Modification of Horse Heart Cytochrome c with trans-2-Hexenal

702

Chem. Res. Toxicol. 1997, 10, 702-710

Modification of Horse Heart Cytochrome c with trans-2-Hexenal Luka´sˇ Zˇ ´ıdek, Petr Dolezˇel,† Josef Chmelı´k,† Andrew G. Baker, and Milos Novotny* Department of Chemistry, Indiana University, Bloomington, Indiana 47405-4001 Received July 11, 1996X

Horse heart cytochrome c reacting with trans-2-hexenal was used as a simple model of the nonspecific interactions of proteins with 2-alkenals. The reaction mixtures containing relatively high concentrations of the protein and aldehyde were characterized using visible spectrophotometry, fluorescence, and circular dichroism measurements, capillary isoelectric focusing, sizeexclusion chromatography, polyacrylamide gel electrophoresis, and mass-spectrometric techniques. The mass-spectrometric data indicate that cytochrome c becomes modified with one or two molecules of hexenal as the major reaction product. The modified species with a correspondingly lowered isoelectric point were detected through capillary isoelectric focusing. The results of proteolytic studies indicate nonspecific modifications. Significant quantities of the oligomeric forms of hexenal-modified protein were also observed electrophoretically.

Introduction Since the 1970s, when various carbonyl compounds were first recognized as the major lipid peroxidation products (1), the toxicological properties of aldehydes have been extensively studied. The substances formed during the free-radical degradation of polyunsaturated fatty acids are sufficiently stable to diffuse from the site of their origin and attach to various target biomolecules which may be quite distant from the initial free-radical event (2). The simplest of them, malondialdehyde, is often used as a marker of lipid peroxidation, as it can easily be determined through its reaction with thiobarbituric acid (3) or its derivatives (4). However, 4-hydroxy2-alkenals are currently being recognized to be far more potent than malondialdehyde and thus responsible for most of the cytotoxic effects (2, 5-7). Elevations in these and related compounds were also determined in plasma and various organs under conditions of oxidative stress (8) and diabetes (9). Both alkenals and hydroxyalkenals represent a group of highly reactive agents containing two electrophilic reaction centers. A partially positive carbon 1 or 3 in such molecules can attack a nucleophile. They readily react with sulfhydryl and amino groups of different amino acids (10), peptides (11), and nucleotides (12), the imidazole ring of histidine (13), etc. However, the chemical events of this kind appear quite complex, and most products of such reactions still remain unidentified. The studies of the effect of 4-hydroxy-2-nonenal on human low-density lipoproteins revealed changes in the electrophoretic mobility in agarose gels (14). These are most likely caused by modification of the -amino group within the lysine side chains or modifications of the amino acids (after hydrolysis). Recently, several types of hydroxyalkenal-modified entities were identified in proteins (13, 15-17). In such studies, mass spectrometry (16, 17) and immunochemical detection (18) were shown * Address correspondence to this author. Tel: 812-855-4532. Fax: 812-855-8300. E-mail: [email protected]. † Present address: Institute of Analytical Chemistry, Academy of Sciences of the Czech Republic, Veverˇ´ı 97, 61142 Brno, Czech Republic. X Abstract published in Advance ACS Abstracts, May 15, 1997.

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to be particularly useful. Additional very abundant lipidperoxidation agents, 2-alkenals (19), have been less frequently studied as protein-modifying agents. Recently, the reaction of trans-2-octenal with bovine serum albumin was investigated, albeit the published results are contradictory (cf. refs 13 and 20). Lipid peroxidation in biological samples is also accompanied by the formation of fluorescent products with emission maxima in the range of 430-470 nm (2, 21). However, the two described (21) fluorophores, dihydropyridine (λex ≈ 400 nm; λem ≈ 450-470 nm) and 1-amino3-iminopropene (λex ≈ 360-400 nm; λem ≈ 450-470 nm) derivatives, do not adequately explain the fluorescence spectra widely measured in association with lipid peroxidation. Recently, a new fluorophore (2-hydroxy-1,2pyrrol-3-one), which corresponds to the readily observed fluorescence at lower wavelengths, was described (22). The present study has focused on the modification of lysine-rich proteins with 2-alkenals. We have chosen a strategy of accumulating large amounts of an alkenalmodified protein. While the aldehyde concentration was much higher than its level determined in physiological fluids (2), this approach allowed us to study the products of protein modification directly by mass spectroscopy and to facilitate a general description of the seemingly complex modification processes. Additional experimental techniques were employed to investigate conformational changes and side-chain modifications during the incubation. Specificity of locating these modifications has been addressed by proteolytic studies followed by a massspectrometric analysis. A simple system (trans-2-hexenal and horse heart cytochrome c) was chosen in this study because both reactive components are sufficiently stable and soluble in water. Additionally, the lack of free SHgroups in the cytochrome c molecule has simplified considerations of protein “oligomerization”.

Experimental Section Materials. Horse heart cytochrome c, trans-2-hexenal, trypsin (TPCK treated, type XIII from bovine pancreas), R-chymotrypsin (type II from bovine pancreas), hydroxypropylmeth-

© 1997 American Chemical Society

Reactions of Cytochrome c with 2-Hexenal ylcellulose, all electrophoretic buffers, and additives were purchased from Sigma (St. Louis, MO). 2,5-Dihydroxybenzoic acid and R-cyano-4-hydroxycinnamic acid were obtained from Aldrich (Milwaukee, WI); carboxypeptidase Y was received as a kit from PerSeptive Biosystems (Framingham, MA). The broad molecular mass standard, Coomassie brilliant blue R-250, and Bio-Gel P-60 were from Bio-Rad Laboratories (Richmond, CA). Servalyt 3-10 was a product of Serva (Heidelberg, Germany). The pI markers, 2,6-bis((N,N′-di(1-piperidyl)amino)methyl)-4-nitrophenol (pI 10.4), 2,6-bis((N,N′-di(1-(4-methylpiperazinyl))amino)methyl)-4-nitrophenol (pI 8.6), and 4,6-bis((N,N′di(4-morpholinyl)amino)methyl)-2-nitrophenol (pI 7.5), were produced by Tessek (Prague, Czech Republic). “Omnisolv” methanol from EM Science (Gibbstown, NJ), acetonitrile UV from Baxter (Muskegon, MI), “Biochemika” trifluoroacetic acid from Fluka (Ronkonkoma, NY), and glacial acetic acid (Reagent ACS) from Fisher Scientific (Fair Lawn, NJ) were used as solvents. The other common chemicals were the products of J. T. Baker, Inc. (Phillipsburg, NJ) and Mallinckrodt, Inc. (Paris, KY). Methods. The standard mixture for protein modification contained 0.8 mM horse heart cytochrome c and 0.2% (v/v), i.e., 17 mM, aqueous trans-2-hexenal. Incubation was performed at 37 °C in the absence of buffers or additional compounds. In all cases, the control experiments were performed in the absence of trans-2-hexenal. In contrast to the modifications reported below, no changes (fluorescence or dimer formation, occurrence of species with different mass or isoelectric point) were found in the control samples due to native cytochrome c. During the proteolytic studies, 2 mM cytochrome c was incubated with 0.2% trans-2-hexenal for 24 h at 37 °C followed by washing with 0.2 M ammonium bicarbonate in Microcon 10 microconcentrators. A 5-µL volume of the enzyme solution (5 mg of trypsin or chymotrypsin/mL of 0.2 M ammonium bicarbonate) was added to a 50-µL aliquot, incubated for 24 h at 37 °C, and diluted 25 times prior to chromatography. Sodium dodecyl sulfate-polyacrylamide electrophoresis (SDSPAGE1) was performed according to Laemmli (23-25) in the Bio-Rad Mini-Protean apparatus. The gel composition was T4.3C5.7 (stacking) and T14.9C1 (separation). Coomassie brilliant blue R-250 stain was used to visualize the protein bands. A laboratory-made instrument was employed for capillary isoelectric focusing with electroosmotic zone displacement (CIEF) (26, 27). The system consisted of an uncoated fused-silica gel capillary (90 cm in length, 75-µm i.d.; Polymicro Technologies, Phoenix, AZ) combined with a SpectraFocus fast-scanning multiwavelength detector with a capillary flow cell (Spectra Physics, Mountain View, CA) and a Spellman high-voltage DC supply (Spellman High Voltage Electronics Corp., Plainview, NY). The effective separation distance was 70 cm; 10 mM phosphoric acid and 20 mM NaOH containing 0.075% hydroxypropylmethylcellulose (HPMC) were used as the anolyte and catholyte, respectively. Prior to each run, the capillary was rinsed for 5 min with 0.1 M NaOH and then for 5 min with catholyte. A 2% solution of Servalyte 3-10 was used as a carrier ampholyte, in which both modified cytochrome c (0.15 mg/mL) and pI markers (28, 29) (0.1 mg/mL) were dissolved. The sample was introduced by elevating the inlet of the capillary by 60 cm, for 4 min. All experiments were performed at 20 kV. Cytochrome c (Fe3+) concentrations were determined spectrophotometrically (30). Spectrophotometric measurements were performed on Shimadzu UV-160 (Shimadzu, Kyoto, Japan) or Perkin-Elmer GC55 (Norwalk, CT) instruments. Circular dichroism spectra were recorded with a Jasco J-20 CD-spectrophotometer (Jasco, Tokyo, Japan). An Aminco SPF-500 spectrofluorometer (Urbana, IL) was used for fluorescence studies. Quartz cuvettes (1 cm) were used for all spectrophotometric measurements. The samples were diluted 100-fold with distilled 1 Abbreviations: SDS, sodium dodecyl sulfate; HPMC, hydroxypropylmethylcellulose; PAGE, polyacrylamide gel electrophoresis; CIEF, capillary isoelectric focusing; ESI-MS, electrospray ionization mass spectrometry; MALDI-TOF-MS, matrix-assisted laser-desorption/ ionization time-of-flight mass spectrometry.

Chem. Res. Toxicol., Vol. 10, No. 6, 1997 703 water for absorbance and fluorescence measurements and 500fold for circular dichroism spectral recording. ESI-MS studies were performed using an Analytica of Branford (Branford, CT) electrospray source interfaced to a Finnigan 4600 mass spectrometer (San Jose, CA). A Teknivent (St. Louis, MO) computer system was used for data acquisition. Mass spectra were deconvoluted into the mass domain using an algorithm developed by Mann et al. (31). The operating conditions were optimized for horse heart cytochrome c (1.0 mg/mL). The protein solutions were diluted to 1 mg/mL with water: acetonitrile:acetic acid (50:50:1) and delivered to the electrospray unit at 4 µL/min using a syringe pump (KP Scientific, Boston, MA). Acetonitrile at 4 µL/min was utilized as a sheath liquid. A countercurrent of warm (ca. 80 °C) nitrogen facilitated desolvation. Reversed-phase chromatography was performed using a 480 × 0.53-mm i.d., fused-silica gel capillary (Polymicro Technologies) packed with Vydac C18 5-µm particles and a modified Pharmacia (Uppsala, Sweden) SMART liquid-chromatographic system; 0.1% aqueous trifluoroacetic acid was used as the mobile phase at 12 µL/min flow with 0-40% acetonitrile gradient in 1.7 mL. Absorbance was recorded at 214, 280, and 410 nm. In order to measure fluorescence, a helium-cadmium laser (325 nm, Omnichrome Series 56; Chino, CA) beam was focused, under 45°, into a window burned in the coating of a fused-silica gel capillary (1125 × 0.1-mm i.d.). The emitted light was collected by a fiber optic and detected through a 389-nm cutoff filter by a photomultiplier (R928, Hamamatsu Photonics Corp., Hamamatsu, Japan), with a EG&G (Trenton, NJ) lock-in amplifier (Model 7220). Photomultiplier voltage of 600 V and amplifier sensitivity of 10 nA were used through the measurements. A more sensitive MALDI-TOF-MS supplanted ESI-MS to identify HPLC-separated peptides. A Voyager workstation (PerSeptive Biosystems, Framingham, MA) with a 1.2-m linear flight tube, one-stage ion source, and nitrogen 337-nm laser provided sufficient accuracy for the assignment of almost all major peaks in the chromatograms with a high degree of confidence. 2,5-Dihydroxybenzoic acid was the standard matrix in the study. Complementary measurements with R-cyano-4hydroxycinnamic acid were performed when matrix signals interfered with the mass signals of certain samples. Data were collected at 28 125 V and 9.3 µJ/pulse laser intensity. Theoretical molecular mass calculations were based on the known tryptic and chymotryptic cleavage sites (32-34). A comparison of retention volumes with predicted chromatographic behavior (GPMAW 2.13a software, Lighthouse Data, Odense, Denmark) was utilized to rule out incorrect assignments: the calculated HPLC factors were plotted against the real retention volumes of unambiguously assigned peptides. (The correlation coefficients were 0.91 and 0.89 for the tryptic and chymotryptic digests, respectively.) Subsequently, retention volumes of the peptides allowing two possible assignments were compared with their calculated HPLC factors. If one possibility resulted in a point which was far off the diagonal set of correlating values, this assignment was excluded from further considerations. Because several peptides whose assignment required a partial cleavage by a residual chymotryptic activity were found in the tryptic digest, the chymotryptic cleavage sites were taken into account in the interpretation of all trypsintreated samples. Similarly, a possibility of a residual tryptic activity was considered when interpreting the chymotryptic digests data. Size-exclusion chromatographic experiments were performed using a Bio-Rad delivery system (Model 2800), an HPLC gel filtration column Bio-Sil SEC-250 (600 × 7.5 mm), and a UVvis-205 linear absorbance detector. Aliquots (20-µL) of the incubation mixture were diluted 20-fold with water and injected; 0.05 M phosphate buffer (pH 6.8), at a flow rate of 1.0 mL/min, was used as the mobile phase.

Results and Discussion General Observations. Alkenals are highly reactive substances that could modify a protein in numerous

704 Chem. Res. Toxicol., Vol. 10, No. 6, 1997

ways. To account for various modifications, different techniques were used to investigate the reaction mixtures. First, circular dichroism spectroscopy was employed to determine any possible gross conformational changes of cytochrome c due to the reaction with trans2-hexenal. The spectra were recorded during the reaction time course, and visible absorbance spectra were measured simultaneously. Increasing intensity of the negative circular dichroism peaks at 208 and 222 nm (data not shown) was identical to the well-known (35) changes seen during reduction of the heme iron. Similar conclusions could be drawn from the recorded visible absorbance spectra (the appearance of the R and β bands and shift of the Soret absorption maximum to higher wavelengths in agreement with reference 30, data not shown). A slow recovery in the original (i.e., oxidized) sample spectrum upon the exposure to oxygen denotes reversible heme reduction. No reoxidation was observed under argon. The results of these rough spectral measurements seem to merely reflect the redox chemistry typical for cytochrome c without any evidence for significant conformational changes after reaction with trans-2-hexenal. Side-Chain Modifications. The absence of free sulfhydryls in cytochrome c (30) leaves only hydroxyls and amine groups (including the imidazole nitrogen of histidine) as the expected targets of modification by trans2-hexenal. The lysines are the most prevalent nucleophiles in cytochrome c (19 residues per molecule (30)). The Michael adducts at carbon 3 and the Schiff bases formed after a nucleophilic attack at the carbonyl carbon were thus the major expected modification products, and the electrospray ionization mass spectrometry was used to examine the products of such reactions. The charging inherent in electrospray ionization mass spectrometry allows high mass species (>>2000 mass units) to be analyzed using conventional quadrupole mass spectrometers. Molecular mass information about these high-mass species is obtained by deconvoluting the charge state envelope (m/z domain) into the mass domain using an algorithm developed by Mann et al. (31). This deconvolution algorithm enables an accurate (0.01%) determination of a molecular mass for even large molecules. The mass differences between the condensation (Schiff-base) products and adducts (protein + 80 mass units for the condensation product vs protein + 98 mass units for the Michael adduct) studied here are resolvable at this level of accuracy. The mass spectra of cytochrome c at various time points during modification are shown in Figure 1. At very short incubation times, the signals corresponding to attachment of 2-hexenal to the protein via the Schiffbase linkages dominated the spectra. A similarly rapid formation of such adducts with 2-unsaturated ketones is observed in contrast to the much lower reactivity of saturated aldehydes and ketones (A. Baker and M. Novotny, unpublished experiments). After dialysis aiming at removal of the unreacted 2-hexenal and the reversible Schiff bases, other additional signals appeared in the acquired spectra. Among them, 98-, 160-, and 196mass unit additions became highly significant. The addition of aldehyde’s molecular mass to a protein was observed previously (16). However, a transient formation of species corresponding to the condensation products has thus far not been reported. A kinetic study comparing the reactivities of various carbonyl compounds aimed at the explanation of this phenomenon is in progress. Upon removal of 2-hexenal by ultrafiltration (Figure 1), the signal due to the Schiff-base-modified

Z ˇ ı´dek et al.

protein vanished completely and a signal due to the unmodified protein was recovered. In contrast, the signals due to the Michael adducts, and also those of 160mass increments, remained visible. A possibility that the Schiff-base condensation products may be formed in the electrospray process due to droplet evaporation was tested by mixing the solutions of cytochrome c and hexenal directly at the inlet of the electrospray needle. A small amount of modified protein was observed under this condition; however, the extent of modification was strongly dependent on the time when the solutions were in contact. If the adducts are formed mostly with the lysine residues, the isoelectric point of the protein should be influenced by such modifications. CIEF was used to verify the corresponding changes. Accordingly, the analysis of cytochrome c after a 7-day incubation with trans2-hexenal showed several peaks due to the modified cytochrome c within the pH range of 9.3-7.5 (Figure 2), while the control sample remained unchanged, i.e, the original peaks due to cytochrome c were observed. This indicates a multiple lowering of the isoelectric point through modification, as expected for the modification of various lysines. The modified species can differ in the number of modifications and in their chemical nature, since the reaction of hexenal with a single amino group containing peptides can give several products (36). In the complex reaction mixture, only the simplest and the most abundant species could be observed directly as mass adducts. However, the initial modification products are still likely to be considerably reactive species. With a simple peptide model system, we have recently found that some reaction products with two or more incorporated aldehyde molecules can also be formed (36). Additional, perhaps minor, reactions lead to formation of the already mentioned fluorescent derivatives (2, 21). Their characteristic emission maxima at 430-470 nm enable a general, nonselective monitoring of the advanced reactions through application of appropriate spectroscopic techniques. With cytochrome c, a strong absorbance of the heme near these fluorophores’ excitation wavelengths is an obvious complication. In spite of this drawback, we were able to observe a time-dependent increase of fluorescence with a modified cytochrome c at the excitation wavelength of 360 nm. The emission maximum was found at 445 nm (Figure 3). Considering the influence of a strongly absorbing Soret band at 415 nm, the emission maximum was reasonably close to the value described for the fluorescence related to lipid peroxidation (emission maximum at 430 nm) (2, 21, 22) rather than those for dihydropyridine or 1-amino-3-iminopropene derivatives (470 nm) (21). The native fluorescence of tryptophan at 280-nm excitation was not significantly affected by the modification, and its time course was within the range of experimental error (Figure 4). Location of the Modified Residues. Mass spectrometry of the modified protein molecules, as discussed in the previous section, provided information about the stoichiometry of various modified species. However, it cannot distinguish protein molecules differing in the position of residues which have been modified. The protein can be modified either specifically at certain easily accessible residues or randomly at different aminoacid side chains throughout the macromolecule. Proteolytic digestions followed by a chromatographic separation and analysis of the resulting peptides were used to investigate location of the protein modifications. Trypsin- and chymotrypsin-treated samples were sepa-

Reactions of Cytochrome c with 2-Hexenal

Chem. Res. Toxicol., Vol. 10, No. 6, 1997 705

Figure 1. Portion of the charge state envelope from the positive ion electrospray ionization mass spectra of cytochrome c. Raw data (A) were deconvoluted as described in the Experimental Section (B): scan a, unmodified cytochrome c; scan b, 1 min of reaction with trans-2-hexenal; scan c, modified cytochrome c, after 30 min of reaction; and scan d, modified cytochrome c after 30 min of reaction and a subsequent removal of hexenal (sample was diluted 10-fold with water, centrifuged in Microcon 10 microconcentrators, and dialyzed against distilled water).

rated chromatographically on a reversed-phase capillary column. The comparative chromatograms of the tryptic peptides from both control and hexenal-modified cytochrome c are shown in Figure 5 (A vs B). After the modification, three new peaks appeared in the chromatogram (see arrows in Figure 5B). The chromatograms of a control (Figure 6A) and hexenal-modified (Figure 6B) chymotryptic digests differed only in the intensities of some peaks: the modification seemed to suppress formation of certain peptides during digestion. A possible interpretation of these results is that the modification may be a random process: a large number of modified peptides are formed, but their quantities are usually too small to be noticed as distinct chromatographic entities. In order to obtain more information about the location of modified residues, mass spectra of the individual fractions collected from the respective chromatograms were measured through MALDI-TOF-MS. The peptide maps (i.e., the sets of mass signals detected in chromato-

graphic fractions) of the control vs hexenal-modified digests were compared. As expected from the apparent similarities of these chromatograms, most signals were found in both control and modified samples. These were easily interpreted as unmodified peptides. Occasionally, mass signals found in the control digest were not identified in the corresponding modified fraction. This was particularly evident with the chymotryptic digests, presumably due to the quantitative changes in chromatograms: some minor peptides accompanying an abundant peptide were not collected when the intensity of the major peak was lowered. However, the number of mass signals found in the modified fractionssbut absent in the control sampleswas much higher. Among these signals, possible assignments for modified peptides were sought. From the mass of an unidentified signal, mass increments corresponding to the most probable modification were subtracted, and the resulting masses were compared to the molecular mass of identified or theoretically predicted peptides; 80 and 98 mass units were subtracted to search


Z ˇ ı´dek et al.

Figure 2. CIEF of the modified cytochrome c (recorded at 410 nm). Three pI markers are indicated by their pI’s (10.4, 8.6, and 7.5). The remaining peaks correspond to cytochrome c (confirmed by visible absorbance spectra).

Figure 4. Time course of fluorescence intensity during the modification reaction. Fluorescence intensities due to the modified cytochrome c are indicated with open circles (λex ) 280 nm; λem ) 350 nm) and filled circles (λex ) 360 nm; λem ) 440 nm). Triangles represent fluorescence (λex ) 360 nm; λem ) 440 nm) of the blank trans-2-hexenal sample (at the same concentration). Table 1. Comparison of the Number of Mass Signals Obtained through MALDI-TOF-MS from the Chromatographic Fractions of Enzymatically Digested Control and Hexenal-Modified Cytochrome c Samples fractions control

modified

both

1 0 1

39 3 42

0 15 15

3 c 3

3 1 4

41 3 44

0 12 12

8 c 8

Peptides of Tryptic Digest predicted from theoretical cleavage 2 identifieda identified ambiguouslyb 0 total number 2 unpredicted signals unidentified 1 proposed as modified 0 total number 1

Figure 3. Changes in the fluorescence spectra of cytochrome c during modification with trans-2-hexenal. Spectra were recorded immediately after mixing (a, b) and after 24-h incubation (c, d) at the excitation wavelengths of 280 nm (a, c) and 360 nm (b, d).

for condensation and addition products, respectively. A probability of additional modifications within one peptide was considered as negligible. Other subtracted values, i.e., 158 and 160 mass units, correspond to the pyridinium structures found as typical products of the reaction of N-acetyl-glycyl-lysine methyl ester with trans2-hexenal (36). Deviation of (2 mass units due to the experimental error was allowed for a positive assignment. The retention volumes of the proposed modified peaks were checked. (An increase in retention volume, in comparison with the unmodified peptide, is expected because the modifying aldehyde introduces a hydrophobic moiety.) A statistical summary of the numbers of mass signals in individual peptide maps and their assignments is given in Table 1 (a complete list of the mass signals is

Peptides of Chymotryptic Digest predicted from theoretical cleavage identifieda 4 identified ambiguouslyb 1 total number 5 unpredicted signals unidentified 3 proposed as modified 2 total number 5

a Assigned as a single peptide or two indistinguishable peptides differing in C- vs N-terminal position of one amino-acid residue (e.g., KTEREDLIAYLK vs TEREDLIAYLKK). b Assignments to two different isobaric peptides possible (e.g., KKTEREDL vs GRKTGQAPGF). c No search was performed for modification of the signals observed in both control and modified samples.

available on request). The obviously incorrect predictions of modification for the control signals (the second column of Table 1) demonstrate the probability of false positive assignment. It is clearly lower than the number of proposed modified peptides in the hexenal-treated fractions, especially in the tryptic digest. The proposed assignments of modified peptides are listed in Tables 2 (tryptic digest) and 3 (chymotryptic digest). Approximately two-thirds of them can be derived from the peptides found in the control digests. The other assignments may reflect changes of a cleavage pattern


Figure 5. Reversed-phase liquid chromatograms of control (A) and hexenal-modified (B) tryptic digests of cytochrome c, obtained as described in the Experimental Section. Absorbance at 214 nm (upper trace) and laser-induced fluorescence chromatograms are shown. New peaks in the absorbance chromatograms of the modified sample are marked with arrows. Collected fractions are indicated in the middle (the double arrows identify uncollected interspace in the chromatograms).

due to the modification. The mass signals of 923, 1195, 1268, and 1525, listed in Table 2, were found in the earlier mentioned new chromatographic peaks of the hexenal-treated tryptic digest (see Figure 5B). An occasional cleavage at modified lysines may be attributed to a residual chymotryptic activity of the enzyme. In spite of the uncertainties with the assignment based solely on mass comparison, the number of proposed peptide modifications with the mass increased by approximately 160 units is in good agreement with the results of a model study (36) implicating pyridinium salts as major reaction products. A short, modified peptide present in the chymotryptic digest provided evidence that the mass increase of 160 units is due to a modification at a single lysine residue (Figure 7). A signal with the molecular mass of 566.8 Da was found in the modified chymotryptic digest only. Treatment with carboxypeptidase Y (37) revealed phenylalanine as the C-terminal residue. A comparison with the amino acid sequence of


Figure 6. Reversed-phase liquid chromatograms of control (A) and hexenal-modified (B) chymotryptic digests of cytochrome c obtained as described in the Experimental Section. Absorbance at 214 nm (upper trace) and laser-induced fluorescence chromatograms are shown. Collected fractions are indicated in the middle (the double arrows identify uncollected interspace in the chromatograms).

cytochrome c correlates with the molecular mass of peptide Lys-Ile-Phe (residues 8-10 of the cytochrome c polypeptide chain), found in the control chymotryptic fraction 17, which has been increased by 159.3 mass units. It is very unlikely that this peptide involves a reaction site other than a single lysine amino group. Figure 8 shows that the proposed modified peptides are spread along the entire cytochrome c sequence. Since the peptides usually contain more than one nucleophilic residue, the exact location of the targets of modification is not possible. Nevertheless, the number of proposed modified peptides shows nonspecific modification in at least one-half of the possible target residues. Numerous peaks were also recorded with the laser-induced fluorescence detector (see the lower traces in Figures 5 and 6). This also supports the hypothesis that there is a large number of nonspecific reaction sites within the protein molecule. Among various nucleophilic targets in the protein molecule, the 19 lysine residues probably play the major role. Three histidine residuessclaimed as the


Z ˇ ı´dek et al.

Table 2. List of Molecular Mass Signals Found in the Hexenal-Modified, But Not Control, Tryptic Digesta found

possible assignment

MF

mass

12 13

1525.2 1062.4

APGFTYTDANKNK @GDVEKGKK or HKTGPNLH

14

25

1568.8 935.4 952.5 1552.6 923.0 1195.3 1267.8

TGQAPGFTYTDANK @GDVEKGK TDANKNK TGQAPGFTYTDANK KIFVQK MIFAGIKKK TGPNLHGLFGR or TEREDLIAY

26 27

1640.2 1249.5

KTEREDLIAYLK TGPNLHGLFGR or EDLIAYLKK

31

1594.4 1786.4 2370.5

HKTGPNLHGLFGR EETLMEYLENPKK GITWKEETLMEYLENPKK or KYIPGTKMIFAGIKKKTER

16 22

corresponding peptide

residue

mass

CF

MA

43-55 1-8 26-33 40-53 1-7 49-55 40-53 8-13 80-88 28-38 90-97 88-99 28-38 92-100 26-38 61-73 56-73 73-91

1427.5 903.0 904.0 1471.6 774.9 790.8 1471.6 763.0 1036.4 1169.3 1110.2 1479.7 1169.3 1093.3 1434.4 1624.8 2210.7 2210.8

NI 3

97.7 159.4 158.4 97.2 160.5 161.7 81.5 160.0 158.9 98.5 157.6 160.5 80.2 156.2 160.0 161.6 159.8 159.7

9 NI NI 9 7 NI 21 17 22 21 22 19 26 28

a MF, number of the fraction in the chromatogram of the modified tryptic digest (Figure 5B) where the signal was found; CF, number of the fraction in the chromatogram of the control tryptic digest (Figure 5A) where the corresponding peptide was found; MA, proposed mass addition; NI, not identified in the control digest; and @, acetyl group. Mass of the molecular ions (M + H)+, expressed in mass units, is listed.

Table 3. List of Molecular Mass Signals Found in the Hexenal-Modified, But Not Control, Chymotryptic Digesta found

possible assignment

MF

mass

4 7 10

670.0 748.5 806.1

@GDVEK @GDVEK GRKTGQ or AGIKKK

18

849.2

KEETLM or KKATNE

21

1178.9

24

926.7

25

753.2 779.5 1526.8 911.3 1861.9

27 28

32

566.8

corresponding peptide

KKTEREDL or GRKTGQAPGF KTGPNLH or HKTGPNL GKKIF KEETL KKTEREDLIAY KEETLM KCAQCMTVEK + heme or LENPKKKYIPGTKMIF or MIFAGIKKKTEREDL KIF

Residue

Mass

CF

MA

1-5 1-5 37-42 83-88 60-65 99-104 87-94 37-46 27-33 26-32 6-10 60-64 87-97 60-65 13-22 68-82 80-94 8-10

589.6 589.6 646.7 644.8 751.0 690.8 1019.1 1019.1 766.9

3 3 NI NI 15 NI 11

80.4 158.9 159.4 161.3 98.2 158.4 159.8

8

159.8

592.7 619.7 1366.6 751.0 1763.9 1780.2 1780.1 407.5

NI 9 19 15 NI NI NI 17

160.5 159.8 160.2 160.3 98.0 81.7 81.8 159.3

a

MF, number of the fraction in the chromatogram of the modified chymotryptic digest (Figure 6B) where the signal was found; CF, number of the fraction in the chromatogram of the control chymotryptic digest (Figure 6A) where the corresponding peptide was found; MA, proposed mass addition; NI, not identified in the control digest; and @, acetyl group. Mass of the molecular ions (M + H)+, expressed in mass units, is listed.

only 4-hydroxy-2-nonenal reactive residues in apo-myoglobin by Bolgar and Gaskell (17)sdo not seem to explain the observed complexity of the modification products. The exclusive reactivity of histidine seen in ref 17 might reflect a different reaction system and especially the presence of phosphate, which is known to catalyze alkenal addition to primary amines (8). Oligomerization. An intermolecular cytochrome c cross-linking by hexenal represents yet another effect of alkenals on proteins. SDS-PAGE of the modified cytochrome c compared with the Bio-Rad broad molecular mass standard showed minor bands corresponding to the dimer and trimer of cytochrome c (Figure 9). The dimeric species were also observed using size-exclusion chromatography on the BioSil SEC-250 column (Figure 10). The Bio-Rad protein test mixture was used for mass vs retention time calibration. This technique was used quantitatively to illustrate the kinetics of dimerization. The initial lag, reflecting formation of an intermediate required for dimerization, is clearly observable (see the inset in Figure 10). Based on the stability of dimers in

SDS-PAGE and also on the data from dilution studies by size-exclusion chromatography (not shown), the bond forming the dimeric species is presumably covalent. Two peptide molecules cross-linked with three hexenal molecules, found among the products of a reaction of Nacetyl-glycyl-lysine methyl ester with trans-2-hexenal (36), may serve as a model for such a linkage.

Conclusions This study demonstrates the great complexity of the reactions between proteins and 2-alkenals. Two partially positively charged carbons of the aldehyde in combination with the multiple nucleophiles present in a protein molecule can react in numerous ways with different kinetics. Several interesting steps of this process were monitored in the course of our modification. ESI-MS was used to observe directly mass changes in cytochrome c in the initial phase of modification. The species corresponding to condensation of one or more aldehyde molecules with the protein (i.e., those requiring



Figure 9. SDS-PAGE of 0.8 mM cytochrome c (lane N, 1 µL/ well), 0.8 mM cytochrome c after trans-2-hexenal treatment (lane M, 1 µL/well), and Bio-Rad broad molecular mass standards (lane S). The procedure is described in the Experimental Section. Electrophoresis was performed in the T14.9C1 polyacrylamide gel at 80 V for 2.5 h. Figure 7. MALDI-TOF mass spectra of the hexenal-modified chymotryptic fraction 32 (cf. Figure 6B). Aliquots (0.5-µL) were mixed with 0.5 µL of water or carboxypeptidase Y solution on the sample plate and left to dry; 0.5 µL of R-cyano-4-hydroxycinnamic acid matrix (saturated solution in 50:50:0.3 acetonitrile:water:trifluoroacetic acid) was added to each well. The mass spectra of dry samples were acquired at the laser intensity of 6.9 µJ/pulse, as described in the Experimental Section: (a) 0.5 µL of water + 0.5 µL of carboxypeptidase Y, dilution 1; (b) 0.5 µL of fraction 32 + 0.5 µL of water; (c) 0.5 µL of fraction 32 + 0.5 µL of carboxypeptidase Y, dilution 3; and (d) 0.5 µL of fraction 32 + 0.5 µL of carboxypeptidase Y, dilution 1.

Figure 10. Time course of the dimerization of cytochrome c during modification with trans-2-hexenal. Peak heights of the cytochrome c dimer separated on the size-exclusion Bio-Sil SEC250 column (absorbance detection at 280 nm) are plotted in the graph. The initial lag of the progress curve is enlarged in the inset. Figure 8. Location of the proposed modified peptides. Unambiguous assignments are shown as dashed lines, and the peptides allowing assignments to additional unmodified “parent” peptides are shown as dotted lines. Tryptic and chymotryptic data are placed above and below the cytochrome c sequence, respectively. The standard letter symbols are used for the amino-acid residues, except for @, indicating acetyl group.

dehydration) were formed abundantly during the first minutes. A comparison with the reactions of other carbonyl compounds indicated carbon 1 of trans-2-hexenal as the reaction center. The Schiff base is the most probable structure of the early products. A more detailed mechanistic study on this aspect is in progress. Later, the amount of condensation products decreased, while a variety of other modified species resulted in complex mass spectra. The adducts of aldehyde molecules to the protein and the product(s) of molecular mass increased by approximately 160 mass units were observed as the most prevalent species in the modified sample. The increase of mass by 160 units can be interpreted as formation of a pyridinium salt, which was further elucidated in a model study (36). Location of these modifications was studied using the proteolytic enzymes.

Several new mass signals, allowing a tentative assignment to the modified peptides, were found in the modified digests. While no specific site of modification was discovered, some general conclusions can be drawn from these results. The modifications seem to be randomly scattered along the polypeptide chain according to the proposed assignments. The formation of species with lower isoelectric points supported the assumption that lysines are the major targets for modification. The circular dichroism spectra showed that if any conformational changes takes place due to the modification, they are negligible in comparison to the difference between two oxidation states of cytochrome c. Fluorescent species, easily observable at high sensitivity, were formed as the products of presumably complex reactions. The amounts formed did not allow any structural investigations. Additionally, this fluorescence was used as an independent modification marker in the proteolytic experiments. Strongly fluorescent chromatograms of the modified digests support the idea of nonselective modification at various amino-acid residues. Within a longer time scale, an “oligomerization” of the modified cytochrome c was observed. Judging from the


stability of these oligomers under denaturating conditions of SDS-PAGE, the molecules of cytochrome c are probably cross-linked covalently. Similar species were also found in the model study with a simple peptide (36). The ability of trans-2-hexenal to modify efficiently cytochrome c supports the hypothetical role of alkenals in the toxic effects of lipid peroxidation (e.g., inhibition of enzymes). The nonspecificity of this modification indicates that this system may serve as a model for modification of more relevant proteins under real physiological conditions.

Acknowledgment. This work was supported by Grant No. R01 DK44347 from the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Department of Health and Human Services. Partial support by the Grant Agency of the Czech Republic (Grant No. 203/94/1003) for the isoelectric focusing studies is also acknowledged. The authors wish to thank Dr. D. Wiesler for fruitful discussions.

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TX960118M

Modification of Horse Heart Cytochrome c with trans-2-Hexenal

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