerosis. Oxidized LDL that accumulates in atherosclerotic plaques is known to exhibit a characteristic fluorescence with excitation and emission near 360 and 430 nm, respectively. (E)-4-Hydroxy-2-nonenal (HNE), formed during LDL oxidation, is capable of modifying LDL to generate the same fluorescent signature. The HNE-derived fluorophore was shown by us to possess a 2-hydroxy-2-pentyl-1,2-dihydropyrrol-3-one iminium (HPDPI) structure. We herein report the synthesis of the HPDPI-derived lysine-lysine cross-link needed as a standard reference for HPLC quantitation of the cross-link in protein hydrolysates. The main focus of the current work, however, is the design and development of two polyclonal antibodies against the HPDPI epitope. Utilizing these antibodies, levels of the HPDPI epitope were estimated in HNE-treated bovine serum albumin and in copper-oxidized LDL by an enzyme-linked immunosorbent assay. Our results are consistent with the premise that the fluorescent HPDPI cross-link is a key contributor to the fluorescence exhibited by atherosclerotic lesions.

Introduction Over the last two decades, much evidence suggesting that lipid peroxidation plays a crucial role in the pathogenesis of atherosclerosis has accumulated (1, 2). It is believed that lipid peroxidation is involved in the oxidative modification of human low-density lipoprotein (LDL)1 (1), which accumulates in the arterial wall, ultimately leading to the formation of atherosclerotic plaques (3). A strong fluorescence with excitation (ex) and emission (em) near 360 and 430 nm, respectively, is associated with the oxidation of LDL, and the measurement of this fluorescence is used to estimate the extent of LDL oxidation. Of the complex mixture of reactive aldehydes generated during peroxidation of LDL lipids (1, 4), an especially cytotoxic aldehyde, (E)-4-hydroxy-2-nonenal (HNE), is created by cleavage of peroxidized arachidonic and linoleic esters (5). There is increasing evidence that HNE not only induces aggregation of LDL (3-10) and crosslinking of cytoskeletal proteins (11-13) but also contributes to the ex/em 360/430 nm fluorescence seen in atherosclerotic plaques and age-related lipofuscin granules (7, 13-15). HNE-cross-linked, fluorescent protein has been shown to be resistant to proteolysis and acts * To whom correspondence should be addressed: Department of Chemistry, Case Western Reserve University, Cleveland, OH 44106. Phone: (216) 368-3704. Fax: (216) 368-3006. E-mail: [email protected]. 1 Abbreviations: LDL, human low-density lipoprotein; HNE, (E)4-hydroxy-2-nonenal; HPDPI, 2-hydroxy-2-pentyl-1,2-dihydropyrrol3-one iminium; ELISA, enzyme-linked immunosorbent assay; HOSu(SO3)Na, N-hydroxysulfosuccinimide sodium salt; oxLDL, oxidized LDL; KLH, keyhole limpet hemocyanin; BSA, bovine serum albumin; RNase, ribonuclease A; ONE, (E)-4-oxo-2-nonenal; BCA, bicinchoninic acid; COA, chicken ovalbumin; PBS, phosphate-buffered saline.

as a potent noncompetitive inhibitor of the multicatalytic protease-proteasome, a proteolytic complex involved in the degradation of oxidatively modified proteins (13, 16). Thus, HNE modification likely contributes to the accumulation of aggregated protein observed during aging and the progression of several age-related degenerative diseases. HNE-induced intra- and intermolecular protein crosslinking was initially postulated to arise from Lys Schiff base formation at the C-1 carbonyl and Michael addition of Lys, Cys, or His nucleophiles at C-3 (17). However, in the modeling of this possibility for Lys, the 1:2 HNEamine Schiff base Michael adducts were found to be formed reversibly and, upon isolation by removal of excess amine, were found to undergo complete reversal to HNE when dissolved in neutral aqueous phosphate buffer (18). The fact that verification of the presence of the 1:2 Schiff base Michael adducts in solution could be achieved only by reduction with NaBH4 (18, 19) led us to conclude that such Lys-Lys cross-link would be stable only in select protein microenvironments and thus was an unlikely candidate for the stable cross-links which form upon treatment of proteins with HNE. Recently, we reported that the major ex/em 360/430 nm fluorophore formed from HNE or (E)-4-hydroxy-2hexenal (HHE) and primary amines was a 2-hydroxy-2alkyl-1,2-dihydropyrrol-3-one iminium 1:2 adduct, representing a four-electron oxidative Lys-Lys cross-link (20, 21). Parallel, independent work by Itakura et al. (22) led them to propose the free base form of the same fluorophore formed from HNE and NR-hippuryllysine. Upon treatment of proteins with HNE, the extent of

10.1021/tx990200s CCC: $19.00 © 2000 American Chemical Society Published on Web 04/19/2000

Immunochemical Detection of an HNE Cross-Link

cross-linking and fluorescence development responded in parallel to changing reaction conditions, suggesting that the identified fluorophore is the major HNE-derived protein cross-link (21, 23). However, our attempt to directly characterize this fluorophore in HNE-modified protein upon standard acid hydrolysis (6 N HCl for 24 h at 110 °C) failed due to its rapid decomposition under these conditions (24).2 Complete enzymatic proteolysis and subsequent HPLC analysis and/or on-line LC/MS analysis may provide an alternative method for direct quantitation of the HNEderived fluorophore in proteins. As a forerunner to such studies, we describe herein the synthesis in good yield of the HPDPI-based lysine-lysine cross-link that would serve as an HPLC reference standard. At the same time, immunochemical methods required for in situ localization studies can also provide the basis of convenient and reliable quantification in cases where the antibodies recognize discrete structures with a high degree of specificity. We thus report herein two approaches to obtaining polyclonal antibodies that specifically recognize the HPDPI fluorescent cross-link. Following immunoaffinity purification, these antibodies were used to detect and quantify the HPDPI epitope created in HNE-treated protein and in copper-oxidized LDL. This provides the first evidence for the existence of the protein-bound HPDPI epitope in LDL oxidized in vitro, and sets the stage for the assessment of this epitope in biological samples.

Experimental Procedures Materials. Keyhole limpet hemocyanin (KLH) was obtained from Calbiochem (La Jolla, CA). Bovine serum albumin (BSA), ribonuclease A (RNase A), chicken ovalbumin (COA), disodium p-nitrophenyl phosphate, cyanogen bromide (CNBr)-activated Sepharose 4B, Immunopure (G) immobilized protein G gel, IgG binding buffer, and IgG elution buffer were obtained from Sigma Chemical Co. (St. Louis, MO). Spectrapor membrane tubing for standard dialysis had a Mr cutoff of 12000-14000. N-Hydroxysulfosuccinimide sodium salt [HOSu(SO3)Na] was obtained from Pierce (Rockford, IL). Alkaline phosphatase-conjugated goat anti-rabbit IgG was from Boehringer-Mannheim (Indianapolis, IN). HNE and 4-oxo-2-nonenal (ONE) were prepared as described previously (21). BSA-bound HNE-N-acetyl-Lhistidine (24) and 4-oxononanal-BSA (25) were from previous studies. Phosphate-buffered saline (PBS) was prepared from a pH 7.4 stock solution containing 0.2 M NaH2PO4/Na2HPO4, 3.0 M NaCl, and 0.02% NaN3 (w/w). This solution was diluted 20fold as needed. Native human LDL (500 µg of apoB-100/mL), isolated according to the general procedure (26), was generously supplied by H. Hoff (Cleveland Clinic Foundation, Cleveland, OH) and was dialyzed for 5 h at 4 °C against 0.2 M PBS (pH 7.4). All other reagents and chemicals were AR or ACS grade. General Methods. 1H NMR (300 MHz) and 13C NMR (75.1 MHz) spectra were recorded on a Varian Gemini 300 instrument. In all cases, Me4Si or the solvent peak served as the internal standard for reporting chemical shifts. In the 13C NMR line listings, attached proton test (APT) designations are given as (+) or (-) following the chemical shift. When desirable, the samples for proton NMR spectroscopy were exchanged three times with CD3OD or D2O under N2. High-resolution mass spectra were obtained at 20 eV on a Kratos MS-25A instrument. Electrospray ionization mass spectra were analyzed on an Extrel Benchmar mass spectrometer equipped with a prototype elec2 HNE-derived fluorophores such as N,N′-dibutyl-2-hydroxy-2-pentyl-1,2-dihydropyrrol-3-one iminium and N,N′-bis(5,5-carboxypentyl)2-hydroxy-2-pentyl-1,2-dihydropyrrol-3-one iminium are stable in 2 N HCl at room temperature but are labile under standard HCl hydrolysis conditions.

Chem. Res. Toxicol., Vol. 13, No. 5, 2000 407 trospray interface (ABB Extrel, Pittsburgh, PA) and a 20 cm (4 mm inscribed radius) quadrupole mass filter with a m/z range of 0-2000. UV spectra were obtained with a Perkin-Elmer model Lambda 3B spectrophotometer, and fluorescence spectra were recorded on an SLM8100C spectrofluorometer. For all enzyme-linked immunosorbent assays (ELISAs), duplicates of each sample were run on the same plate. All protein concentrations were determined with the bicinchoninic acid (BCA) assay using BSA as the standard (24) (kit obtained from Pierce). HPLC. The HPLC system (Shimadzu) consisted of two LC6A pumps, a model SCL-6A controller, a model C-R3A integrator, and a model SPD-6A variable-wavelength UV detector. Spectral monitoring was accomplished by substituting a HewlettPackard diode array detector (HP 1050). Preparative-scale separations were accomplished with two 8 mm × 100 mm Waters RCM Radial PAK cartridges in series (packing, Resolve C18, 10 µm), with gradient elution by a binary system consisting of 0.1% aqueous CF3COOH (A) and MeOH (B) at a flow rate of 1.5 mL/min. Analytical and preparative thin-layer chromatography was performed using Merck silica gel 60 plates with a 254 nm indicator. N,N′-Bis[5-(tert-butoxycarboxamido)-5-carboxypentyl]2-hydroxy-2-pentyl-1,2-dihydropyrrol-3-one Iminium (1). To an aqueous solution (10 mL) containing NR-(tert-butoxycarbonyl)lysine (NR-t-BOC-lysine) (985 mg, 4 mmol), Na2HPO4 (26 mg), and CuCl2‚H2O (2 mg, 0.01 mmol) was added ONE (154 mg, 1 mmol) in CH3CN (10 mL). The reaction mixture turned brown immediately and was kept stirring for 16 h, and then concentrated. Separation of desired product 1 (138 mg, 22%) was achieved by semipreparative HPLC (0.1% TFA and MeOH as the eluent). Compound 1 was also isolated according to the same semipreparative HPLC conditions in 0.8% yield from the 24 h reaction of HNE (390 mg, 2.5 mmol) with NR-t-BOC-lysine (4.9 g, 20 mmol) in 100 mL of phosphate buffer (pH 7.4)/CH3CN (3:1, v/v): 1H NMR (D2O) δ 0.87 (t, J ) 5.00 Hz, 3 H), 0.870.92 (m, 2 H), 1.21-1.31 (m, 6 H), 1.42 (s, 18 H), 1.61-1.85 (m, 10 H), 1.84-2.10 (m, 2 H), 3.30-3.39 (m, 2 H), 3.45-3.51 (m, 2 H), 3.95-4.00 (m, 2 H), 5.47 (d, J ) 2.51 Hz, 1 H), 8.26 (d, J ) 2.60 Hz, 1 H); 13C NMR (D2O) δ 14.34 (-), 22.91 (+), 23.36 (+), 23.68 (+), 24.02 (+), 28.85 (-), 29.12 (+), 29.66 (+), 32.33 (+), 33.50 (+), 33.57 (+), 37.21 (+), 45.38 (+), 46.66 (+), 56.33 (-), 56.39 (+), 80.17 (+), 86.86 (-), 97.10 (+), 157.74 (+), 167.64 (-), 176.13 (+), 178.93 (+), 179.23 (+); ESI-MS m/z calcd for C31H55N4O9 (M+) 627.4, found 627.3. N,N′-Bis(5-amino-5-carboxypentyl)-2-hydroxy-2-pentyl1,2-dihydropyrrol-3-one Iminium (2). Dissolution of the t-BOC derivative 1 in CF3COOH, followed by evaporation, yielded the HPDPI lysine-lysine cross-link 2 as its trifluoroacetate salt: 1H NMR (D2O) δ 0.85 (t, J ) 5.00 Hz, 3 H), 0.850.94 (m, 2 H), 1.23-1.29 (m, 6 H), 1.61-1.85 (m, 10 H), 2.002.15 (m, 2 H), 3.30-3.51 (m, 4 H), 3.95-4.00 (m, 2 H), 5.49 (d, J ) 2.52 Hz, 1 H), 8.21 (d, J ) 2.61 Hz, 1 H); ESI-MS m/z calcd for C21H39N4O5 (M+) 427.5, found 427.4. N,N′-Bis(5-carboxypentyl)-2-hydroxy-2-pentyl-1,2-dihydropyrrol-3-one Iminium (3). To an aqueous solution (10 mL) containing 6-aminocaproic acid (524 mg, 4 mmol), Na2HPO4 (26 mg), and CuCl2‚H2O (2 mg, 0.01 mmol) was added ONE (154 mg, 1 mmol) in CH3CN (10 mL). The reaction mixture turned brown immediately and was kept stirring for 16 h, and then concentrated. Separation of desired product 3 (103 mg, 26%) was achieved by semipreparative HPLC (0.1% TFA and MeOH as the eluent): 1H NMR (D2O) δ 0.82 (t, J ) 6.66 Hz, 3 H), 0.860.97 (m, 2 H), 1.21-1.27 (m, 4 H), 1.33-1.44 (m, 4 H), 1.581.70 (m, 6 H), 1.70-1.82 (m, 2 H), 1.75-1.91 (m, 1 H), 2.062.22 (m, 1 H), 2.39 (t, J ) 7.14 Hz, 2 H), 2.40 (t, J ) 7.14 Hz, 2 H), 3.41 (t, J ) 6.60 Hz, 2 H), 3.51 (t, J ) 7.43 Hz, 2 H), 5.47 (d, J ) 3.12 Hz, 1 H), 8.19 (d, J ) 3.12 Hz, 1 H); 13C NMR (D2O) δ 14.28 (-), 22.19 (+), 22.84 (+), 24.91 (+), 25.04 (+), 26.48 (+), 26.72 (+), 28.62 (+), 29.07 (+), 31.57 (+), 34.74 (+), 34.83 (+), 36.64 (+), 45.21 (+), 46.27 (+), 89.05 (-), 96.57 (+), 168.02 (-), 178.0 (+), 178.10 (+), 180.10 (+); HRMS m/z calcd for C21H37N2O5 397.2704, found 397.2711.

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N,N′-Bis(4,4-diethoxybutyl)-2-hydroxy-2-pentyl-1,2-dihydropyrrol-3-one Iminium (6). An aqueous solution (15 mL) containing 4-aminobutyraldehyde diethylacetal (500 mg, 3.1 mmol) and CuCl2‚H2O (2 mg, 0.01 mmol) was neutralized to pH 8.3 with solid NaH2PO4. Then, freshly prepared ONE (154 mg, 1 mmol) in 10 mL of CH3CN was added to the amine solution dropwise. Within a few minutes, strong fluorescence with ex and em at 360 and 430 nm, respectively, had developed. After 16 h, the crude mixture was concentrated and the resulting brown residue was purified by semipreparative HPLC (CH3CN containing 0.05% TFA as the eluent) to afford the desired product 6 (91 mg, 20%): 1H NMR (CD3OD) δ 0.90 (t, J ) 7.53 Hz, 3 H), 1.17 (t, J ) 7.05 Hz, 6 H), 1.18 (t, J ) 6.69 Hz, 6 H), 1.22-1.38 (m, 6 H), 1.58-1.73 (m, 6 H), 1.73-1.90 (m, 2 H), 2.22-2.32 (m, 2 H), 3.38-3.43 (m, 2 H), 3.45-3.59 (m, 6 H), 3.61-3.72 (m, 4 H), 4.53 (m, 2 H), 5.49 (m, 1 H), 8.30 (m, 1 H); 13C NMR (CD OD) δ 14.26 (-), 14.40 (-), 22.92 (+), 23.38 (+), 3 24.77 (+), 25.52 (+), 31.97 (+), 32.37 (+), 34.78 (+), 37.20 (+), 45.43 (+), 46.59 (+), 62.90 (+), 63.03 (+), 88.93 (-), 97.10 (+), 104.10 (-), 167.87 (-), 174.19 (+); HRMS m/z calcd for C25H49N2O5 457.3644 (MH+), found 457.3637; HRMS m/z calcd for C25H48N2O5 456.3565 (M+), found 456.3559. HPDPI-Butyl-KLH Antigen. Compound 6 (21.0 mg, 45 µmol) was treated with 2 N HCl (10 mL) for 16 h; then, the solvent was removed completely with a stream of nitrogen. The resulting yellow residue was dissolved in 0.1 M sodium phosphate buffer (5 mL, pH 7.4) containing KLH (20.94 mg). To this solution was added NaCNBH3 (22.5 mg, 360 µmol), and the reaction mixture was incubated at room temperature for 4 h followed by two successive 12 h dialyses against 0.1 M sodium phosphate buffer (500 mL, pH 7.4). The concentration of KLHbutyl-linked HPDPI fluorophore (86.5 nmol/mg of KLH) was estimated by measuring ∆A360 (360 ) 1.3 × 104 M-1 cm-1). HPDPI-Butyl-BSA Coating Agent. Compound 6 (4.6 mg, 10 µmol) was hydrolyzed by acid treatment (5 mL of 2 N HCl for 16 h); the resulting mixture was concentrated, and the residue (3.1 mg) was dissolved in 2 mL of a 4:1 (v/v) mixture of 0.1 M sodium phosphate buffer (pH 7.4) and MeOH. To this solution was added BSA (5.5 mg, 0.085 µmol); the resulting mixture was stirred for 10 min at room temperature, and NaBH3CN (5 mg, 80 µmol) was added. After 6 h, a small amount of precipitated BSA was removed by filtration, and the reaction mixture was dialyzed twice against PBS buffer (pH 7.4, 500 mL) for 12 h each time. The final concentration of soluble BSA was 0.96 mg/mL. The concentration of BSA-butyl-linked HPDPI (106 nmol/mg of BSA) was estimated by measuring ∆A360 (360 ) 1.3 × 104 M-1 cm-1). ONE-RNase Antigen. A solution of ONE (2.5 mM final concentration), RNase A (41.1 mg), and CuCl2‚H2O (1 mg, 5 µmol) in 3 mL of a 5:1 mixture of 0.1 M sodium phosphate buffer (pH 7.4) and MeOH was incubated at room temperature for 16 h. The mixture was dialyzed twice against 0.1 M PBS buffer (500 mL, pH 7.4) for 12 h each time. After solubilization of aggregates by addition of 250 µL of 6.5 mM SDS, the concentration of the RNase-bound HPDPI fluorophore (10.9 nmol/mg of RNase A) was estimated by measuring ∆A360 (360 ) 1.3 × 104 M-1 cm-1). ONE-BSA Coating Agent. A solution of ONE (2.5 mM final concentration) and BSA (45 mg) in 3 mL of a 5:1 mixture of 0.1 M sodium phosphate buffer (pH 7.4) and MeOH was incubated at room temperature for 16 h. A small amount of precipitated BSA was removed by filtration. The mixture was dialyzed twice against 0.1 M PBS buffer (500 mL, pH 7.4) for 12 h each time. The concentration of the BSA-bound HPDPI fluorophore (21.1 nmol/mg of BSA) was estimated by measuring ∆A360 (360 ) 1.3 × 104 M-1 cm-1). The final concentration of the ONE-BSA coating agent was 14.5 mg/mL. Immunization. Polyclonal antibodies were generated in New Zealand white rabbits. The two immunogens that were used were HPDPI-KLH [86.5 nmol of fluorophore/mg of KLH, 4.19 mg/mL of KLH in PBS (pH 7.4)] and ONE-RNase [10.9 nmol of fluorophore/mg of RNase, 13.7 mg/mL of RNase in PBS (pH

Xu et al. 7.4)]. Both immunogens (400 µL) were emulsified to homogeneity with an equal volume of Freund’s complete adjuvant (400 µL) using a syringe fitted with a three-way valve. One rabbit each was inoculated intradermally in several locations on the back (125 µL total) as well as the rear leg (125 µL total). Thereafter, booster injections (250 µL) of both immunogens in Freund’s incomplete adjuvant were given every 21 days. Test bleeds (2-5 mL) were obtained 10 days after each inoculation, and the antibody titer was monitored with an ELISA. The final bleed was obtained only after reaching a convincing plateau of the titer. ELISA Determination of Antibody Titer. To monitor the HPDPI-KLH and ONE-RNase antibody levels in rabbit blood serum, the corresponding BSA conjugates (HPDPI-BSA, 106 nmol of fluorophore/mg of BSA or ONE-BSA, 21.1 nmol of fluorophore/mg of BSA) were used as coating agents. HPDPIBSA (100 µL of a 1:100 dilution of the original HPDPI-BSA) or ONE-BSA (100 µL of a 1:100 dilution of the original ONEBSA) was added to each well of two sterilized Baxter ELISA plates. The plate was then incubated at 37 °C for 1 h in a moist chamber. After the coating solution was discarded, each well was washed with PBS (3 × 300 µL), then filled with 1.0% COA in PBS (300 µL), and incubated at 37 °C for 1 h to block remaining active sites on the plastic surface. Each well was washed with 0.1% COA in PBS (300 µL), and then either 100 µL of rabbit serum from each bleed (available up to that point) diluted 1:1000 with 0.2% COA or 100 µL of 0.2% COA in PBS without serum (blank) was dispensed into the sample wells. Normal rabbit (not injected with antigen) serum diluted as described above was employed as a negative response control. The plate was then incubated at room temperature for 1 h with gentle agitation. After the supernatants had been discarded and the wells washed with 0.1% COA (3 × 300 µL), 100 µL of alkaline phosphatase-conjugated goat anti-rabbit IgG diluted 1:1000 with 1.0% COA was added to each well, and the plate was again incubated with gentle agitation at room temperature for 1 h. After the supernatant had been discarded, the wells were washed with 0.1% COA (3 × 300 µL). To each well was then added 100 µL of a solution of disodium p-nitrophenyl phosphate (10 mg) in water (11 mL) adjusted to pH 9.6 with NaOH, containing glycine (50 mM) and MgCl2 (1 mM), and the plate was allowed to develop for 20-30 min at room temperature. This time period resulted in a maximum absorbance of about 1 when the titer plateaued. The development was terminated by adding 3 N NaOH (50 µL) to each well before measuring the final absorbance values using a Bio-Rad 450 dual-wavelength microplate reader, with detection at 405 nm relative to 655 nm. Antibody Purification on a Protein G Column. The crude HPDPI-KLH antiserum from the 93-day bleed of one rabbit contained 36.07 mg/mL protein, and the crude ONE-RNase antiserum from the 111-day bleed of another rabbit contained 39.86 mg/mL protein as determined from the A280 (1.35 for 1 mg/mL) (25). The crude antibodies were purified by using a protein G column. The resulting HPDPI-KLH and ONERNase IgG fractions contained 1.28 and 1.12 mg/mL IgG protein, respectively, as determined from the A280. This corresponds to 14.2 and 10.1%, respectively, of the total protein in these two crude sera. Antibody Purification on an Affinity Column. (1) Hapten-BSA Conjugation. A DMF solution (0.5 mL) containing 3 (4 mg, 10 µmol), 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide methiodide (6 mg, 20 µmol), and N-hydroxysulfosuccinimide (6.6 mg, 30 µmol) was incubated for 2 h at room temperature, and then added to 50 mM sodium phosphate buffer (1.5 mL, pH 7.4) containing BSA (5.31 mg). After 16 h, the mixture was dialyzed twice against 50 mM phosphate buffer (500 mL, pH 7.4). The precipitated protein was pelleted by centrifugation, and the protein concentration in the supernatant was determined to be 2.32 mg/mL by the BCA assay. The HPDPI fluorophore content was estimated to be 0.12 µmol of fluorophore/mg of BSA based on a ∆A360 of 3.76.

Immunochemical Detection of an HNE Cross-Link (2) Binding of a Hapten-BSA Conjugate to Sepharose. Cyanogen bromide (CNBr)-activated Sepharose 4B (2 g) was washed and reswollen on a sintered glass filter using 1 mM HCl (300 mL). Then, the above hapten-BSA conjugate (2 mL, 4.6 mg of modified BSA containing 0.56 µmol of 3) was added to the gel suspension and the mixture incubated for 6 h at room temperature. The remaining active sites on Sepharose were blocked with 0.2 M sodium glycinate buffer (pH 8.0). (3) Column Packing, Sample Loading, and Antibody Elution. The excess unbound hapten-BSA conjugate was removed by washing alternately with 0.1 M sodium bicarbonate buffer (pH 8.3) (containing 0.5 M NaCl) and 0.1 M acetate buffer (pH 4). On the basis of the unbound BSA which was washed off the affinity column (0.5 mg, as determined by the BCA assay), we estimated that 4.1 mg of the hapten-BSA conjugate was bound to Sepharose, corresponding to a loading of 2.05 mg of the hapten-BSA conjugate per gram of CNBr-activated Sepharose. The resulting slurry was equally distributed into two polypropylene columns. Each column was equilibrated with 10 mM Na2HPO4 and 100 mM KCl (pH 7.4) and then loaded with 1 mL of crude rabbit anti-HPDPI-KLH or anti-ONE-RNase sera. The column was allowed to equilibrate at room temperature for 2 h, followed by extensive washings with 10 mM Na2HPO4 and 100 mM KCl (pH 7.4) to ensure complete removal of unbound serum. The affinity column-bound anti-HPDPI-KLH and anti-ONE-RNase antisera were then eluted with 100 mM glycine hydrochloride buffer (pH 2.8). The pH of the antibody solutions was adjusted to 7.4 by adding 1.0 M Tris buffer (pH 8.0). The purified antibodies were dialyzed against 50 mM PBS (2 × 500 mL, pH 7.4) for 24 h. The yields were 0.90 mg of immunopurified HPDPI-KLH antibody (in 2 mL) and 0.95 mg of immunopurified ONE-RNase antibody (in 2 mL) from 1 mL of each crude antiserum. The titers of the immunopurified antibodies were determined relative to the titers of the original crude antisera set as 100%. Competitive Antibody Binding Inhibition Studies. For antibody binding inhibition studies to measure cross-reactivities, HPDPI-BSA and ONE-BSA were used as coating agents for anti-HPDPI-KLH and anti-ONE-RNase antibodies, respectively. Aliquots (100 µL) of the respective coating agent were added to each well of a 96-well microtiter plate, which was then incubated at 37 °C for 1 h in a moist chamber. After the supernatant was discarded, each well was washed with phosphate-buffered saline (PBS, 3 × 300 µL), and then filled with 1.0% chicken ovalbumin (COA) in PBS (300 µL, pH 7.4), and the plate was incubated at 37 °C for 1 h to block the remaining sites on the plastic surface. The supernatant was discarded, and each well was washed with 0.1% COA in PBS (3 × 300 µL). Eight serial dilutions (1:10) of each inhibitor (HPDPI-BSA, ONE-BSA, HNE-BSA, BSA-bound HNE-N-acetyl-L-histidine, ON-BSA, and BSA) in PBS (150 µL, pH 7.4) were incubated in test tubes at 37 °C for 1 h with solutions of either the antiHPDPI-KLH antibody or the anti-ONE-RNase antibody [150 µL of 1:100 dilutions of purified antisera in PBS (pH 7.4) containing 0.2% COA]. The antibody-antigen complex solutions (100 µL) were then added in duplicate to each well of the plate, and the plate was then incubated at room temperature with gentle agitation on a shaker for 1 h. The remaining procedure, including application of the secondary antibody and final absorbance development, was identical to that described above for the ELISA determination of the antibody titer. Absorbance values for duplicate assays were averaged and scaled, and the data were curve-fitted as previously described (25). Incubation of BSA with HNE. BSA (226 mg, 3.55 µmol) was dissolved in 0.2 M PBS buffer (9 mL, pH 7.4) containing CuSO4 (0.5 µmol). The reaction was initiated by addition of a HNE solution in MeOH (8 mg of HNE/mL) and allowed to proceed at 37 °C for 48 h, and then the mixture was frozen in a freezer. In Vitro Oxidation of LDL. Native LDL (250 µg of apoB100/0.5 mL) was dialyzed against 500 mL of 20 µM CuSO4 in PBS buffer (pH 7.4) at 37 °C for 5 h (27). Oxidation was stopped

Chem. Res. Toxicol., Vol. 13, No. 5, 2000 409 by adding Na2EDTA (1 mg/mL, final concentration) and butylated hydroxytoluene (BHT) (40 µM, final concentration). The resulting solution was dialyzed against PBS (pH 7.4) for 24 h at room temperature.

Results and Discussion Synthesis of the Lysine-Lysine 2-Hydroxy-2pentyl-1,2-dihydropyrrol-3-one Iminium (HPDPI) Cross-Link. The HPDPI-derived cross-links of primary amines (20), NR-hippuryllysine (22), and NR-acetyllysine (28) have been reported, the latter two in very low yields from HNE. However, there have been no reports of the free lysine-lysine HPDPI cross-link that would be needed as a reference standard for HPLC detection of the fluorophore in protein hydrolysates. The di-t-BOCprotected derivative 1 was obtained in good yield by reaction of NR-t-BOC-L-lysine with 4-oxononenal (ONE), and, following semipreparative HPLC purification, was subsequently converted to the free lysine derivative 2 using CF3COOH (Scheme 1). Scheme 1

Design and Synthesis of Antigens for 4-HNEDerived Fluorophore. To incorporate the HNE-derived fluorophore into the carrier protein, we first considered EDC coupling of the diacid fluorophore derivative 3, derived from 6-aminocaproic acid and ONE (Scheme 2), which was synthesized and purified as in the case of 1. However, the major protein modification using this approach would be expected to be monoadduct 4 instead of cross-link 5. Thus, most of the resulting antibodies recognizing this HPDPI derivative would be recognizing an anionic carboxylate “tail”, which is predicted to be especially immunogenic (29). In fact, while our work was in progress, Tsai et al. reported on a polyclonal antiserum obtained by carbodiimide coupling of the NR-acetyllysine analogue of 2 (28), also expected to result mainly in monoadduction and thus recognition by most of the antibodies of the carboxylate “tail”. Although their antiserum was found to recognize the HNE-induced fluorescent cross-link in biological samples, we re-designed our approach to generate antisera that would possess minimal cross-reactivity to monotethered HPDPI fluorophores. Scheme 2

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Chem. Res. Toxicol., Vol. 13, No. 5, 2000

Xu et al.

the structures of such side reactions could be removed by immunoaffinity purification using an HPDPI-modified column.

Our strategy was to couple the preformed HPDPI core to the protein using reductive alkylation of an HPDPIderived dialdehyde (Scheme 3) rather than carbodiimide coupling of an HPDPI-derived diacid. Incubation of ONE with 4-aminobutyraldehyde diethylacetal afforded N,N′bis(4,4-diethoxybutyl)-2-hydroxy-2-pentyl-1,2-dihydropyrrol-3-one iminium (6) in 20% yield. The bisacetal 6 was hydrolyzed in 2 N HCl, and the resulting aldehyde (without isolation) was conjugated onto KLH using NaBH3CN. Even though this coupling strategy was still expected to generate both monoadduct 7 and diadduct 8, the neutral alcohol tail in the monoadduct should be less immunogenic relative to the carboxylate in monoadduct 4. Scheme 3a

a (a) CH CN/sodium phosphate buffer (pH 8.3), CuCl ; (b) 2 N 3 2 HCl, 16 h; (c) KLH, NaBH3CN/sodium phosphate buffer (pH 7.4).

A second approach was to form the fluorescent crosslink directly on the protein. To optimize the level of HPDPI fluorophore incorporated into the immunogen, we employed ONE, which in model studies with amines gave 20-30% of the fluorescent cross-link compared to