Pyrrolization and Antioxidant Function of Proteins Following Oxidative

582

Chem. Res. Toxicol. 2001, 14, 582-588

Pyrrolization and Antioxidant Function of Proteins Following Oxidative Stress Francisco J. Hidalgo, Manuel Alaiz, and Rosario Zamora* Instituto de la Grasa, Consejo Superior de Investigaciones Cientı´ficas, Avenida Padre Garcıá Tejero 4, 41012-Sevilla, Spain Received October 10, 2000

The consequences of oxidative stress on microsomal proteins were analyzed by studying their pyrrolization and the antioxidative activity of the modified proteins produced. The microsomal system consisted of freshly prepared trout muscle microsomes, which were oxidized in the presence of 5 µM Cu2+, 1 mM Fe3+/5 mM ascorbate, or 1 mM Cu2+/10 mM H2O2. Pyrroles on proteins were detected by forming Ehrlich adducts with p-(dimethylamino)benzaldehyde and by determination of -N-pyrrolylnorleucine (Pnl) by capillary electrophoresis. Their antioxidative activity was studied by testing two model pyrrolized proteins (dimeric and monomeric modified bovine serum albumin: DBSA and MBSA, respectively), which were produced in the reaction of BSA and 4,5(E)-epoxy-2(E)-heptenal. These proteins were assayed at a concentration of 1040 µg/mL, which was selected because at this concentration both DBSA and MBSA had a concentration of Pnl similar to the Pnl concentration produced in oxidized microsomes. Both DBSA and MBSA significantly (p < 0.05) protected against lipid peroxidation, assessed by the formation of thiobarbituric acid reactive substances (TBARS), and protein damage, evaluated by amino acid analysis, for the three systems assayed, and this protection was always higher than that exhibited by BSA, which was used as control. The order of effectiveness was DBSA > MBSA > BSA and was parallel to the Pnl content in the assayed proteins. These results suggest that antioxidative activity of BSA may also be related to its ability to react with lipid oxidation products and to produce modified BSA with antioxidative activity. This mechanism may also be contributing to the antioxidative activity exhibited by many proteins.

Introduction Reactive oxygen species (ROS)1 and reactive nitrogen species (RNS) are unavoidably and continuously produced under normal metabolic conditions because they are critically involved in normal metabolism, especially in inter- and intracellular signaling (1). However, ROS/ RNS can directly modify DNA, lipids, and proteins, which results in mutations, strand breaks, aldehyde formation, LDL oxidation, and alterations in enzyme activities and signal transduction pathways (2-4). To minimize these deleterious effects, aerobic organisms have evolved elaborate antioxidant systems, which consist of low molecular weight antioxidants and antioxidant enzymes (5, 6). Under physiological conditions, the range of antioxidant defenses available within the cells is adequate to protect them against oxidative damage. However, the protective balance can be lost because of overproduction of free radicals that overwhelm the antioxidant defenses or by inadequate intake of nutrients that contribute to the defense system. This imbalance, namely oxidative stress, * To whom the correspondence should be addressed. Phone: +34 95 461 1550. Fax: +34 95 461 6790. E-mail: [email protected]. 1 Abbreviations: BHT, butylated hydroxytoluene; BSA, bovine serum albumin; DBSA, dimeric modified BSA; DMAB, p-(dimethylamino)benzaldehyde; EH, 4,5(E)-epoxy-2(E)-heptenal; HPCE, highperformance capillary electrophoresis; HPLC, high-performance liquid chromatography; KRPB, Krebs-Ringer phosphate buffer; MBSA, monomeric modified BSA; OLAARPs, oxidized lipid/amino acid reaction products; PI, protection index; Pnl, -N-pyrrolylnorleucine; RNS, reactive nitrogen species; ROS, reactive oxygen species; TBARS, thiobarbituric acid reactive substances.

can produce many of the above cited major interrelated derangements of cell metabolism. As a part of the cell defense system, previous studies from this laboratory have pointed out that oxidative stress is also, in some extent, delayed by the oxidized lipid/amino acid reaction products (OLAARPs) that are produced as an ultimate step in the lipid peroxidation process, suggesting that this might be a protective mechanism by which highly toxic aldehydes are blocked and endogenous antioxidants are generated (7). As a continuation of those studies, the present investigation was undertaken to analyze the potential physiological role of OLAARP-containing proteins by studying their in vitro formation as a consequence of oxidative stress and their antioxidant activity. Although their physiological functions are unknown at present, OLAARP-containing proteins have been detected in the sera of healthy individuals (8), and they are likely to be the most important source of OLAARPs in living beings. Among these OLAARPs, pyrroles have been shown to be produced in the reaction of amino groups with many lipid oxidation products, including 4,5-epoxy-2-alkenals (9), 4-hydroxy-2-alkenals (10, 11), unsaturated epoxyoxo fatty acids (12), and lipid hydroperoxides (13). Therefore, protein pyrrolization appears to be an important step in the lipid peroxidation process initiated by the oxidative stress. In addition, pyrrolized proteins have been found in vivo in human plasma and vasculature as levuglandin E2-protein adducts (14).

10.1021/tx000215m CCC: $20.00 © 2001 American Chemical Society Published on Web 05/21/2001

Antioxidant Function of Proteins

To test antioxidative activity of pyrrolized proteins, two model purified proteins were prepared from a bovine serum albumin/4,5(E)-epoxy-2(E)-heptenal (BSA/EH) mixture, which was selected for a variety of reasons. BSA has been suggested to act as antioxidant (15, 16) and to protect apolipoprotein B from damage by sequestering lipid peroxidation products (17). In addition, it represents a major class of animal proteins; it is free of prosthetic groups and other complicating factors; and its primary, secondary, and tertiary structure has been well characterized in the literature (18). Among the many different aldehydes which can be formed during lipid peroxidation and that pyrrolize proteins, EH was selected because it is highly reactive with the lysine amino groups (19), and because the mechanisms involved in these reactions are now well-known (9, 20).

Materials and Methods Materials. EH was prepared from 2(E),4(E)-heptadienal analogously to 4,5(E)-epoxy-2(E)-decenal (13). 2(E),4(E)-Heptadienal and 3-chloroperoxybenzoic acid were purchased from Aldrich Chemical Co. (Milwaukee, WI). Essentially fatty acid free BSA, Sephacryl S-200-HR, PD-10 columns packed with Sephadex G-25 medium, and butylated hydroxytoluene (BHT) were purchased from Sigma Chemical Co. (St. Louis, MO). Other reagents and solvents used were analytical grade and were purchased from reliable commercial sources. Preparation of OLAARP-Containing Proteins. BSA (30 mg) was dissolved in 3 mL of 50 mM sodium phosphate buffer, pH 7.4, and treated with 10 mM EH. The mixture was allowed to react in a closed vessel for 19 h at 37 °C, and, then, the brown solution was fractionated chromatographically on a Sephacryl S-200-HR column. One milliliter of the protein solution was injected in the column (1 × 56 cm) and eluted with 50 mM sodium phosphate buffer, pH 7.4, at a flow rate of 11 mL/h. Fivehundred microliter fractions were collected and tested for protein by absorption at 280 nm. This sequence was used repeatedly until the whole sample was fractionated. Fractions corresponding to monomeric and dimeric modified BSA (MBSA and DBSA, respectively) were pooled, desalted using a PD-10 column, and freeze-dried. This procedure yielded 16.2 mg of MBSA and 4.7 mg of DBSA. OLAARP presence in MBSA and DBSA was analyzed by determining OLAARP product -Npyrrolylnorleucine (Pnl) by high-performance capillary electrophoresis (HPCE) after basic hydrolysis as described previously (21). Preparation and Oxidation of Trout Muscle Microsomes. Muscle microsomes from freshly killed rainbow trout (Salmo gairdnerii) were prepared by differential centrifugation according to a procedure of Parkin and Hultin (22) as described previously (21). Washed microsomes were dispersed in KrebsRinger phosphate buffer (KRPB) (23), and protein concentration, measured according to Bradford’s method using BSA as standard (24), was adjusted to 3.0 mg/mL for experiments. Microsomes (50 µL of the stock solution) were suspended in 5 mL of KRPB and incubated at 37 °C in the presence of a ROS generating system. Three different procedures were used to generate ROS. Procedure 1 consisted in the treatment with 5 µM CuCl2. Procedure 2 consisted in the treatment with 1 mM FeCl3 and 5 mM ascorbic acid. Procedure 3 consisted in the treatment with 1 mM CuCl2 and 10 mM H2O2. These systems were very efficient to produce lipid and protein oxidation in incubated microsomes, and they were used previously to test antioxidant activities for unbound OLAARPs (7). Protein Pyrrolization in Incubated Microsomes. The production of OLAARP-containing proteins in incubated microsomes was analyzed by both: detection of pyrrolized proteins with the Ehrlich reagent and determination of OLAARP product Pnl after basic hydrolysis. Pyrrole determination was carried out by using p-(dimethylamino)benzaldehyde (DMAB) under

Chem. Res. Toxicol., Vol. 14, No. 5, 2001 583 acid conditions as described previously (25). Briefly, incubated microsomes (2 mL) were treated with 320 µL of a 2% solution of DMAB in 3.5 N HCl:ethanol (4:1), and the resulting solution was incubated at 45 °C for 30 min. The protein was precipitated with 1.2 mL of 30% trichloroacetic acid at 0 °C for 1 h and, then, centrifuged at 2250g for 15 min. The pellet was washed with ethanol:ethyl acetate (1:1), and, finally, treated with 2 mL of 6 M guanidine HCl containing 20 mM potassium phosphate: trifluoroacetic acid, pH 2.3, for 30 min at 37 °C with vortexing. This treatment partially solubilized the pyrrolized proteins and their absorption spectra could be obtained. Pnl was determined as described previously (21). Briefly, incubated samples (15 mL) were centrifuged at 100000g for 1 h and the resulting pellets, which contained the oxidized microsomes, were suspended in 1 mL of deionized water and submitted to basic hydrolysis with 2 N NaOH at 120 °C for 18 h. Pnl was determined by capillary electrophoresis after derivatization with diethyl ethoxymethylenemalonate and using homoarginine as standard. Model pyrrolized microsomes were obtained by incubating microsomes (50 µL of the stock solution suspended in 5 mL of KRPB) at 37 °C for 3 h in the presence of 1 mM EH. Effect of OLAARP-Containing Proteins on Oxidative Damage Induced by ROS in Microsomes. Microsomes (50 µL of the stock solution) were suspended in 5 mL of KRPB and incubated at 37 °C in the presence of the ROS generating system and the protein tested as antioxidant. At different incubation times, lipid peroxidation was assessed by the formation of thiobarbituric acid reactive substances (TBARS) using a procedure of Kosugi et al. (26), which was modified as described previously (7). At the end of the incubation period, incubated samples (1.8 mL) were hydrolyzed with 6 N HCl for 20 h (110 °C) and, then, submitted to amino acid analysis by highperformance liquid chromatography (HPLC) using a previously described procedure (27). For comparison purposes, when differences were significant, a protection index (PI) was calculated according to the following equation:

PI ) 100 × [1 - ((x - y)/(a - b))] where a is the microsomes with ROS, b is the microsomes without ROS, x is the microsomes plus modified proteins treated with ROS, and y is the untreated microsomes plus the modified proteins. Statistical Analysis. All results are expressed as mean values of three experiments unless otherwise indicated. Statistical comparisons between two groups were made using Student’s t-test. With several groups, ANOVA was used. When significant F values were obtained, group differences were evaluated by the Student-Newman-Keuls test (28). All statistical procedures were carried out using Primer of Biostatistics: The Program (McGraw-Hill, Inc., New York). Significance level is p < 0.05 unless otherwise indicated.

Results Characterization of Nonenzymatically Browned Proteins. The incubation of BSA with EH rapidly produced browning and fluorescence in the protein (9). Figure 1 shows the gel filtration chromatogram on Sephacryl S-200-HR obtained for a BSA/EH incubation mixture. Two main fractions, corresponding to dimeric and monomeric modified BSA (DBSA and MBSA, respectively) were observed. Both fractions, which exhibited fluorescence at 440 nm when excited at 350 nm (data not shown), were isolated and studied for amino acid composition. Table 1 collects the amino acid composition of BSA, MBSA, and DBSA. In accordance with previous studies (9, 29), the reaction of EH with BSA mainly modified the

584

Chem. Res. Toxicol., Vol. 14, No. 5, 2001

Hidalgo et al.

Figure 1. Gel filtration chromatogram of a EH/BSA reaction mixture on Sephacryl S-200-HR. BSA was incubated at 37 °C in the presence of 10 mM EH for 19 h and injected in the column. Fractions were collected and tested for protein by absorption at 280 nm. Two fractions were isolated, which mainly corresponded to dimeric and monomeric modified BSA (DBSA and MBSA, respectively). Table 1. Amino Acid Composition of Nonenzymatically Browned Proteinsa amino acid

BSA

MBSA

DBSA

Ala Arg Asxb cystine Glxb Gly His Ile Leu Lys Met Phe Ser Thr Tyr Val

0.68 ( 0.02 (9.1) 0.29 ( 0.03 (3.9) 0.83 ( 0.03 (11.1) 0.14 ( 0.01 (1.9) 1.16 ( 0.03 (15.4) 0.27 ( 0.02c (3.6) 0.16 ( 0.02 (2.1) 0.18 ( 0.01c (2.4) 0.89 ( 0.03 (11.9) 0.82 ( 0.03c (10.9) 0.06 ( 0.01c (0.8) 0.38 ( 0.01c (5.1) 0.40 ( 0.01c (5.3) 0.47 ( 0.01c (6.3) 0.26 ( 0.01 (3.5) 0.52 ( 0.01 (6.9)

0.74 ( 0.04 (10.0) 0.29 ( 0.02 (3.9) 0.79 ( 0.04 (10.7) 0.14 ( 0.01 (1.9) 1.17 ( 0.02 (15.8) 0.35 ( 0.01d (4.7) 0.15 ( 0.01 (2.0) 0.22 ( 0.01d (3.0) 0.90 ( 0.02 (12.1) 0.45 ( 0.02d (6.1) 0.07 ( 0.01c (0.9) 0.37 ( 0.01c (5.0) 0.47 ( 0.01d (6.3) 0.52 ( 0.02d (7.0) 0.24 ( 0.01 (3.2) 0.54 ( 0.02 (7.3)

0.74 ( 0.02 (9.7) 0.28 ( 0.02 (3.7) 0.78 ( 0.02 (10.2) 0.16 ( 0.02 (2.1) 1.21 ( 0.01 (15.9) 0.36 ( 0.01d (4.7) 0.18 ( 0.01 (2.4) 0.21 ( 0.02d (2.8) 0.93 ( 0.01 (12.2) 0.43 ( 0.01d (5.6) 0.09 ( 0.01d (1.2) 0.41 ( 0.02d (5.4) 0.47 ( 0.02d (6.2) 0.56 ( 0.02e (7.3) 0.24 ( 0.01 (3.1) 0.57 ( 0.03 (7.5)

a Values are mean ( SD for three experiments and are given in micromoles per milligram protein (amino acid percentage is shown in parentheses). b Asx, aspartic acid + asparagine; Glx, glutamic acid + glutamine. c,d,e Means in the same row with different superscripts are significantly different (p < 0.05).

amino acid lysine, which decreased about 45% after acid hydrolysis. A similar decrease in this amino acid was observed for both MBSA and DBSA. These losses are a consequence of the reactivity of EH for the amino groups. Thus, EH modified some of the -amino groups of lysine residues into pyrrole amino acids (19). The reaction scheme has been summarized in Figure 2. When EH reacts with the -amino group of lysine, two different pyrrole derivatives are produced: 1-(5′-amino-5′-carboxypentyl)-2-(1′′-hydroxypropyl)pyrrole (I) and -N-pyrrolylnorleucine (Pnl) (II). The hydroxyalkylpyrrole derivative I is unstable and polymerizes spontaneously (both intraand intermolecularly) producing lipofuscin-like polymers, which are the responsible for the brown color and fluorescence of MBSA and DBSA (9, 20). However, Pnl

Figure 2. Reaction scheme for protein pyrrolization produced by EH. P represents the protein. Polymerization is produced both intra- and intermolecularly. Table 2. OLAARP Formation in Nonenzymatically Browned Proteinsa amino acid

BSA

MBSA

DBSA

Arg Lys Pnl

0.35 ( 0.02 0.75 ( 0.01b

0.32 ( 0.02 0.46 ( 0.01c 0.09 ( 0.01b

0.33 ( 0.02 0.46 ( 0.02c 0.12 ( 0.01c

a Values are mean ( SD for three experiments and are given in micromoles per milligram protein. b,c Means in the same row with different superscripts are significantly different (p < 0.05).

is stable enough to be determined by HPCE after basic hydrolysis (21). Table 2 shows arginine, lysine, and Pnl composition of BSA, MBSA, and DBSA determined after basic hydrolysis. As expected (9), the decreases observed in the amino acid lysine of MBSA and DBSA were partially compensated by the formation of the new amino acid Pnl. According to HPCE, about 40% of lysine residues were lost in BSA upon EH treatment and the 13% of these residues appeared as Pnl. The other 27% of lysine residues should be converted, in a first step, into hydroxyalkylpyrroles and, later, into polymers. Although both MBSA and DBSA exhibited the same losses of lysine after acid and basic hydrolysis, Pnl was not produced in the same proportion in both modified proteins and DBSA exhibited a higher proportion of Pnl residues than MBSA (p ) 0.021). Pyrrolization of Microsomal Proteins Produced by ROS. A pyrrolization of microsomal proteins analogous to that observed for the reaction with EH was also observed following their exposition to ROS. Figure 3 shows the UV spectra obtained for the Ehrlich adducts produced with three pyrrolized proteins: (A) BSA incubated in the presence of EH; (B) microsomes treated with


Chem. Res. Toxicol., Vol. 14, No. 5, 2001 585

Figure 3. UV spectra of Ehrlich adducts obtained from pyrrolized proteins. Ehrlich adducts were produced in the reaction of DMAB with (A) BSA incubated overnight with 1 mM EH; (B) microsomes incubated for 3 h in the presence of 1 mM EH; (C) microsomes oxidized for 3 h in the presence of the Fe3+/ ascorbate system; (D) control microsomes. Spectra B, C, and D were taken using the same conditions and, therefore, are comparable. Spectrum A was reduced to be incorporated into the figure.

EH; and (C) microsomes oxidized with Fe3+/ascorbate. These three spectra exhibited similar absorption maxima, which was characteristic of the chromophore pyrrole/ DMAB (25) and that was not shown by control microsomes (unoxidized microsomes treated with DMAB, Figure 3D), suggesting that oxidative stress produced protein pyrrolization analogously to the treatment with EH. A further confirmation of this modification of microsomal proteins was obtained by determination of Pnl by HPCE after basic hydrolysis. Figure 4 shows the electropherogram obtained from hydrolyzed unoxidized microsomes, and microsomes oxidized for 3 h in the presence of Fe3+/ascorbate. Pnl concentration in control microsomes was 0.5 ( 0.4 µM, and this concentration increased significantly (p < 0.001) when microsomes were incubated in the presence of the Fe3+/ascorbate system. Pnl concentration in these oxidized microsomes was 2.3 ( 0.6 µM. Effect of Nonenzymatically Browned Proteins on Oxidative Damage Induced by ROS in Microsomes. The nonenzymatically browned proteins MBSA and DBSA protected microsomes against both lipid peroxidation and protein damage. The concentrations of the proteins used to test this protection were selected to obtain a Pnl concentration in the added proteins that were similar to Pnl concentration produced after Fe3+/ ascorbate treatment. Thus, a concentration of 40 µg/mL of MBSA or DBSA corresponded to about 4 µM Pnl, and therefore, this was the maximum concentration used in these studies. Microsomal protection of lipid peroxidation by MBSA and DBSA was evaluated by studying TBARS production in microsomes incubated in the presence of Cu2+, Fe3+/ ascorbate, and Cu2+/H2O2. Previous studies showed that the best incubation periods for the Cu2+, Fe3+/ascorbate,

Figure 4. Electropherograms obtained for (A) control microsomes; (B) microsomes oxidized for 3 h in the presence of the Fe3+/ascorbate system. Peaks corresponding to amino acids Pnl, arginine, and lysine are indicated in the figure.

and Cu2+/H2O2 systems were 24, 3, and 1 h, respectively (7). Figure 5 shows TBARS values obtained in microsomes oxidized in the presence of 20 µg/mL of BSA, MBSA, and DBSA as a function of the incubation time. When the microsomes were incubated in the presence of 5 µM Cu2+, a significant decrease in TBARS production was observed for DBSA after 6 h and for BSA and MBSA after 24 h (Figure 5A). At the end of the incubation period (24 h) the PI observed were 15, 19, and 32 for BSA, MBSA, and DBSA, respectively. Analogous results were obtained for the Fe3+/ascorbate and Cu2+/H2O2 systems (Figure 5, panels B and C, respectively). In the treatment with Fe3+/ascorbate, both MBSA and DBSA decreased significantly TBARS production after 1 min, and BSA after 15 min. At the end of the incubation period (3 h) the PI observed were 6, 16, and 27 for BSA, MBSA, and DBSA, respectively. In the treatment with the Cu2+/H2O2 system, DBSA exhibited a significant decrease in TBARS production after 15 s and for MBSA and BSA after 1 min. At the end of the incubation period (1 h) the PI observed were 6, 12, and 23 for BSA, MBSA, and DBSA, respectively. This protection of lipid peroxidation depended on the concentration of the tested OLAARP-containing protein and increased when its concentration also increased. Figure 6A shows the effect of protein concentration on TBARS production in microsomes treated with Cu2+. In the treatment with Cu2+, the PI obtained at the three tested concentrations (10, 20, and 40 µg/mL) were, respectively, 3, 15, and 21 for BSA, 7, 19, and 40 for MBSA, and 12, 32, and 54 for DBSA. Analogous results

586


Figure 5. TBARS production in microsomes incubated with ROS and nonenzymatically browned proteins. Microsomes were suspended in KRPB and incubated at 37 °C in the absence (O) or in the presence of 20 µg/mL of BSA (4), MBSA (3), or DBSA ()), and oxidized with (A) 5 µM Cu2+; (B) 1 mM Fe3+/5 mM ascorbate; (C) 1 mM Cu2+/10 mM H2O2. Control microsomes with or without proteins added (0) are included in graphs for comparison purposes.

were obtained for the Fe3+/ascorbate and the Cu2+/H2O2 systems when the proteins were tested at 20 or 40 µg/ mL, respectively (Figure 6, panels B and C). Addition of modified proteins to microsomes also induced a protection against protein damage as could be determined by amino acid analysis after acid hydrolysis. Table 3 shows the amino acid analysis obtained for microsomes treated with Cu2+ when 40 µg/mL of BSA, MBSA, or DBSA was added. This table only includes the amino acids that were destroyed in microsomes following Cu2+ oxidation (7). Addition of BSA induced a slight protection in histidine and lysine residues but not in arginine, cyst(e)ine, or tyrosine. BSA had negative PI for these last three amino acids. These results suggest that BSA did not protect arginine, cyst(e)ine and tyrosine residues in microsomes, and a part of these residues in the added BSA were also destroyed by the ROS generating system. Addition of 40 µg/mL of MBSA was significantly more protective of all the amino acids, except cyst(e)ine. Finally, addition of 40 µg/mL of DBSA exhibited an almost complete protection, and all the amino acids were recovered almost quantitatively after the 24 h incubation period. Analogous results were obtained for the Fe3+/ascorbate and the Cu2+/H2O2 systems (Tables 4 and 5, respectively). However, the protection exhibited depended on the amino acid studied and the ROS generating system employed. As a general rule, and with independence of the treat-

Hidalgo et al.

Figure 6. TBARS production in microsomes incubated with ROS and nonenzymatically browned proteins at different concentrations. Microsomes were suspended in KRPB at 37 °C in the absence (stripped bars) or in the presence of BSA (open bars), MBSA (crosshatched bars), or DBSA (horizontally striped bars), and oxidized with (A) 5 µM Cu2+; (B) 1 mM Fe3+/5 mM ascorbate; (C) 1 mM Cu2+/10 mM H2O2. The incubation time depended on the ROS generating system used and was 24 h for Cu2+, 3 h for Fe3+/ascorbate, and 1 h for Cu2+/H2O2. Bars with different letters are significantly different (p < 0.05).

ment and the amino acid studied, DBSA was always more protective than MBSA, and both of them were much more protective than BSA. Thus, for example, lysine damage was protected by 8, 38, and 50% in the treatment with Fe3+/ascorbate, and -17, 53, and 62%, in the treatment with Cu2+/H2O2. These two systems were slightly different from Cu2+ because some amino acids were not protected at the tested concentrations of MBSA and DBSA. These were cyst(e)ine and methionine for the Fe3+/ ascorbate system, and cyst(e)ine, methionine, and tyrosine for the Cu2+/H2O2 system. Nevertheless, the PI also increased in these cases in the order BSA < MBSA < DBSA.

Discussion The results obtained in this study suggest that one consequence of oxidative stress is the pyrrolization of proteins. This effect, which was previously observed in other systems (see, for example, ref 25), seems to be general with independence of the lipids implicated. Protein pyrrolization has been determined by using two different procedures. The first of them consisted in the treatment of oxidized microsomes with the Ehrlich reagent. The Ehrlich adducts obtained for oxidized microsomes were analogous to those obtained for microsomes incubated in the presence of EH and for BSA


Chem. Res. Toxicol., Vol. 14, No. 5, 2001 587

Table 3. Effect of Nonenzymatically Browned Proteins on Amino Acid Composition of Microsomes Incubated with Cu2+ none

amino acid

control

treated

control

Arg cystine His Lys Tyr

1.16 ( 0.06b 0.15 ( 0.04b 0.53 ( 0.07b 2.00 ( 0.10b 0.68 ( 0.03b

1.11 ( 0.03c 0.09 ( 0.01c 0.35 ( 0.06c 1.65 ( 0.06c 0.59 ( 0.02c

1.51 ( 0.03b 0.39 ( 0.01b 0.80 ( 0.05b 2.97 ( 0.07b 1.00 ( 0.02b

BSA treated 1.45 ( 0.02c 0.25 ( 0.02c 0.67 ( 0.06c 2.68 ( 0.04c 0.85 ( 0.03c

MBSA treated

PI

control

-20 -133 28 17 -67

1.29 ( 0.01b 0.30 ( 0.01b 0.68 ( 0.01b 2.20 ( 0.02b 0.84 ( 0.01b

1.28 ( 0.01b 0.24 ( 0.01c 0.65 ( 0.01c 2.07 ( 0.01c 0.80 ( 0.01c

PI

control

100 0 83 63 56

1.27 ( 0.01b 0.28 ( 0.01b 0.66 ( 0.02b 2.15 ( 0.01b 0.82 ( 0.01b

DBSA treated 1.27 ( 0.01b 0.27 ( 0.01b 0.65 ( 0.01b 2.09 ( 0.01c 0.81 ( 0.01b

a

PI 100 100 100 83 100

a Values (in µmol/mg of microsomal protein) are mean ( SD for three independent experiments and are given for the amino acids which decreased significantly in microsomes following Cu2+ treatment (7). When control and treated samples were significantly different (p < 0.05), percentages of inhibition (PI) were calculated as described in the Materials and Methods. When control and treated samples were not different, PI was equal to 100. b,c Means for control and treated samples with different superscripts are significantly different (p < 0.05).

Table 4. Effect of Nonenzymatically Browned Proteins on Amino Acid Composition of Microsomes Incubated with Fe3+/Ascorbatea none

amino acid

control

treated

control

Ala Arg Asxd cystine Glxd Gly His Ile Leu Lys Met Phe Ser Thr Tyr Val

2.54 ( 0.13b 1.16 ( 0.06b 3.84 ( 0.20b 0.15 ( 0.04b 3.00 ( 0.16b 2.42 ( 0.17b 0.53 ( 0.07b 1.74 ( 0.10b 2.50 ( 0.13b 2.00 ( 0.10b 0.90 ( 0.19b 1.19 ( 0.06b 1.69 ( 0.08b 1.80 ( 0.15b 0.68 ( 0.03b 2.50 ( 0.13b

2.43 ( 0.18b 0.97 ( 0.11c 3.63 ( 0.19c 0.12 ( 0.02c 2.79 ( 0.14c 2.19 ( 0.18c 0.27 ( 0.07c 1.54 ( 0.14c 2.27 ( 0.19c 1.52 ( 0.10c 0.74 ( 0.15b 1.02 ( 0.10c 1.53 ( 0.13c 1.54 ( 0.14c 0.54 ( 0.05c 2.27 ( 0.18c


BSA treated 3.37 ( 0.06b 1.37 ( 0.08c 4.93 ( 0.07b 0.29 ( 0.01c 4.26 ( 0.06b 2.72 ( 0.06b 0.56 ( 0.01c 1.61 ( 0.04c 3.42 ( 0.03b 2.53 ( 0.03c 0.76 ( 0.01c 1.51 ( 0.02c 2.01 ( 0.02c 2.41 ( 0.04b 0.83 ( 0.04c 3.00 ( 0.06b

PI

control

100 26 100 -233 100 100 8 20 100 8 -38 35 31 100 -21 100


MBSA treated 2.91 ( 0.01b 1.17 ( 0.04b 4.34 ( 0.06b 0.21 ( 0.01c 3.72 ( 0.09b 2.35 ( 0.05b 0.41 ( 0.02c 1.55 ( 0.03c 3.02 ( 0.03b 1.90 ( 0.03c 0.41 ( 0.04c 1.37 ( 0.01c 1.85 ( 0.02c 2.20 ( 0.03b 0.76 ( 0.03c 2.55 ( 0.04b

PI

control

100 37 100 -200 100 100 -4 35 100 38 -238 82 38 100 43 100


DBSA treated 2.97 ( 0.06b 1.18 ( 0.03c 4.38 ( 0.09b 0.23 ( 0.01c 3.76 ( 0.06b 2.42 ( 0.08b 0.56 ( 0.03c 1.58 ( 0.04c 3.01 ( 0.06b 1.91 ( 0.03c 0.50 ( 0.05c 1.36 ( 0.02b 1.83 ( 0.03c 2.22 ( 0.06b 0.75 ( 0.04c 2.59 ( 0.07b

PI 100 53 100 -67 100 100 62 60 100 50 -138 100 50 100 50 100

a Values (in µmol/mg of microsomal protein) are mean ( SD for three independent experiments. When control and treated samples were significantly different (p < 0.05), percentages of inhibition (PI) were calculated as described in the Materials and Methods. When control and treated samples were not different, PI was equal to 100. b,c Means for control and treated samples with different superscripts are significantly different (p < 0.05). d Asx, aspartic acid + asparagine; Glx, glutamic acid + glutamine.

Table 5. Effect of Nonenzymatically Browned Proteins on Amino Acid Composition of Microsomes Incubated with Cu2+/H2O2 a none

amino acid

control

treated

control

Ala Arg Asxd cystine Glxd Gly His Ile Leu Lys Met Phe Ser Thr Tyr Val

2.54 ( 0.13a 1.16 ( 0.06a 3.84 ( 0.20a 0.15 ( 0.04a 3.00 ( 0.16a 2.42 ( 0.17a 0.53 ( 0.07a 1.74 ( 0.10a 2.50 ( 0.13a 2.00 ( 0.10a 0.90 ( 0.19a 1.19 ( 0.06a 1.69 ( 0.08a 1.80 ( 0.15a 0.68 ( 0.03a 2.50 ( 0.13a

1.80 ( 0.16c 0.67 ( 0.08c 2.82 ( 0.29c 0.00 ( 0.00c 2.23 ( 0.25c 1.66 ( 0.28c 0.16 ( 0.04c 1.12 ( 0.15c 1.44 ( 0.21c 0.96 ( 0.21c 0.26 ( 0.22c 0.02 ( 0.02c 1.05 ( 0.16c 1.21 ( 0.20c 0.00 ( 0.00c 1.79 ( 0.22c


BSA treated 2.91 ( 0.12c 1.11 ( 0.04c 4.36 ( 0.07c 0.00 ( 0.00c 3.96 ( 0.06c 2.08 ( 0.06c 0.25 ( 0.02c 1.52 ( 0.04c 2.61 ( 0.05c 1.75 ( 0.04c 0.00 ( 0.00c 0.10 ( 0.04c 1.54 ( 0.06c 1.88 ( 0.05c 0.00 ( 0.00c 2.58 ( 0.07c

PI

control

57 18 57 -160 40 55 -49 60 17 -17 -16 -30 9 36 -47 55


MBSA treated 2.95 ( 0.04a 1.16 ( 0.01c 4.36 ( 0.02a 0.00 ( 0.00c 3.76 ( 0.03a 2.21 ( 0.04c 0.27 ( 0.02c 1.57 ( 0.03c 2.94 ( 0.04a 1.71 ( 0.02c 0.00 ( 0.00c 0.56 ( 0.05c 1.74 ( 0.01c 2.18 ( 0.05a 0.00 ( 0.00c 2.59 ( 0.07a

PI

control

100 73 100 -100 100 71 -11 82 100 53 -48 28 67 100 -24 100


DBSA treated 2.97 ( 0.06a 1.15 ( 0.02c 4.37 ( 0.11a 0.00 ( 0.00c 3.78 ( 0.08a 2.41 ( 0.06c 0.46 ( 0.03c 1.70 ( 0.03a 3.00 ( 0.06a 1.75 ( 0.02c 0.00 ( 0.00c 0.56 ( 0.02c 1.74 ( 0.02c 2.21 ( 0.06a 0.00 ( 0.00c 2.67 ( 0.06a

PI 100 76 100 -87 100 100 46 100 100 62 -38 30 73 100 -21 100

a Values (in µmol/mg of microsomal protein) are mean ( SD for three independent experiments. When control and treated samples were significantly different (p < 0.05), percentages of inhibition (PI) were calculated as described in the Materials and Methods. When control and treated samples were not different, PI was equal to 100. b ,c Means for control and treated samples with different superscripts are significantly different (p < 0.05). d Asx, aspartic acid + asparagine; Glx, glutamic acid + glutamine.

also treated with this aldehyde, suggesting that all of these processes produced analogous pyrroles. Other studies from this laboratory have shown that the same Ehrlich adducts were also obtained in the oxidation of serum proteins with Fe3+/ascorbate or in the treatment of serum proteins with lipid hydroperoxides, but not in the treatment of these proteins with hydroxyalkenals (25). The second procedure consisted in the determination of OLAARP product Pnl. Pnl was produced in microsomes oxidized with Fe3+/ascorbate. After 3 h, the Pnl concentration in these microsomes was 2.3 ( 0.6 µM, a concentration that was high enough to delay the oxidative stress process (7).

When model OLAARP-containing proteins, with a Pnl concentration similar to that produced in oxidized microsomes, were tested for antioxidant activity in the microsomal system, these proteins efficiently protected microsomes against lipid peroxidation and protein damage induced by three different ROS generating systems. This protection, which was parallel to the concentration of the assayed protein and the Pnl content in the protein, was much higher than that exhibited by the unmodified protein. These results suggest that OLAARP-containing proteins, which are produced as a final step in the lipid peroxidation process, are delaying the whole oxidative stress by competing effectively with other nucleophiles

588


Figure 7. Antioxidant effect of OLAARP-containing proteins produced as a consequence of oxidative stress. ROS produce both lipid and protein oxidation, but oxidized lipids react with proteins to form OLAARP-containing proteins with antioxidant properties.

in reacting with the ROS produced. In this process, all different OLAARPs produced (9, 29) may participate. The whole process is shown in Figure 7. The above results also give a much more complete vision of the oxidative stress process. Proteins are modified following oxidative stress, and this modification is able to induce antioxidative activity in these proteins, which delays the whole process. According to these results, and in addition to the antioxidant properties that some proteins have by themselves, all proteins may also exert an antioxidant effect after reaction with lipid oxidation products, which might either increase their ability to sequester metals or generate new molecules that are antioxidants by themselves. This may be a protective mechanism by which these toxic compounds are removed and endogenous antioxidants are produced, constituting a general pathway for an additional antioxidative effect of proteins. This protective mechanism may also be acting in parallel with the endogenous antioxidant activities suggested for some amino acid residues, like methionine (30).

Acknowledgment. This study was supported in part by the Comisioń Interministerial de Ciencia y Tecnologıá (CICYT) of Spain (Project ALI97-0358) and the Junta de Andalucıá (Project AGR 0135). We are indebted to Unioń Piscıćola Navarra, S. L. (Granada, Spain) for the gift of the trouts used in this study, and to Mr. J. L. Navarro for the technical assistance.

References (1) Halliwell, B., and Gutteridge, J. M. (1999) Free Radicals in Biology and Medicine, 3rd ed., Oxford, U.K. (2) Ogino, T., Packer, L., and Traber, M. C. (1999) Oxidant stress and host oxidant defense mechanisms. In Nutritional Oncology (Heber, D., Blackburn, G. L., and Go, V. L. W., Eds.) pp 253275, Academic Press, San Diego, CA. (3) Keyse, S. M., Ed. (2000) Stress Response. Methods and Protocols, Humana Press, Totowa, NJ. (4) Rice-Evans, C., and Bruckdorfer, K. R., Eds. (1995) Oxidative Stress, Lipoproteins and Cardiovascular Dysfunction, Portland Press, London, U.K. (5) Scandalios, J. G., Ed. (1997) Oxidative Stress and the Molecular Biology of Antioxidant Defenses, Cold Spring Harbor Lab., Plainview, NY. (6) O ¨ zben, T., Ed. (1998) Free Radicals, Oxidative Stress, and Antioxidants: Pathological and Physiological Significance, Plenum Press, New York.

Hidalgo et al. (7) Zamora, R., Alaiz, M., and Hidalgo, F. J. (1997) Feed-back inhibition of oxidative stress by oxidized lipid/amino acid reaction products. Biochemistry 36, 15765-15771. (8) Kotani, K., Maekawa, M., Kanno, T., Kondo, A., Toda, N., and Manabe, M. (1994) Distribution of immunoreactive malondialdehyde-modified low-density lipoprotein in human serum. Biochim. Biophys. Acta 1215, 121-125. (9) Hidalgo, F. J., and Zamora, R. (2000) Modification of bovine serum albumin structure following reaction with 4,5(E)-epoxy-2(E)heptenal. Chem. Res. Toxicol. 13, 501-508. (10) Xu, G., Liu, Y., Kansal, M. M., and Sayre, L. M. (1999) Rapid cross-linking of proteins by 4-ketoaldehydes and 4-hydroxy-2alkenals does not arise from the lysine-derived monoalkylpyrroles. Chem. Res. Toxicol. 12, 855-861. (11) Salomon, R. G., Kaur, K., Podrez, E., Hoff, H. F., Krushinsky, A. V., and Sayre, L. M. (2000) HNE-derived 2-pentylpyrroles are generated during oxidation of LDL, are more prevalent in blood plasma from patients with renal disease or atherosclerosis, and are present in atherosclerotic plaques. Chem. Res. Toxicol. 13, 557-564. (12) Hidalgo, F. J., and Zamora, R. (1995) In vitro production of long chain pyrrole fatty esters from carbonyl-amine reactions. J. Lipid Res. 36, 725-735. (13) Zamora, R., and Hidalgo, F. J. (1995) Linoleic acid oxidation in the presence of amino compounds produces pyrroles by carbonyl amine reactions. Biochim. Biophys. Acta 1258, 319-327. (14) Salomon, R. G., Subbanagounder, G., O’Neil, J., Kaur, K., Smith, M. A., Hoff, H. F., Perry, G., and Monnier, V. M. (1997) Levuglandin E2-protein adducts in human plasma and vasculature. Chem. Res. Toxicol. 10, 536-545. (15) Peters, T., Jr. (1996) All About Albumin. Biochemistry, Genetics and Medical Applications, pp 238-239, Academic Press, San Diego, CA. (16) Das, D. K., and Maulik, N. (1994) Antioxidant effectiveness in ischemia-reperfusion tissue injury. Methods Enzymol. 233, 601610. (17) Deigner, H. P., Friedrich, E., Sinn, H., and Dresel, H. A. (1992) Scavenging of lipid peroxidation products from oxidizing LDL by albumin alters the plasma half-life of a fraction of oxidized LDL particles. Free Radical Res. Commun. 16, 239-246. (18) Brown, J. R. (1977) Structure and evolution of serum albumin. In Albumin Structure, Biosynthesis, Function (Peters, T., and Sjoholm, I., Eds.) Vol. 50, pp 1-10, Pergamon Press, New York. (19) Zamora, R., and Hidalgo, F. J. (1994) Modification of lysine amino groups by the lipid peroxidation product 4,5(E)-epoxy-2(E)heptenal. Lipids 29, 243-249. (20) Hidalgo, F. J., and Zamora, R. (1993) Fluorescent pyrrole products from carbonyl-amine reactions. J. Biol. Chem. 268, 16190-16197. (21) Zamora, R., Navarro, J. L., and Hidalgo, F. J. (1995) Determination of lysine modification product -N-pyrrolylnorleucine in hydrolyzed proteins and trout muscle microsomes by micellar electrokinetic capillary chromatography. Lipids 30, 477-483. (22) Parkin, K. L., and Hultin, H. O. (1982) Fish muscle microsomes catalyze the conversion of trimethylamine oxide to dimethylamine and formaldehyde. FEBS Lett. 139, 61-64. (23) De Luca, H. F. (1972) Methods for preparation and study of tissues and enzymes. In Manometric and Biochemical Techniques (Umbreit, W. W., Burris, R. H., and Stauffer, J. F., Eds.) pp 133147, Burgess Publishing, Minneapolis, MN. (24) Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254. (25) Hidalgo, F. J., Alaiz, M., and Zamora, R. (1998) A spectrophotometric method for the determination of proteins damaged by oxidized lipids. Anal. Biochem. 262, 129-136. (26) Kosugi, H., Kojima, T., and Kikugawa, K. (1989) Thiobarbituric acid-reactive substances from peroxidized lipids. Lipids 24, 873881. (27) Alaiz, M., Navarro, J. L., Giron, J., and Vioque, E. (1992) Amino acid analysis by high-performance liquid chromatography after derivatization with diethyl ethoxymethylene malonate. J. Chromatogr. 591, 181-186. (28) Snedecor, G. W., and Cochran, W. G. (1980) Statistical Methods, 7th ed., Iowa State University Press, Ames, Iowa. (29) Zamora, R., Alaiz, M., and Hidalgo, F. J. (1999) Modification of histidine residues by 4,5-epoxy-2-alkenals. Chem. Res. Toxicol. 12, 654-660. (30) Levine, R. L., Mosoni, L., Berlett, B. S., and Stadtman, E. R. (1996) Methionine residues as endogenous antioxidants in proteins. Proc. Natl. Acad. Sci. U.S.A. 93, 15036-15040.

TX000215M

Pyrrolization and Antioxidant Function of Proteins Following Oxidative

Recommend Documents