Modification of Histidine Residues by 4,5-Epoxy-2-alkenals - Chemical

654

Chem. Res. Toxicol. 1999, 12, 654-660

Modification of Histidine Residues by 4,5-Epoxy-2-alkenals Rosario Zamora, Manuel Alaiz, and Francisco J. Hidalgo* Instituto de la Grasa, Consejo Superior de Investigaciones Cientı´ficas, Avenida Padre Garcı´a Tejero, 4, 41012 Sevilla, Spain Received September 22, 1998

The reactions of 4,5(E)-epoxy-2(E)-heptenal with 4-methylimidazole and NR-acetyl-L-histidine methyl ester were studied to characterize the adducts produced in the modification of histidine residues by epoxyalkenals and to develop a methodology for the determination of these adducts in protein hydrolysates. The reaction products, which were isolated and characterized, resulted in the Michael adducts produced in the addition of one of the imidazolic nitrogens to the carboncarbon double bond of the epoxyalkenal. Only some of the theoretical isomers were produced. Thus, in the reaction with 4-methylimidazole, the main product was 4,5-epoxy-3-(4-methylimidazol-1-yl)heptanol (88%), although the formation of 4,5-epoxy-3-(5-methylimidazol-1-yl)heptanol (12%) was also observed. On the other hand, the reaction with NR-acetyl-L-histidine methyl ester produced exclusively NR-acetyl-1-[1′-(1′′,2′′-epoxybutyl)-3′-hydroxypropyl]-L-histidine methyl ester. This last compound was used to develop a procedure for the determination of 4,5(E)-epoxy-2(E)-heptenal-histidine adducts in protein hydrolysates. When this procedure was applied to the analysis of bovine serum albumin treated with 0.01-10 mM 4,5(E)-epoxy2(E)-heptenal, the formation of the adduct was observed and its concentration increased with the concentration of the aldehyde and the incubation time, and was parallel to the histidine losses observed in the protein after acid hydrolysis as well as to the formation of protein carbonyls. In addition, the number of histidine residues lost in the protein was very similar to the number of adduct residues produced, suggesting that the addition reaction is the major mechanism for histidine losses suffered by proteins following their reaction with epoxyalkenals.

Introduction Oxidative stress and exposure to xenobiotic substances generate reactive compounds, including aldehydes, which have been implicated in some of the pathophysiological effects associated with the oxidative damage produced in cells and tissues (1-3). These effects are believed to be, at least partially, a consequence of the modification of proteins and other biomolecules by aldehydes produced during the lipid peroxidation process (4-7). Therefore, during the past decade an important effort has been dedicated to study these reactions, and presently, the mechanisms of some of them are well-known. However, the mechanisms of many others and the relative contributions of all of them to the oxidative damage produced by lipids are only beginning to be understood (8). Among the many different aldehydes which can be formed during lipid peroxidation and that modify proteins, the most intensively studied have been malonaldehyde and 4-hydroxy-2-alkenals. In addition to these aldehydes, many others are also produced in tissues, cells, and subcellular fractions, because there are different polyunsaturated fatty acids and several routes of formation. In particular, studies from this laboratory have pointed out to 4,5-epoxy-2-alkenals as a family of aldehydes with a high reactivity with respect to amino acids, peptides, and proteins (9, 10). * To whom correspondence should be addressed: Instituto de la Grasa, Avda. Padre Garcı´a Tejero, 4, 41012 Sevilla, Spain. Fax: +34 95 461 6790. E-mail: [email protected]. Phone: +34 95 461 1550.

4,5-Epoxy-2-alkenals result from the decomposition of intermediate epoxyhydroperoxy fatty acids by a mechanism which seems to be common for the different polyunsaturated fatty acids. Thus, when starting from the n - 6 linoleic acid, the epoxyalkenal that is obtained is 4,5(E)-epoxy-2(E)-decenal by decomposition of an intermediate 12,13(E)-epoxy-9-hydroperoxy-10-octadecenoic acid (11). Analogously, the 4,5(E)-epoxy-2(E)-heptenal is the product of oxidation of the n - 3 polyunsaturated fatty acids. These epoxyalkenals, which are supposed to be intermediates in the formation of 4,5-dihydroxydecenal by peroxidation of liver microsomal lipids (12), have been detected in several systems (13). These aldehydes are able to modify very rapidly the lysine residues producing pyrroles that, by means of a polymerization reaction, are responsible for the color and fluorescence production in these reactions (14). In addition to lysine residues, the reaction of epoxyalkenals with proteins also produces the modification of the other basic amino acid residues in the protein (15). However, the structure of these adducts has not been elucidated yet. As a continuation of these studies, this investigation was undertaken to characterize the products of the reaction between histidine residues and epoxyalkenals, and to assess their formation in proteins modified by these aldehydes.

Materials and Methods General. All chemicals were purchased from Aldrich (Milwakee, WI), Sigma (St. Louis, MO), Fluka (Buchs, Switzerland), or Merck (Darmstadt, Germany). 4,5(E)-Epoxy-2(E)-heptenal

10.1021/tx980218n CCC: $18.00 © 1999 American Chemical Society Published on Web 07/01/1999

Modification of Histidines by 4,5-Epoxy-2-alkenals Scheme 1. Reaction of 4,5(E)-Epoxy-2(E)-heptenal with 4-Methylimidazole and Nr-Acetyl-L-histidine Methyl Estera

Chem. Res. Toxicol., Vol. 12, No. 7, 1999 655 δ 9.73 (q, C7), 13.45 (q, CH3), 24.60 (t, C6), 33.34 (t, C2), 54.65 (d, C3), 56.42 (t, C1), 58.08 (d, C5), 59.82 (d, C4), 113.70 (d, C5′), 136.18 (d, C2′), 138.09 (s, C4′); GC/MS data identical to those of isomer 3a; retention time 23.24 min. Isomer 4a: 1H NMR (CDCl3) δ 0.95 (t, J6,7 ) 7.4 Hz, 3H, H7), 1.56 (m, 2H, H6), 2.06 (m, 2H, H2), 2.18 (s, 3H, CH3), 2.44 (td, J4,5 ) 2.1 Hz, J5,6 ) 5.5 Hz, 1H, H5), 3.00 (dd, J3,4 ) 5.0 Hz, J4,5 ) 2.1 Hz, 1H, H4), 3.32 (m, 1H, H1a), 3.65 (m, 1H, H1b), 6.75 (bs, 1H, H4′) (the signals corresponding to H3 and H2′ were hidden by other signals in the spectrum and could not be identified); 13C NMR (CDCl3) δ 9.73 (q, C7), 13.45 (q, CH3), 24.57 (t, C6), 34.80 (t, C2), 54.10 (d, C3), 56.73 (t, C1), 57.87 (d, C5), 59.67 (d, C4), 125.50 (d, C4′), 125.89 (s, C5′), 135.07 (d, C2′); GC/ MS data identical to those of isomer 3a; retention time 25.44 min.

a R is CH in compounds 2-4 and CH(NHCOCH )CO CH in 3 3 2 3 compounds 5 and 6.

was prepared in a manner analogous to that of 4,5(E)-epoxy2(E)-decenal (10). NR-Acetyl-L-histidine methyl ester was prepared by reaction of NR-acetyl-L-histidine with diazomethane (16). 1H and 13C NMR spectra at 300 and 75.4 MHz, respectively, were determined in a Bruker AC-300P instrument (Karlsruhe, Germany), with Me4Si as an internal standard. Two-dimensional NMR was used to assign the 13C NMR spectra. GC/MS analysis was conducted with a Hewlett-Packard 5890 series II gas chromatograph (Hewlett-Packard, Palo Alto, CA) interfaced, via an open coupling system, to an AEI-MS/70VG mass spectrometer (VG Analytical, Manchester, U.K.) operating at 70 eV. A DB-5 fused-silica capillary column (J&W Scientific, Folsom, CA; 30 m × 0.25 mm i.d.) was used in GC/MS experiments. The column temperature was programmed from 100 (2 min) to 280 °C at a rate of 4 °C/min. Reaction of 4,5(E)-Epoxy-2(E)-heptenal with 4-Methylimidazole. A solution of 4,5(E)-epoxy-2(E)-heptenal (95 mg, 0.75 mmol) (1) and 4-methylimidazole (123 mg, 1.5 mmol) (2) in chloroform (7 mL) was incubated overnight at 37 °C. After this time, the products were reduced with NaBH4 (21 mg) for 30 min, and fractionated by column chromatography on silica gel using chloroform/methanol (9:1) as an eluent. The major product of the reaction was characterized as an isomeric mixture of 4,5-epoxy-3-[4(5)-methylimidazol-1-yl]heptanol, according to 1H and 13C NMR and GC/MS. The structures of the products are shown in Scheme 1. Four isomers (3a, 3b, 4a, and 4b) were detected by both NMR and GC/MS in the following proportions: 55, 33, 9, and 6%, respectively. The proportion was calculated by 1H NMR and GC. Both techniques gave very similar results. The four isomers exhibited the same mass spectra, but significant differences were observed by 1H and 13C NMR. Their spectral data were as follows. Isomer 3a: 1H NMR (CDCl3) δ 0.96 (t, J6,7 ) 7.4 Hz, 3H, H7), 1.56 (m, 2H, H6), 2.06 (m, 2H, H2), 2.16 (s, 3H, CH3), 2.58 (td, J4,5 ) 2.1 Hz, J5,6 ) 5.5 Hz, 1H, H5), 2.97 (dd, J3,4 ) 5.0 Hz, J4,5 ) 2.1 Hz, 1H, H4), 3.32 (m, 1H, H1a), 3.65 (m, 1H, H1b), 4.28 (ddd, J2a,3 ) 5.0 Hz, J2b,3 ) 10.0 Hz, J3,4 ) 5.0 Hz, 1H, H3), 6.67 (bs, 1H, H5′), 7.21 (d, J2′,5′ ) 0.9 Hz, 1H, H2′); 13C NMR (CDCl3) δ 9.73 (q, C7), 13.45 (q, CH3), 24.50 (t, C6), 34.65 (t, C2), 54.83 (d, C3), 56.38 (t, C1), 58.04 (d, C5), 59.82 (d, C4), 113.76 (d, C5′), 136.18 (d, C2′), 137.83 (s, C4′); GC/MS (relative intensity, ion structure) of the trimethylsilyl derivative m/z 282 (11, M+), 267 (5, M+ - CH3), 166 [27, M+ + H - CH2CH2OSi(CH3)3], 137 (100, 166 - CH2CH3), 96 (15, dimethylimidazole), 73 [82, Si(CH3)3]; retention time 24.24 min. Isomer 3b: 1H NMR (CDCl3) δ 0.94 (t, J6,7 ) 7.4 Hz, 3H, H7), 1.56 (m, 2H, H6), 2.06 (m, 2H, H2), 2.17 (s, 3H, CH3), 2.79 (td, J4,5 ) 2.1 Hz, J5,6 ) 5.6 Hz, 1H, H5), 2.96 (dd, J3,4 ) 4.6 Hz, J4,5 ) 2.1 Hz, 1H, H4), 3.32 (m, 1H, H1a), 3.65 (m, 1H, H1b), 4.35 (ddd, J2a,3 ) 4.3 Hz, J2b,3 ) 10.6 Hz, J3,4 ) 4.3 Hz, 1H, H3), 6.71 (bs, 1H, H5′), 7.26 (d, J2′,5′ ) 1.1 Hz, 1H, H2′); 13C NMR (CDCl3)

Isomer 4b: 1H NMR (CDCl3) δ 0.91 (t, J6,7 ) 7.4 Hz, 3H, H7), 1.56 (m, 2H, H6), 2.06 (m, 2H, H2), 2.21 (s, 3H, CH3), 2.75 (td, J4,5 ) 2.1 Hz, J5,6 ) 5.5 Hz, 1H, H5), 2.96 (dd, J3,4 ) 5.0 Hz, J4,5 ) 2.1 Hz, 1H, H4), 3.32 (m, 1H, H1a), 3.65 (m, 1H, H1b) (the signals corresponding to H3, H4′, and H2′ were hidden by other signals in the spectrum and could not be identified); 13C NMR (CDCl3) δ 9.73 (q, C7), 13.45 (q, CH3), 33.94 (t, C2), 56.00 (t, C1), 57.60 (d, C5), 59.07 (d, C4) (the signals corresponding to C6, C3, C4′, C2′, and C5′ were hidden by other signals in the spectrum and could not be identified); GC/MS data identical to those of isomer 3a; retention time 24.55 min. Reaction of 4,5(E)-Epoxy-2(E)-heptenal with Nr-AcetylMethyl Ester. A solution of 4,5(E)-epoxy-2(E)heptenal (75 mg, 0.6 mmol) (1) and NR-acetyl-L-histidine methyl ester (253 mg, 1.2 mmol) (5) in chloroform (10 mL) was incubated overnight at 37 °C. After this, the products were reduced with NaBH4 (30 mg) for 1 h, and fractionated by column chromatography on silica gel using chloroform/methanol (9:1) as an eluent. The major product of the reaction was characterized as an isomeric mixture of NR-acetyl-1-[1′-(1′′,2′′-epoxybutyl)3′-hydroxypropyl]-L-histidine methyl ester, according to 1H and 13C NMR and GC/MS. Two isomers (6a and 6b) were detected by both NMR and GC/MS in approximately the same proportion. Both of them exhibited the same mass spectra, but significant differences were observed by 1H and 13C NMR. The retention times in GC for the two isomers were 44.0 and 47.4 min, respectively. Their spectral data were as follows. L-histidine

Isomer 6a: 1H NMR (CDCl3) δ 0.96 (t, J6′′,7′′ ) 7.5 Hz, 3H, H7′′), 1.57 (m, 2H, H6′′), 2.00 (s, 3H, CH3CO), 2.07 (m, 2H, H2′′), 2.57 (td, J4′′,5′′ ) 2.0 Hz, J5′′,6′′ ) 5.5 Hz, 1H, H5′′), 3.00 (dd, J3′′,4′′ ) 4.9 Hz, J4′′,5′′ ) 2.0 Hz, 1H, H4′′), 3.01 (bs, 2H, H1′), 3.28 (m, 1H, H1′′a), 3.60 (m, 1H, H1′′b), 3.67 (s, 3H, OCH3), 4.01 (bs, 1H, OH), 4.38 (ddd, J2′′a,3′′ ) 4.9 Hz, J2′′b,3′′ ) 9.8 Hz, J3′′,4′′ ) 4.9 Hz, 1H, H3′′), 4.74 (dt, J1′,2′ ) 6.0 Hz, J1′,NH ) 7.7 Hz, 1H, H2′), 6.80 (bs, 1H, H5), 7.48 (d, J2,5 ) 1.3 Hz, 1H, H2), 7.53 (m, 1H, NH); 13C NMR (CDCl ) δ 9.60 (q, C7′′), 22.89 (q, CH CO), 24.35 (t, 3 3 C6′′), 29.88 (t, C1′), 34.62 (t, C2′′), 52.10 (q, CH3O), 52.39 (d, C2′), 54.82 (d, C3′′), 56.60 (t, C1′′), 57.93 (d, C5′′), 59.49 (d, C4′′), 115.24 (d, C5), 136.98 (d, C2), 137.13 (s, C4), 170.28 (s, ester), 171.92 (s, amide); GC/MS (relative intensity, ion structure) of the trimethylsilyl derivative m/z 411 (19, M+), 352 (25, M+ - CH3OCO), 310 (8), 281 [8, M+ - CH(COOCH3)(NHCOCH3)], 207 (25), 165 (26), 77 (100), 73 [70, Si(CH3)3]. Isomer 6b: 1H NMR (CDCl3) δ 0.96 (t, J6′′,7′′ ) 7.5 Hz, 3H, H7′′), 1.57 (m, 2H, H6′′), 2.00 (s, 3H, CH3CO), 2.07 (m, 2H, H2′′), 2.51 (td, J4′′,5′′ ) 2.0 Hz, J5′′,6′′ ) 5.5 Hz, 1H, H5′′), 3.00 (dd, J3′′,4′′ ) 5.0 Hz, J4′′,5′′ ) 2.0 Hz, 1H, H4′′), 3.01 (bs, 2H, H1′), 3.28 (m, 1H, H1′′a), 3.60 (m, 1H, H1′′b), 3.68 (s, 3H, OCH3), 4.01 (bs, 1H, OH), 4.31 (ddd, J2′′a,3′′ ) 5.0 Hz, J2′′b,3′′ ) 10.0 Hz, J3′′,4′′ ) 5.0 Hz, 1H, H3′′), 4.74 (dt, J1′,2′ ) 6.0 Hz, J1′,NH ) 7.7 Hz, 1H, H2′), 6.80 (bs, 1H, H5), 7.47 (d, J2,5 ) 1.2 Hz, 1H, H2), 7.53 (m, 1H, NH); 13C NMR (CDCl3) δ 9.63 (q, C7′′), 22.92 (q, CH3CO), 24.44 (t, C6′′), 29.81 (t, C1′), 34.56 (t, C2′′), 52.10 (q, CH3O), 52.47 (d, C2′), 54.25 (d, C3′′), 56.73 (t, C1′′), 57.57 (d, C5′′), 59.56 (d, C4′′), 115.36 (d, C5), 136.80 (d, C2), 136.94 (s, C4), 170.28 (s, ester), 171.81 (s, amide); GC/MS data identical to those of isomer 6a.

656 Chem. Res. Toxicol., Vol. 12, No. 7, 1999 Determination of 4,5(E)-Epoxy-2(E)-heptenal-Histidine Adducts in Protein Hydrolysates. A procedure was developed to determine the adduct of 4,5(E)-epoxy-2(E)-heptenal with histidine residues in protein hydrolysates. Compound 6 was obtained by hydrolysis with 6 M HCl for 19 h, and then derivatized with diethyl ethoxymethylenemalonate. The resulting product was assessed by high-performance liquid chromatography with a 300 mm × 3.9 mm i.d. reverse-phase column (Nova-Pack C18, 4 µm, Waters) using a previously described gradient for amino acid analysis (17). Standard plots of peak area ratio of the adduct to the internal standard (R-aminobutyric acid) versus its concentration were obtained, and linear leastsquares regression was performed to determine the slope, y-intercept, and correlation coefficient of the best-fit line. Amino acid concentrations in unknown samples were calculated using results of the regression analysis. Reaction of 4,5(E)-Epoxy-2(E)-heptenal with Bovine Serum Albumin. A solution of 1 mg/mL bovine serum albumin in 50 mM sodium phosphate buffer (pH 7.4) was incubated for different periods of time at 37 °C and in the presence of several concentrations of 4,5(E)-epoxy-2(E)-heptenal. Incubated samples were analyzed by HPLC after acid hydrolysis for histidine losses, and by reaction with 2,4-dinitrophenylhydrazine for production of protein carbonyls. Histidine losses after acid hydrolysis were determined by HPLC after derivatization with diethyl ethoxymethylenenmalonate by using a previously described procedure (17). Formation of protein carbonyls was assessed by using a procedure of Levine et al. (18). Briefly, 500 µL of the incubated mixture was treated with 500 µL of 12.5 mM 2,4-dinitrophenylhydrazine in 2.5 M HCl, and the mixture was incubated for 1 h at room temperature. After that time, the protein was precipitated with 500 µL of 30% (w/v) trichloroacetic acid at 0 °C for 1 h and, then, centrifuged at 2200g for 15 min. The pellet that was produced was washed four times with a 1:1 mixture of ethanol/ethyl acetate, then dissolved in 1 mL of 6 M guanidine hydrochloride with 20 mM phosphate buffer/trichloroacetic acid (pH 2.3), and, finally, left for 30 min at 37 °C with vortexing. Any insoluble materials were removed by centrifugation, and the reactive carbonyl content was calculated from its peak absorption at 370 nm using a molar absorption coefficient () of 22 000 M-1. In addition, incubated samples (600 µL) were treated with 60 µL of 0.5 M NaBH4 in 0.1 N NaOH for 15 min and then the pH was changed to neutral with diluted HCl. Proteins in these reduced samples were purified by using PD-10 columns, then dried under nitrogen, and, finally, hydrolyzed with 6 N HCl (1 mL) for 19 h. The content of 4,5(E)-epoxy-2(E)-heptenalhistidine adducts was determined by HPLC after derivatization with diethyl ethoxymethylenemalonate as described above.

Results Characterization of 4,5(E)-Epoxy-2(E)-heptenal4-Methylimidazole Reaction Products. The reaction of 4,5(E)-epoxy-2(E)-heptenal with 4-methylimidazole produced the addition of the imidazole ring to the carbon-carbon double bond, as could be easily observed by NMR. However, the isolation of the reaction products was not easy because the reaction seemed to be reversible and the spectra of the products were very complex. This last complexity was likely a consequence of the existence of an equilibrium among different species produced between the carbonyl and epoxy groups. The isolation and complete characterization of the reaction products could only be carried out after reduction of the aldehydic group with sodium borohydride. Four isomers were produced, which were a consequence of the formation of a new chiral center in the molecule and the tautomerism in the imidazole ring. The 4,5(E)-epoxy-2(E)-heptenal was obtained from 2(E),4(E)-heptadienal by a syn epoxidation.

Zamora et al.

Figure 1. Total ion chromatogram of GC/MS analysis for the trimethylsilyl derivatives of 4,5-epoxy-3-[4(5)-methylimidazol1-yl]heptanol. Peaks have been numbered according to the number given in Materials and Methods.

Figure 2. Partial proton NMR (300 MHz) of 4,5-epoxy-3-[4(5)methylimidazol-1-yl]heptanol in CDCl3. The signals shown in the figure corresponded to proton H5 of isomers 3b, 4b, 3a, and 4a, which appeared at δ 2.79, 2.75, 2.58, and 2.44, respectively.

Therefore, the 4,5(E)-epoxy-2(E)-heptenal should be a mixture of R,R and S,S enantiomers. Because the addition reaction produced a new chiral center, the formation of two pairs of diastereomers could be expected: R,R,R and R,R,S and their corresponding enantiomers. In addition, and because of the tautomerism in the imidazole ring, the addition could be produced by any of the two nitrogen atoms of the imidazole ring, therefore forming the 1,4- and 1,5-disubstituted imidazole derivatives. The presence of the four isomers (each one being a mixture of enantiomers) could be easily observed by GC and 1H and 13C NMR. Figure 1 shows the chromatogram obtained for the mixture. Each peak corresponded to one isomer, and all of them had the same mass spectra. Figure 2 shows a portion of the 1H NMR spectrum of the isomeric mixture. Although most of the signals were different for each proton in each isomer, one of the clearest differences among the four isomers was observed in the signals corresponding to proton H5, which are shown in the figure. The assignment of the NMR absorbances to signals in the 1H NMR spectrum was first carried out according to the relative abundances determined for isomers 3a, 3b, 4a, and 4b, by both 1H NMR and GC. These assignments were confirmed by irradiation or two-dimensional experiments. Assigned 1H NMR spectra were then used to assign the 13C NMR spectrum by means of two-dimensional experiments. The identification of each isomer was

Modification of Histidines by 4,5-Epoxy-2-alkenals

Figure 3. Partial carbon NMR (75.4 MHz) of 4,5-epoxy-3-[4(5)methylimidazol-1-yl]heptanol in CDCl3. The signals shown in the figure correspond to the heterocyclic carbons of isomers 3a, 3b, and 4a. Signals were assigned as follows: δ 138.09 (C4′, 3b), 137.83 (C4′, 3a), 136.18 (C2′, 3a and 3b), 135.07 (C2′, 4a), 125.89 (C5′, 4a), 125.50 (C4′, 4a), 113.76 (C5′, 3a), and 113.70 (C5′, 3b).

better carried out by studying its 13C NMR spectrum. This technique produces two different patterns of signals for the 1,4- and 1,5-disubstituted imidazoles. Thus, the spectrum of 1,4-dimethylimidazole exhibited signals at δ 116.2, 136.5, and 138.0 ppm for carbons 5, 2, and 4, respectively, and the spectrum of 1,5-dimethylimidazole exhibited signals at δ 125.6, 126.7, and 136.4 ppm for carbons 4, 5, and 2, respectively (19). According to these data, the major isomers produced in the reaction of 4,5(E)-epoxy-2(E)-heptenal with 4-methylimidazole reaction were characterized as 1,4-disubstituted imidazoles because the carbon signals of their imidazole ring appeared at 113.76, 136.18, and 137.83 ppm for isomer 3a and 113.70, 136.18, and 138.09 ppm for isomer 3b (Figure 3). In addition, isomer 4a was characterized as a 1,5disubstituted imidazole because the carbon signals of its imidazole ring appeared at 125.50, 125.89, and 135.07 ppm. Although the carbon signals of the isomer 4b were hidden in the spectrum, the structure of this isomer should correspond to the second diastereomer of the 1,5disubstituted imidazole. Characterization of 4,5(E)-Epoxy-2(E)-heptenalNr-Acetyl-L-histidine Methyl Ester Reaction Products. Like the reaction between 4,5(E)-epoxy-2(E)heptenal and 4-methylimidazole, the reaction of 4,5(E)epoxy-2(E)-heptenal and NR-acetyl-L-histidine methyl ester also produced the addition of the imidazole ring to the carbon-carbon double bond, and the latter reduction of the aldehyde stabilized the products and facilitated the interpretation of the spectra. In this reaction, the presence of several isomers should also be expected. Thus, the reaction between the racemic (R,R and S,S) 4,5(E)epoxy-2(E)-heptenal and the S enantiomer of the NRacetyl-L-histidine methyl ester and the creation of a new chiral center in the reaction should produce four diastereomers, R,R,S,R, R,R,S,S, S,S,S,R, and S,S,S,S, in addition to the formation of the 1,4- and 1,5-disubstituted imidazoles. Surprisingly, the gas chromatogram and the 1H and 13C NMR spectra of this isomeric mixture were simpler than those corresponding to the 4,5(E)-epoxy2(E)-heptenal-4-methylimidazole adducts, and only two of the eight possible isomers were detected. Figure 4 shows the 1H NMR portion of the spectrum corresponding to H5′′. In addition, both isomers corresponded to 1,4disubstituted imidazoles because the imidazole carbons appeared at 115.24, 136.98, and 137.13 ppm and 115.36, 136.80, and 136.94 ppm for carbons 5, 2, and 4 of isomers 6a and 6b, respectively. For isomers 6a and 6b, NMR absorbances were assigned to signals in the 1H and 13C NMR spectra in a manner similar to the procedure

Chem. Res. Toxicol., Vol. 12, No. 7, 1999 657

Figure 4. Partial proton NMR (300 MHz) of NR-acetyl-1-[1′(1′′,2′′-epoxybutyl)-3′-hydroxypropyl]-L-histidine methyl ester in CDCl3. The signals shown in the figure correspond to proton H5′′ of isomers 6a and 6b, which appeared at δ 2.57 and 2.51, respectively.

Figure 5. Elution pattern of N-[2,2-bis(ethoxycarbonyl)vinyl]amino derivatives of (A) 4,5(E)-epoxy-2(E)-heptenal-histidine adducts after acid hydrolysis and (B) a standard mixture of amino acids, including the adducts. Peaks are labeled with single-letter notations for amino acids. I.S. represents D,L-Raminobutyric acid (internal standard). The asterisk indicates the peak of the adduct used for its determination.

described above for the assignment of absorbances of isomers 3a, 3b, 4a, and 4b. However, the relative abundances were not useful in the 6a/6b mixture. Determination of 4,5(E)-Epoxy-2(E)-heptenalHistidine Adducts in Protein Hydrolysates. The formation of the adduct between 4,5(E)-epoxy-2(E)-heptenal and histidine residues in proteins could be easily assessed by high-performance liquid chromatography after acid hydrolysis. The hydrolysis of compound 6 with HCl and its later derivatization with diethyl ethoxymethylenemalonate produced two main peaks (Figure 5A). This result is in agreement with the presence of two diastereomers in compound 6. The first of these two peaks could be easily separated by HPLC from the peaks corresponding to the other amino acids (Figure 5B) and, therefore, could be used for the determination of this adduct in protein hydrolysates. The peak area ratio between the first peak obtained in the hydrolysis of compound 6 and the internal standard was plotted against the concentration of compound 6 in the solution injected into the chromatograph, and the response was linear in the studied range of 2-50 µM. The calibration curve (r ) 0.9997, p ) 0.000329) was defined by the equation

par ) 0.00382c + 0.0038

658 Chem. Res. Toxicol., Vol. 12, No. 7, 1999

Figure 6. Effect of incubation time on the histidine residues recovered in a bovine serum albumin incubated in the presence of 0 (0), 1 (O), and 10 mM (4) 4,5(E)-epoxy-2(E)-heptenal. Table 1. Effect of the Concentration of 4,5(E)-Epoxy-2(E)-heptenal on Histidine Losses and Production of Protein Carbonyls in Bovine Serum Albumina epoxyaldehyde concentration (mM)

histidine recovered (no. of residues)b

protein carbonyls (nmol/mg of protein)

0 0.01 0.1 0.25 0.5 1 5 10

17 17 16 16 16 16 13 8

1.24 1.60 3.70 6.57 16.18 20.14 >57.84c >77.58c

a The protein was incubated for 19 h at 37 °C. b The number of residues was rounded to the nearest integer. c The derivatized protein was only partially soluble.

where par is the peak area ratio and c the concentration in micromolar of compound 6 in the solution injected into the chromatograph. Reaction of 4,5(E)-Epoxy-2(E)-heptenal with Bovine Serum Albumin. A modification analogous to that produced by 4,5(E)-epoxy-2(E)-heptenal in 4-methylimidazole and N-acetylhistidine methyl ester was also observed in the reaction of 4,5(E)-epoxy-2(E)-heptenal with bovine serum albumin. Thus, the incubation of bovine serum albumin with several concentrations of 4,5(E)-epoxy-2(E)-heptenal and different periods of time produced the modification of some histidine residues in the protein, which were converted into 1-[1′-(1′′,2′′epoxybutyl)-2′-formylethyl]-L-histidines, and the formation of protein carbonyls. Figure 6 shows the histidine residues recovered after acid hydrolysis as a function of the incubation time. A concentration of 1 mM of 4,5(E)epoxy-2(E)-heptenal only produced small losses in the histidine recovered after acid hydrolysis. However, these losses were much higher when the protein was incubated in the presence of 10 mM epoxyalkenal. By using this concentration, 24% of the histidine residues were lost after 4 h, and these losses increased to 52% of the histidine residues after 8 h. After that time, no higher losses were observed. The effect of the concentration of the epoxyalkenal on histidine losses produced in a bovine serum albumin incubated for 19 h is shown in Table 1. Although small losses were observed in the incubations with 0.1-1 mM 4,5(E)-epoxy-2(E)-heptenal, high losses were only observed at 5 and 10 mM. At these concentrations of

Zamora et al.

Figure 7. Effect of incubation time on the formation of protein carbonyls in a bovine serum albumin incubated in the presence of 0 (0), 1 (O), and 10 mM (4) 4,5(E)-epoxy-2(E)-heptenal.

epoxyaldehyde, 29 and 53%, respectively, of the histidine residues were lost. Histidine losses were parallel to the production of carbonyl derivatives in the protein. Figure 7 shows the protein carbonyl production in the protein after incubation with 1 or 10 mM 4,5(E)-epoxy-2(E)-heptenal. At a concentration of 1 mM, the level of protein carbonyls increased for the first 4 h, and then remained unchanged for the rest of the incubation time. A similar behavior was also observed for the protein incubated in the presence of 10 mM 4,5(E)-epoxy-2(E)-heptenal. However, the protein incubated with this concentration of epoxyalkenal was so damaged that the protein precipitated with trichloroacetic acid could not be solubilized if it had been incubated for more than 3 h in the presence of the epoxyalkenal. If it is supposed that all protein carbonyls in bovine serum albumin are only produced with histidine residues, the maximum theoretical concentration of protein carbonyls would be 254 nmol/mg. This means that about 8% of the histidine residues produced protein carbonyls when the bovine serum albumin was treated with 1 mM 4,5(E)-epoxy-2(E)-heptenal, and this value increased to 31% in the first 3 h when the bovine serum albumin was treated with 10 mM epoxyalkenal. The effect of the concentration of 4,5(E)-epoxy-2(E)heptenal on the formation of protein carbonyls in bovine serum albumin incubated for 19 h is shown in Table 1. The values obtained at 5 and 10 mM are lower than those actually obtained because the protein was so damaged that it was only partially soluble. The level of protein carbonyls increased linearly with the concentration of aldehyde in the range of 0-1 mM (r ) 0.965, p ) 0.0018). When the adducts of the reaction of bovine serum albumin and 4,5(E)-epoxy-2(E)-heptenal were reduced with sodium borohydride before being hydrolyzed, the losses of histidine residues in the protein were similar to that observed in the unreduced protein. In addition, the formation of 4,5(E)-epoxy-2(E)-heptenal/histidine adducts could also be assessed. The incubation of the protein in the absence of 4,5(E)-epoxy-2(E)-heptenal did not produce any histidine losses or adduct formation (Figure 8). However, when the bovine serum albumin was incubated in the presence of 1 mM 4,5(E)-epoxy-2(E)heptenal, 13% of the histidine residues were lost in the first 2 h of incubation and this percentage increased to 19% in the next 22 h (Figure 8A). Most of the histidine residues that were lost during the incubation (about two or three residues) appeared as 4,5(E)-epoxy-2(E)-hepte-

Modification of Histidines by 4,5-Epoxy-2-alkenals

Chem. Res. Toxicol., Vol. 12, No. 7, 1999 659 Table 2. Histidine Losses and Formation of Protein Carbonyls and 4,5(E)-Epoxy-2(E)-heptenal-Histidine Adducts in Bovine Serum Albumin Incubated in the Presence of 4,5(E)-Epoxy-2(E)-heptenala without reduction

after reduction

epoxyaldehyde His carbonylsb His adducts concentration (mM) recovered (%) (%) recovered (%) (%) 1 10

92 54

8 >31c

85 53

10 51

a The protein was incubated for 8 h in the presence of the aldehyde. b The percentage was calculated considering the theoretical maximum concentration of carbonyls in bovine serum albumin when these are produced exclusively with the histidine residues (254 nmol/mg). c The derivatized protein was only partially soluble.

Figure 8. Effect of incubation time on 4,5(E)-epoxy-2(E)heptenal-histidine adduct formation in bovine serum albumin in the presence of 0 (0), 1 (O), 5 (3), and 10 mM (4) 4,5(E)epoxy-2(E)-heptenal: (A) loss of histidine residues and (B) appearance of 4,5(E)-epoxy-2(E)-heptenal-histidine adduct residues after reduction with sodium borohydride and acid hydrolysis.

nal-histidine adducts (Figure 8B). Similar results were observed when the protein was incubated in the presence of 5 or 10 mM 4,5(E)-epoxy-2(E)-heptenal, but higher histidine losses were observed when a higher concentration of the aldehyde was used. Thus, the incubation of bovine serum albumin in the presence of 5 mM 4,5(E)epoxy-2(E)-heptenal produced the loss of 32% of the histidine residues after 2 h, and these losses increased to 38% in the next 22 h (Figure 8A). This loss of five or six histidine residues was parallel to the formation of four or five residues of 4,5(E)-epoxy-2(E)-heptenal-histidine adducts. When the bovine serum albumin was incubated in the presence of 10 mM 4,5(E)-epoxy-2(E)-heptenal, a loss of 44% of the histidine residues was observed after the first 2 h of incubation and it increased to 47% after 24 h. This loss of seven or eight histidine residues was parallel to the formation of eight or nine residues of 4,5(E)-epoxy-2(E)-heptenal-histidine adducts. Table 2 summarizes most of the above results by comparison of the percentages of residues of histidine recovered and the formation of residues containing carbonyls or adducts. The reduction of the protein did not change to a great extent the percentage of histidine recovered after acid hydrolysis. In addition, the percentage of residues containing carbonyl groups was quite similar to the percentage of residues containing adducts, and both percentages were very close to the percentage of histidine that was lost during the incubation of bovine serum albumin with 4,5(E)-epoxy-2(E)-heptenal.

Discussion Modification of proteins and other biomolecules by lipid peroxidation products is believed to play a central role

in many of the pathophysiological conditions associated with free radical damage, including cardiovascular diseases, cancer, neurological diseases, and aging (1, 20, 21). This modification, which is also able to delay some of the damage produced by oxidative stress as a consequence of the antioxidative activity of the compounds produced (22), has been mostly associated with the modification of lysine residues by the lipid oxidation products. However, modification of other amino acid residues has also been described, and all these modifications seem to be related to the lower susceptibility of modified proteins to be degraded by proteases and, therefore, to have important implications for the accumulation of altered protein and fluorescent material in vivo (23-25). The results obtained in this study are in agreement with the histidine losses observed by amino acid analysis when proteins are exposed to epoxyalkenals (15). In addition, these results have allowed for identification of the structure of the adducts that were produced, which is similar to the structure of the adducts produced in the reaction between 4-hydroxy-2-nonenal and histidine residues (26-28). The above results suggest that, when the reaction is carried out with epoxyalkenals, the formation of the 1,4-disubstituted heterocycle is preferred to the 1,5derivative, and for histidine, the 1,4-isomer is the only adduct that is produced. In addition to the formation of these two kinds of isomers, the reaction between the imidazole derivatives and epoxyalkenals is more complex because of the presence of two chiral centers in the aldehyde. This induced the production of diastereomers after creation of the new chiral center. In addition, the diastereomers that were produced were not equally favored, suggesting a stereoselectivity of the reaction. No attempts were carried out to determine the absolute configuration of the products. When the reaction was carried out with NR-acetyl-Lhistidine methyl ester, the reaction was much more specific than when the reaction was carried out with 4-methylimidazole and only two of the eight theoretical products were produced. The adduct produced in the reaction between 4,5(E)epoxy-2(E)-heptenal and NR-acetyl-L-histidine methyl ester could also be used to develop a procedure for the identification of the 4,5(E)-epoxy-2(E)-heptenal-histidine adducts in protein hydrolysates. The procedure consisted of the derivatization of amino acids with diethyl ethoxymethylenemalonate and their separation by reversephase high-performance liquid chromatography. By using this procedure, and with different concentrations of the aldehyde, the number of histidine residues which were transformated into adducts was very similar to the

660 Chem. Res. Toxicol., Vol. 12, No. 7, 1999

number of histidine residues that were lost upon incubation, therefore suggesting that the addition reaction characterized in this study is the major cause of histidine losses produced in proteins following their reaction with epoxyalkenals. The reaction between the histidine residues and the epoxyalkenal also induced the formation of carbonyl groups in the protein. These protein carbonyls, which should be distinguished from the protein carbonyls formed during protein oxidation (29), were produced to the same extent as 4,5(E)-epoxy-2(E)-heptenal-histidine adducts, suggesting that the formation of carbonyls in proteins upon reaction with epoxyalkenals is mainly due to histidine residues. Different from that of 4-hydroxy2-alkenals, where the addition of lysine has been described (23, 24), the reaction of lysine with 4,5-epoxy-2alkenals, and possibly with other compounds with the same 4,5-epoxy-1-oxo-2-pentene system, seems to produce exclusively pyrrole rings (9, 30). All these results confirm that the reaction of histidine residues with epoxyalkenals produces changes in protein charge and conformation, and that histidine residues compete efficiently with lysine residues for the scavenging of these reactive aldehydes.

Acknowledgment. This study was supported in part by the Comisio´n Interministerial de Ciencia y Tecnologı´a of Spain (Project ALI97-0358) and the Junta de Andalucı´a (Project AGR 0135). We thank Mr. J. J. Rı´os for the GC/MS data and Mr. J. L. Navarro and Mrs. M. D. Garcı´a for the technical assistance.

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