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Proteinl -Dopa as an Index of Hydroxyl Radical Attack on Protein Tyrosine
ANALYTICAL BIOCHEMISTRY ARTICLE NO.
263, 232–239 (1998)
AB982766
Protein L-Dopa as an Index of Hydroxyl Radical Attack on Protein Tyrosine Gerald Cohen,1 Svetlana Yakushin, and Dorothy Dembiec-Cohen Department of Neurology and Neurobiology Research Center, Mount Sinai School of Medicine of the City University of New York, New York, New York 10029
Received February 18, 1998
It is widely believed that hydroxyl radicals generated in vivo contribute to damage to macromolecules, such as proteins and DNA. We evaluated methodology based on the transformation of protein tyrosine to L-Dopa, via aromatic ring hydroxylation, as an index of hydroxyl radical attack on proteins. The catechol structure of Dopa makes it amenable to isolation with alumina, followed by HPLC analysis, typically used for the measurement of catecholamines. Because a level of controversy exists about the formation of Dopa by hydroxyl radicals, we conducted a systematic study of the formation of Dopa from tyrosine, tyrosine dipeptides, pure proteins (chymotrypsin and myelin basic protein), and endogenous proteins in tissue homogenates (rat brain), exposed to hydroxylating conditions (Fe21/EDTA/ascorbate at neutral pH). Dopa residues in peptides and proteins were liberated by acid hydrolysis with 6 M HCl at 145°C for 1 h. A marked lability of Dopa in 6 M HCl under hydrolysis conditions was prevented with added phenol; chymotrypsin and precipitated pellets of brain protein were also protective. Overall recoveries (hydrolysis plus purification procedures) averaged 83.4 6 1.7%. This improved analytic procedure may be useful for studying protein damage by hydroxyl radicals. © 1998 Academic Press
A great deal of interest exists in developing methods that detect hydroxyl radicals (•OH) or that measure tissue damage by •OH in living systems. Hydroxyl radicals are produced during inflammatory processes or under conditions of oxidative stress. These free radicals are far too reactive to accumulate and be detected directly. Therefore, indirect methods are employed. A widely used method for assessing formation of •OH in biologic fluids employs salicylate as a competitive scav1
To whom correspondence should be addressed. Fax: (212) 3481310. 232
enger: Salicylate is hydroxylated to generate 2,3- and 2,5-dihydroxybenzoate as relatively stable end products, which are conveniently measured by HPLC with electrochemical detection (1, 2). In order to directly evaluate tissue damage, relatively stable and specific cellular oxidation products are required. A popular method for assessing protein damage by oxidants, such as •OH, is via the formation of protein carbonyl groups (3). This method measures mainly damage to lysine, arginine, proline, and threonine residues. The carbonyl moieties can be generated not only by •OH, but also by other reactive oxidizing species as well (4). We were interested in developing methodology that would be more specific as an indicator for •OH damage to proteins. To this end, we investigated the hydroxylation of L-tyrosine in proteins to form L-Dopa (3,4dihydroxyphenylalanine).2 Although hydroxylation of the aromatic ring in tyrosine forms two products, namely, 2,4-Dopa and 3,4-Dopa, the catechol (orthodihydroxybenzene) grouping in 3,4-Dopa provides a special advantage: It permits the use of alumina (Al2O3) to bind catechols and, hence, isolate the Dopa from potentially interfering contaminants. Therefore, the problem of obtaining an index of •OH attack on proteins reduces to the measurement of Dopa by standard alumina purification and HPLC technology used to study brain catecholamines. Since Dopa is not normally a constituent of mammalian proteins, the formation of Dopa in proteins can be taken as an index of oxidative damage by •OH. Formation of Dopa and other protein products (dimers, organic peroxides, etc.) have previously been observed in pulse radiolysis experiments or under conditions of a Fenton reaction, both of which generate •OH (5, 6). On the other hand, other investigators reported loss of protein tyrosine 2 Abbreviations used: Dopa, 3,4-dihydroxyphenylalanine; TCA, trichloracetic acid; PCA, perchloric acid; DTPA, diethylenetriaminepentaacetate.
0003-2697/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
L-DOPA
IN PROTEINS
without formation of Dopa or failure to hydroxylate phenylalanine or tyrosine in solution (7, 8). In these studies, we confirmed that a Fenton •OHgenerating system is capable of generating Dopa in tyrosine-containing peptides and proteins. Dopa is isolated by hydrolysis of peptide bonds with concentrated HCl vapor, followed by adsorption onto and elution from alumina. These experiments lay the groundwork for further studies of damage to proteins in inflammatory or degenerative disorders (e.g., multiple sclerosis, Parkinson’s disease). Because a prominent site of damage in multiple sclerosis (an inflammatory demyelinating disease) is myelin basic protein, for which protein fragments are excreted in the urine (9, 10), we specifically studied myelin basic protein as a model protein. The method, however, is flexible, requiring only the presence of L-tyrosine in proteins and, therefore, it may prove valuable in a general way for studying a variety of tissues under conditions of oxidative stress. METHODS
Hydroxylation of Substrates Two systems were used to hydroxylate tyrosine (11). One was a Fenton-type reaction between an iron-chelate (Fe21-DTPA) and H2O2, which generates hydroxyl radicals, and the other was the iron/EDTA/ascorbate system, which generates H2O2 and consists of a Fenton-type reaction between ferrous EDTA and H2O2, with recycling of ferric ions to the ferrous state by ascorbate. In general, the yield of Dopa was greater with the iron/EDTA/ascorbate system, which was progressive with time; similar observations had been made previously during the study of hydroxylation of dopamine (3,4-dihydroxyphenylethylamine) to 6-hydroxydopamine (12). Therefore, the iron/EDTA/ascorbate system was preferred. The iron/EDTA/ascorbate system (13) consisted of 50 mM sodium phosphate buffer (pH 7.2), containing 240 mM EDTA and 1 mM ascorbic acid. Tyrosine or the dipeptides were generally present at 100 –500 mM. The reaction was initiated by the addition of Fe21 to a final concentration of 200 mM; the final reaction volume was 1.0 ml. For the preparation of reaction mixtures, it is important that Fe21 be added last (see Discussion). The samples were incubated in wells of a 24-well tissue culture plate (Falcon, 18 mm diameter; Becton–Dickinson, Lincoln Park, NJ) at room temperature; magnetic stirring was used to improve oxygenation (except for initial experiments with tyrosine). Control samples lacked iron, EDTA, and ascorbate; if only the iron was omitted, a low level of hydroxylation occurred, which was attributed to trace amounts of iron present in the phosphate buffer and the EDTA. In experiments with tyrosine, for direct HPLC assay (without isolation of Dopa with alumina), 10- or 50-ml aliquots were quenched by dilution either 100- or 10-
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fold, respectively, into 0.4 M PCA in plastic microcentrifuge tubes; the tubes were capped, mixed, and stored on ice for analyses by HPLC. The contents of the tubes were transferred to glass micro-insert vials used for the automated HPLC sample injector. For evaluation of the hydrolysis and alumina isolation procedures, 10 ml of sample was added to hydrolysis tubes, as described below. For studies with chymotrypsin, protein was added to a concentration of 2.86 mg/ml, and following exposure to iron/EDTA/ascorbate, 0.35 ml (1 mg protein) was taken for analysis. Trichloroacetic acid (TCA, 117 ml, 40% w/v) was added (10% final), and the samples were mixed and centrifuged at 8000g for 15 min. The supernatant was removed and the pellet was taken for assay. Myelin basic protein was hydroxylated at a concentration of 1.43 mg/ml and then 0.35 ml (0.5 mg) with added 0.5 mg chymotrypsin (as a carrier protein) was processed as above. For brain homogenates, rats were decapitated under CO2 anesthesia and 30 mg of cerebral cortex was homogenized in 0.9 ml of buffer, which contained ascorbate and EDTA; iron was added to 200 mM to initiate the reaction. After 60 min, 0.35 ml was processed with TCA. The protein pellet was subsequently washed by resuspension (vortex), followed by recentrifugation three times with fresh TCA in order to remove soluble contaminants, including any endogenous Dopa. Hydrolysis of Dipeptides and Proteins Hydrolysis of dipeptides was conducted in the presence of 1 mg chymotrypsin as a carrier and protective protein (see Results). To 100 ml chymotrypsin (10 mg/ ml) in an amber 0.7-ml glass minivial was added 100 ml of 20% (w/v) TCA; samples were lightly vortexed and centrifuged in carrier 2-ml plastic microcentrifuge tubes at 8000g for 15 min in a microcentrifuge operated in a cold room. The TCA was subsequently aspirated and discarded. To the protein pellet was added 10 ml of sample and 10 ml of 12 M HCl, which contained 10% (v/v) phenol. For studies with chymotrypsin, 10 ml of 6 M HCl containing 5% (v/v) phenol was added to the pelleted protein. For studies with myelin basic protein, chymotrypsin (0.5 mg) was used as a carrier protein for precipitation with TCA. The vials were flushed with argon, sealed with a crimped cap with a Teflon liner, and heated in a sand bath at 145°C; the sand bath was covered with aluminum foil to maintain the hydrolysis temperature. After 60 min, the vials were removed and allowed to cool at room temperature for 5 min. Isolation of Dopa with Alumina Just prior to incubation with alumina, 500 ml of 0.1 M Tris buffer (pH 8.5) was added to the sample, which was mixed by repeated aspiration and release with a
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Pasteur pipet and transferred to a 1.5-ml microcentrifuge tube; a second 500-ml aliquot of Tris was used to rinse the original tube. The samples were still acidic at this point. Ascorbic acid (25 ml) and DTPA (100 ml) were added to final concentrations of 5 and 2.4 mM, respectively; DTPA (metal chelator) and ascorbate (antioxidant) protected the sensitive catechol group in Dopa from oxidation during subsequent binding to alumina at pH 8.5. Alumina (Al2O3) was prepared by washing with HCl solution (14). This procedure removes fine particles that are not easily sedimented; although small in relative weight, the fine particles contribute a disproportionately large surface area for binding and they can induce major losses in Dopa when the supernatant fluid is discarded. The alumina (about 100 g) was washed in a 2-L round-bottom flask, first by swirling with 1 L of distilled water, allowing the heavier particles to settle, and generously discarding the relatively turbid supernatant; this was done twice. The process was then repeated 3– 4 times with 1 M HCl until the remaining particles settled quickly and the supernatant was essentially clear. The alumina particles were subsequently rinsed twice with 1 M HCl in methanol, followed by six washes with distilled water. The final water rinse had a pH between 4 and 5 as judged with pH paper. After two final rinses with pure methanol, the alumina was transferred with methanol to a sintered glass funnel and air dried with a protective cover. To the tube containing the hydrolyzed experimental sample (in Tris containing DTPA and ascorbic acid) was added 50 mg of acid-washed alumina and either 80 or 160 ml of 2 M Tris base, depending on whether 10 ml 6 M HCl (protein) or 20 ml (soluble peptides) was used, which brought the pH to 8.5. As soon as Tris base was added to a tube, it was capped and inverted five times to initiate binding of Dopa to alumina. The binding process was completed by repeated inversion of all tubes for 5 min. Subsequently, the tubes were brought to speed in a microfuge for 10 –15 s, and the supernatant was aspirated and discarded. The alumina was then washed twice with 1.0-ml portions of distilled water by repeated inversion for 30 s, followed by centrifugation and removal of the supernatant. Then, 1.0 ml of 0.4 M perchloric acid (PCA) was added to elute the Dopa. Tubes were repeatedly inverted for 2.5 min and centrifuged (10 –15 s), and the supernatant was taken for HPLC analysis. High-Performance Liquid Chromatography Dopa and Dopa dipeptides were measured by HPLC with electrochemical detection. The detector was a glassy carbon electrode set at 0.65 V vs a Ag/AgCl reference electrode. A C-18 reverse-phase column
(Beckman Ultrasphere IP, 5-mm beads, 4.6 mm 3 15 cm) was used. The mobile phase consisted of 50 mM phosphate buffer containing 1 mM EDTA, adjusted to pH 2.9 (15), and operated at a flow rate of 0.25–1.0 ml/min. Injections of sample (20 ml) were made with a refrigerated autosampler (Waters, Milford, MA). External standards of L-Dopa (0.1 mg/ml) were used for calibration. Reagents The sources of chemicals were as follows: hydrolysis grade 6 M HCl (GFS Chemicals, Powell, OH); 12 M HCl (HCl reagent, ACS grade), disodium EDTA, ferrous ammonium sulfate •6H2O, and L-ascorbic acid (Fisher, Fair Lawn, NJ); chymotrypsin A4 (achymotrypsin, bovine pancreas; Boehringer Mannheim, Indianapolis, IN); myelin basic protein (bovine brain), glycyl-tyrosine, tyrosyl-glycine, and tyrosyltyrosine (Sigma, St. Louis, MO). Solutions of Fe21, prepared in distilled water, were stable to oxidation. Gly-tyr was prepared in water; tyr-gly and tyr-tyr were dissolved in 1 M HCl, neutralized with 1 N NaOH, and diluted into water. RESULTS
Methodological Aspects The analytic procedure consisted of vapor-phase hydrolysis of dipeptides or proteins with concentrated HCl (to release Dopa) followed by HPLC analyses. To protect the sensitive catechol group of Dopa from oxidation by oxygen, samples were flushed with argon prior to heating (140 –145°C) with HCl. Investigation of procedural details showed that L-Dopa was unstable in concentrated HCl (Fig. 1, curve B) compared to 0.4 M PCA as a comparison medium (curve A). However, addition of phenol (5% w/v, 0.6 M) to the HCl sample protected the L-Dopa (curve C). The cause of L-Dopa instability is unknown, but it may be due to the presence of chlorine atoms or chlorine molecules in the concentrated HCl. Phenol, added in relatively high concentration, acts as a competitive substrate (or scavenger) for the L-Dopa-destroying species. Figure 1 shows that a relatively high concentration of phenol (5% w/v) provided complete protection (curve C), while a much lower concentration (0.01% w/v) provided partial protection (curve D). In separate experiments, conducted under hydrolysis conditions (145°C), L-Dopa disappeared completely after 10 min in the absence of phenol. Lastly, in experiments concerning hydroxylation of protein tyrosine residues in chymotrypsin, we noted that the presence of chymotrypsin provided protection for added L-Dopa in the absence of added phenol. Bovine serum albumin, on the other hand, could not substitute for chymotrypsin over 60 min of hydrolysis.
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IN PROTEINS
FIG. 1. Instability of L-Dopa (6 mg/ml) in 6 M HCl at room temperature. Samples were prepared in either 6 M HCl or 0.4 M PCA (control). For analyses, the samples were diluted to 0.1 mg L-Dopa/ml in cold 0.4 M PCA. Curve A, 0.4 M PCA; curve B, 6 M HCl; curve C, 6 M HCl with 5% (w/v) phenol; curve D, 6 M HCl with 0.01% (w/v) phenol.
Based on this experience, chymotrypsin was routinely added to samples to additionally protect Dopa during hydrolysis. Chymotrypsin also served to help pellet myelin basic protein. In addition, TCA rather than PCA was used to pellet the protein. PCA induced some loss of L-Dopa during hydrolysis; this effect may be related to the mild oxidizing properties of perchlorate, which would be amplified at elevated temperature. Hydroxylation of Tyrosine Figure 2 shows experiments carried out with tyrosine. Exposure to iron/EDTA/ascorbate resulted in hydroxylation of tyrosine to Dopa. The accumulation of Dopa was dependent upon both the concentration of tyrosine and the time of incubation. The conversion of tyrosine to Dopa was approximately 10% at 60 min for each concentration of tyrosine. A likely explanation for the lack of linearity with time is that the initial rate reflects the rapid oxidation of ferrous ions by dissolved molecular oxygen to generate H2O2, which supports a Fenton reaction, followed by a slower rate dependent on the diffusion of oxygen into the system. In support of this assumption, separate experiments conducted with magnetic stirring for better aeration produced higher levels of Dopa. For example, with 0.5 mM tyrosine, levels of Dopa without and with stirring, respectively, were 31 and 56 mM at 30 min and 41 and 60 mM at 60 min. In order to test for losses in Dopa during the hydrolysis and alumina isolation procedures used to study
FIG. 2. Formation of Dopa from tyrosine (0.10, 0.25, and 0.50 mM) induced by the iron/EDTA/ascorbate system. The reaction was conducted in 50 mM phosphate buffer at pH 7.2, in the presence of 200 mM Fe21, 240 mM EDTA, and 1.0 mM ascorbic acid. Samples were diluted 10-fold into cold 0.4 M PCA for HPLC analyses. Results are the mean and SE for 4 –12 samples. Where error bars are not visible, they are encompassed by the data point.
dipeptides and proteins, aliquots from tyrosine hydroxylation experiments were analyzed either directly or after the hydrolysis and purification procedures. The results shown in Table 1 tested the procedure over a range of 8.7 to 44.8 mM Dopa (the amounts formed during hydroxylation of pure tyrosine). Recoveries of Dopa were quite good, and ranged from 78.6 to 90.1% (mean 5 83.4% 6 1.7% SE). The small average loss of Dopa (16.7%) consists of losses both during hydrolysis and during isolation of product with alumina.
TABLE 1
Recovery of Dopa after Standard Hydrolysis and Alumina Isolation Procedures Sample (mM tyrosine)
Time (min)
% Recovery a
0.10
30 60 30 60 30 60
90.1 6 2.5 80.7 6 4.1 86.7 6 1.3 78.6 6 0.8 84.2 6 0.9 79.9 6 0.9
0.25 0.50
a Tyrosine was hydroxylated to form Dopa by exposure to iron/ EDTA/ascorbate. Aliquots were quenched with 0.4 M PCA (controls) or they were hydrolyzed with 6 M HCl and purified with alumina (recovery samples). Results are the means of four independent samples (6 SE).
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FIG. 3. Formation of glycyl-Dopa from glycyl-tyrosine (0.10, 0.25, and 0.50 mM) by iron/EDTA/ascorbate. Experiments were conducted as described in the legend to Fig. 2, except that glycyl-tyrosine was used in place of tyrosine. Samples were hydrolyzed in 6 M HCl at 145°C to release Dopa for HPLC analyses.
Hydroxylation of Tyrosine-Containing Dipeptides Figure 3 shows similar experiments carried out with glycyl-tyrosine (gly-tyr). Again the accumulation of Dopa was dependent upon both the concentration of substrate and the time of incubation. As with tyrosine, an initial faster rate was replaced by a slower rate over time. Direct comparison of the data in Figs. 2 and 3 is complicated by the use of stirring to improve oxygenation in Fig. 3. Separate experiments, with and without stirring, showed that without stirring, the yield of Dopa from dipeptides was decreased by 30% at 60 min (similar to the effect seen with tyrosine). When correction is made for the effect of stirring, the amount of Dopa formed with dipeptides was approximately half that seen with tyrosine.
In additional experiments, the concentration of glytyr was raised to 1 and 5 mM, which resulted in higher concentrations of Dopa. With 1 mM gly-tyr, the yields of Dopa were 31.3 6 0.8 mM and 40.2 6 1.6 mM at 30 and 60 min, respectively; the corresponding yields with 5 mM substrate were 50.0 6 1.6 mM and 64 6 1.6 mM, respectively. Table 2 shows data for experiments with two additional tyrosine dipeptides as substrates: tyr-gly and tyr-tyr. The accumulation of Dopa increased with time and concentration of substrate. Similar amounts of Dopa were formed with all three substrates, although the yield with gly-tyr was somewhat greater (P , 0.01). Representative HPLC chromatograms for experiments with gly-tyr and tyr-gly are shown in Fig. 4. After hydroxylation, each of the dipeptides gave rise to an HPLC peak (A and E), which grew with time (B and F). The new peaks, presumably, gly-Dopa (peak 1) and Dopa-gly (peak 2), exhibited somewhat different retention times (legend to Fig. 4). No Dopa was present in the unhydrolyzed samples (A, B, E, and F). Small peaks in the area of DOPA in B, E, and F have a different retention time from DOPA. However, Dopa (peak 3) was present after hydrolysis (C, D, G, and H). Peaks 1, 2, and 3 were absent from control samples (not shown). The broad peak after the initial baseline offset (A) is ascorbic acid. The peak diminishes in size between 30 and 60 min (A vs B and E vs F), reflecting loss of ascorbate. The larger ascorbic acid peaks seen after purification with alumina (C, D, G, and H) reflect the ascorbic acid added during the purification procedure with alumina. Hydroxylation of Proteins Experiments were carried out with two proteins, chymotrypsin and myelin basic protein. Results are shown in Table 3. a-Chymotrypsin has 4 tyrosine residues (16), representing 1.7% of the total of 241 amino acid residues. Human and bovine myelin basic protein have 4 tyrosines out of a total of 170 amino acids (2.4%
TABLE 2
Hydroxylation of Tyrosine in Three Dipeptides by Iron/EDTA/Ascorbate (with Stirring) Dopa formed (mM)a Data at 30 min
Data at 60 min
Dipeptide
0.10 mM
0.25 mM
0.50 mM
0.10 mM
0.25 mM
0.50 mM
Glycyl-tyrosine Tyrosyl-glycine Tyrosyl-tyrosine
6.5 6 0.2 4.3 6 0.3 5.1 6 0.3
12.7 6 0.5 11.0 6 0.2 11.9 6 0.6
20.6 6 0.7 18.2 6 0.4 17.2 6 0.6
8.2 6 0.3 6.9 6 0.2 7.6 6 0.2
18.2 6 0.2 16.2 6 0.4 15.7 6 0.5
28.7 6 0.2 25.9 6 0.8 24.4 6 2.2
a Results are the means 6 SE (n 5 5– 6 per group) in three independent experiments. Dopa levels with glycyl-tyrosine were significantly higher than with the other two dipeptides (P , 0.01, ANOVA followed by Tukey–Kramer multiple comparison test), except for 0.25 mM tyrosyl-tyrosine at 30 min, and both tyrosyl-tyrosine and tyrosyl-glycine at 0.50 mM and 60 min.
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237
FIG. 4. Chromatograms from experiments with 0.5 mM glycyl-tyrosine (A–D) and 0.5 mM tyrosyl-glycine (E–H) exposed to iron/EDTA/ ascorbate. After hydroxylation, new peaks appeared (A and E, at 30 min, no hydrolysis), which grew with time (B and F, 60 min). The new peaks have somewhat different retention times, depending upon the dipeptide studied (peak 1, 28 min, gly-Dopa; and peak 2, 32.5 min, Dopa-gly). The corresponding chromatograms after hydrolysis and purification with alumina are shown in C and D (30 and 60 min) and G and H. Dopa (peak 3) had a retention time of 15 min.
tyrosine) (17). The formation of Dopa in chymotrypsin (MW 24,500) indicated conversion of 0.34% of the available tyrosines in 1 h or 1.4% of the protein molecules (assuming an average of one conversion per chymotrypsin molecule). For myelin basic protein (MW 18,500), the yield was about 0.077% based on tyrosine (or 0.3% of protein molecules) in 1 h. A similar calculation cannot be performed for rat brain because the mean protein tyrosine content is not known. Chromatograms of chymotrypsin hydrolysates are shown in Fig. 5. After hydroxylation and hydrolysis,
chymotrypsin showed a series of peaks on HPLC, including Dopa (A). After purification with alumina (with added ascorbic acid), prominent Dopa and ascorbic acid peaks are seen (B). Chymotrypsin that had not been exposed to iron/EDTA/ascorbate showed a tiny peak in the area of Dopa (C).
TABLE 3
Formation of Dopa Residues in Chymotrypsin, Myelin Basic Protein, and Rat Brain Homogenate (Cortex) pmol Dopa/mg protein Protein
30 min
60 min
Chymotrypsin Myelin basic protein Rat brain
368 6 20 (n 5 6) 124 6 14 (n 5 7)
548 6 35 (n 5 4) 167 6 16 (n 5 7) 137 6 10 (n 5 4)
Note. Following the exposure to iron/EDTA/ascorbate (with stirring), the protein was precipitated with TCA and hydrolyzed, followed by purification with alumina. Results are the means 6 SE.
FIG. 5. HPLC of chymotrypsin hydrolysates. (A) Unpurified (no alumina) hydrolysate after exposure to iron/EDTA/ascorbate for 30 min. Retention times: peak 1, ascorbic acid, 8.5–9.4 min; peak 2, Dopa, 14.3 min. (B) Same sample after purification with alumina; Dopa, 14.4 min. (C) Control sample not exposed to iron/EDTA/ascorbate; Dopa-like peak, 14.4 min.
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DISCUSSION
Ring hydroxylation of aromatic compounds can be used to monitor •OH production in model systems in vitro (1, 18). For in vivo application, methods based on the hydroxylation of salicylate or the conversion of free phenylalanine to o-tyrosine have been utilized to monitor the production of •OH, including application in a variety of clinically relevant circumstances (19 –21). Similarly, the presence of Dopa in proteins can serve as an index of oxidative damage to proteins by •OH. Our experiments were conducted mainly with an iron/EDTA/ascorbate system. It is known that the spontaneous oxidation of Fe21-EDTA chelate by molecular oxygen in phosphate buffer at neutral pH is very rapid; the reaction is complete in less than 1 min (22). The speed of this initial reaction makes it imperative to add the ferrous salt last (i.e., in the presence of substrate). H2O2 produced in the initial burst (Eq. [1]) reacts with remaining Fe21-EDTA in a Fenton-type reaction to produce •OH (Eq. [2]). This accounts for the initial burst in formation of Dopa from free tyrosine or tyrosine-containing dipeptides (Figs. 2 and 3). Subsequently, the reaction is sustained at a slower pace by the reduction of Fe31-EDTA to Fe21-EDTA by ascorbate (AH2, Eq. [3]) and by diffusion of oxygen into the solution. The limiting nature of oxygen diffusion is evident from the higher yield of Dopa when stirring is employed. 2Fe21-EDTA 1 O2 1 2H1 3 2Fe31-EDTA 1 H2O2 [1] Fe21-EDTA 1 H2O2 3 Fe31-EDTA 1 •OH 1 OH2 [2] 2Fe31-EDTA 1 AH2 3 2Fe21-EDTA 1 A 1 2H1
[3]
The initial product of •OH attack on tyrosine is an hydroxycyclohexadienyl radical (•Tyr-OH, Eq. [4]). Formation of stable product (Dopa) requires oxidation (loss of an electron), which is achieved by either Fe31EDTA or molecular oxygen (Eqs. [5] and [6]). Although two hydroxylated products are formed, namely, 2,4Dopa and 3,4-Dopa, in this study we focused on the formation of 3,4-Dopa, which was isolated by binding to alumina. The overall yield of product in the iron/EDTA/ ascorbate system is somewhat suppressed because ascorbate (1 mM) acts as a competitive scavenger for • OH. Other investigators have observed hydroxylation of phenylalanine or tyrosine by pulse or g-radiolysis and in Fenton-type oxidations (23, 24). Although Fenton-type oxidations can also produce ferryl radicals (FeO)21 as reactive intermediates, Maskos et al. (23) suggest that the similar distribution of isomeric hydroxylated products formed from phenylalanine (viz, o-, m-, and p-tyrosine) indicate that the same hydroxy-
cyclohexadienyl radicals (or hydroxyl radical adducts) are formed by both g-radiolysis and Fenton reactions. Tyr 1 •OH 3 •Tyr-OH
[4]
Tyr-OH 1 Fe31-EDTA 3 Dopa 1 Fe21-EDTA 1 H1 [5]
•
•
1 Tyr-OH 1 O2 3 Dopa 1 •O2 2 1 H
[6]
Recovery of Dopa through the hydrolysis and alumina procedures was 78.6–90.1% (mean 83.4%). High recoveries in the described procedure require acid washing of the alumina to remove turbidity and fine particles (see under Methods). Isolation of Dopa with alumina not only eliminates potentially interfering peaks (Fig. 5), but also serves to remove contaminants which may affect column chromatography, particularly for the analysis of tissue extracts. In addition, replacement of air by argon, and the presence of phenol during the vapor state hydrolysis procedure were beneficial; phenol and argon (or nitrogen) have been used in a routine way by others investigators (6, 25) to protect sensitive products during hydrolysis. However, it should be emphasized that phenol is absolutely required to prevent the very rapid loss of L-Dopa in HCl under hydrolysis conditions. In addition, we found that chymotrypsin exerted a protective effect on Dopa formed from tyrosine or dipeptides. Therefore, hydrolysis of soluble reaction products of dipeptides was routinely performed in the presence of pelleted chymotrypsin. Chymotrypsin was also added just prior to protein precipitation in experiments with myelin basic protein; chymotrypsin was not needed for a crude brain homogenate, which possessed endogenous protective proteins. In our studies, external standards were used in order to evaluate absolute recovery in the procedure. Therefore, dilutional effects of fluid remaining in the tube (elution from alumina), systematic losses on handling, and chromatographic effects, such as peak spread, are all included in the overall recovery. For routine use, we recommend use of an internal standard to correct for losses; a convenient standard might be alpha-methyl-Dopa. Conversion of tyrosine to Dopa was also observed with three dipeptides: gly-tyr, tyr-gly, and tyr-tyr. No Dopa was seen before hydrolysis (Figs. 4A, 4B, 4E, and 4F); however, it was seen after hydrolysis (Figs. 4C, 4D, 4G, and 4H). Therefore, Dopa does not arise from scission of the peptide bonds by iron/EDTA/ascorbate, followed by hydroxylation of free tyrosine. The new peaks appearing in Fig. 4 are obviously the corresponding dipeptides gly-Dopa and Dopa-gly, which give rise to free Dopa via hydrolysis. The yields of Dopa with gly-tyr and tyr-gly were similar, although the yield with gly-tyr was moderately higher than tyr-gly at several concentrations and time points (Table 2). A surprising aspect was that the yield for tyr-tyr fell into
L-DOPA
IN PROTEINS
a similar range. An increased yield was anticipated because the available tyrosines are effectively doubled in tyr-tyr, and the overall yield increases with concentration of substrate (Fig. 3). A possible explanation is that the first order rate constant for •OH reacting with tyrosine is k 5 0.6 –2.2 3 1010M21 s21, while that for glycine is only k 5 0.6 –1.0 3 107M21 s21 (26, 27), providing a large competitive advantage to tyrosine. This might mean that attack by •OH on tyr-tyr would not give very different results from that for tyr-gly or gly-tyr. A second possibility is that attack by •OH on glycine residues produces an unstable radical that rearranges electronically to yield hydroxylated tyrosine as a stable end product. Exposure of chymotrypsin, myelin basic protein, or a crude brain homogenate to iron/EDTA/ascorbate produced protein Dopa residues (Table 3). Absolute yields of Dopa with proteins should be affected by several factors: Protein folding and three-dimensional structure will make amino acids either more or less accessible to attack by iron/EDTA/ascorbate, depending upon their positioning. In addition, aromatic amino acids, such as phenylalanine, tryptophan, and histidine, and the thiol group in cysteine, are good competitive substrates for •OH attack (k 5 0.5–1.4 3 1010M21 s21) (27). Lastly, •OH attack on proteins produces a variety of products (e.g., dimers, fragmentation, peroxides, isomeric products). Given these limitations, the overall yield of Dopa in proteins is remarkably high and indicates that protein tyrosine is a good target for oxidative attack (hydroxylation) by •OH. Protein L-Dopa occurs naturally as a posttranslational change in nonmammalian organisms, such as molluscs, annelids, and coelenterates, in insoluble sclerotized structures (25). Dopa residues may be essential for the unusual properties of the water-resistant adhesive produced by mussels (28). Mammalian tyrosinase is also capable of transforming protein tyrosine to L-Dopa (25, 29). Therefore, the presence of protein Dopa as an index of •OH-induced damage should be approached with caution in the study of skin or melanoma. The enzyme tyrosine hydroxylase, present in catecholamine neurons and the adrenal gland, also needs to be investigated for its potential to generate protein L-Dopa. The current study provides an evaluation of methodology for hydrolysis, isolation, and measurement of protein Dopa as an index of oxidative damage. The methodology appears suitable to detect and monitor protein damage by •OH in tissues. ACKNOWLEDGMENTS This work was supported by Grant NS-23017 from the U.S. Public Health Service and by grants from the Multiple Sclerosis Foundation and the National Multiple Sclerosis Society.
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REFERENCES 1. Halliwell, B., Kaur, H., and Ingelman-Sundberg, M. (1991) Free Radical Biol. Med. 10, 439 – 441. 2. Chiueh, C. C., Wu, R. M., Mohanakumar, K. P., Sternberger, L. M., Krishna, G., Obata, T., and Murphy, D. L. (1994) Ann. NY Acad. Sci. 738, 24 –37. 3. Levine, R. L., Williams, J. A., Stadtman, E. R., and Shacter, E. (1994) Methods Enzymol. 233, 346 –357. 4. Berlett, B. S., and Stadtman, E. R. (1997) J. Biol. Chem. 272, 20313–20316. 5. Garrison, W. M. (1987) Chem. Rev. 87, 381–398. 6. Gieseq, S. P., Simpson, J. A., Charlton, T. S., Duncan, M. W., and Dean, R. T. (1993) Biochemistry 32, 4780 – 4786. 7. Guptasarma, P., and Balasubramanian, D. (1992) Biochemistry 31, 4296 – 4303. 8. Stadtman, E. R., and Berlett, B. S. (1991) J. Biol. Chem. 26, 17201–17211. 9. Norton, W. T. (1987) Ann. Neurol. 22, 656 – 657. 10. Whitaker, J. N. (1993) Ann. Neurol. 31, 345–348. 11. Cohen, G. (1985) in CRC Handbook of Methods for Oxygen Radical Research, (Greenwald, R. A., Ed.), pp. 55– 64, CRC Press, Boca Raton, FL. 12. Slivka, A., and Cohen, G. (1985) J. Biol. Chem. 260, 15466 – 15472. 13. Udenfriend, S., Clark, C. T., Axelrod, J., and Brodie, B. B. (1954) J. Biol. Chem. 208, 731–739. 14. Cohen, G., and Goldenberg, M. (1957) J. Neurochem. 2, 71– 80. 15. Morrison, M. E., and Cohen, G. (1983) Biochemistry 22, 5465– 5467. 16. Matthews, B. W., Sigler, P. B., Henderson, R., and Blow, D. M. (1967) Nature 214, 652– 656. 17. Williams, K. A., and Dever, C. M. (1993) Crit. Rev. Clin. Lab. Sci. 30, 29 – 64. 18. Radzik, D. M., Roston, D. A., and Kissinger, P. T. (1983) Anal. Biochem. 131, 458 – 464. 19. Sun, J. Z., Kaur, H., Halliwell, B., Li, X. Y., and Bolli, R. (1993) Circ. Res. 73, 534 –549. 20. Lubec, G., Widness, J. A., Hayde, M., Menzel, D., and Pollak, A. (1997) Pediatrics 100, 700 –704. 21. O’Neill, C., Fu, L. W., Halliwell, B., and Longhurst, J. C. (1996) Am. J. Physiol. 271, H660 –H667. 22. Aisen, P., Cohen, G., and Kang, J. O. (1990) Int. Rev. Exp. Pathol. 31, 1– 46. 23. Maskos, Z., Rush, J. D., and Koppenol, W. H. (1992) Arch. Biochem. Biophys. 296, 521–529. 24. Karam, L. R., Dizdaroglu, M., and Simic, M. G. (1984) Int. J. Radiat. Biol. 46, 715–724. 25. Waite, J. H., and Benedict, C. V. (1984) Methods Enzymol. 107, 397– 413. 26. Anbar, N., and Neta, P. (1967) Int. J. Appl. Radiat. Isotopes 18, 493–523. 27. Dorfman, L. M., and Adams, G. E. (1973) NSRDS, Natl. Bur. Stand. 46, 1–59. 28. Waite, J. H. (1983) J. Biol. Chem. 258, 2911–2915. 29. Ito, S., Kato, T., Shinpo, K., and Fujita, K. (1984) Biochem. J. 222, 407– 411.
263, 232–239 (1998)
AB982766
Protein L-Dopa as an Index of Hydroxyl Radical Attack on Protein Tyrosine Gerald Cohen,1 Svetlana Yakushin, and Dorothy Dembiec-Cohen Department of Neurology and Neurobiology Research Center, Mount Sinai School of Medicine of the City University of New York, New York, New York 10029
Received February 18, 1998
It is widely believed that hydroxyl radicals generated in vivo contribute to damage to macromolecules, such as proteins and DNA. We evaluated methodology based on the transformation of protein tyrosine to L-Dopa, via aromatic ring hydroxylation, as an index of hydroxyl radical attack on proteins. The catechol structure of Dopa makes it amenable to isolation with alumina, followed by HPLC analysis, typically used for the measurement of catecholamines. Because a level of controversy exists about the formation of Dopa by hydroxyl radicals, we conducted a systematic study of the formation of Dopa from tyrosine, tyrosine dipeptides, pure proteins (chymotrypsin and myelin basic protein), and endogenous proteins in tissue homogenates (rat brain), exposed to hydroxylating conditions (Fe21/EDTA/ascorbate at neutral pH). Dopa residues in peptides and proteins were liberated by acid hydrolysis with 6 M HCl at 145°C for 1 h. A marked lability of Dopa in 6 M HCl under hydrolysis conditions was prevented with added phenol; chymotrypsin and precipitated pellets of brain protein were also protective. Overall recoveries (hydrolysis plus purification procedures) averaged 83.4 6 1.7%. This improved analytic procedure may be useful for studying protein damage by hydroxyl radicals. © 1998 Academic Press
A great deal of interest exists in developing methods that detect hydroxyl radicals (•OH) or that measure tissue damage by •OH in living systems. Hydroxyl radicals are produced during inflammatory processes or under conditions of oxidative stress. These free radicals are far too reactive to accumulate and be detected directly. Therefore, indirect methods are employed. A widely used method for assessing formation of •OH in biologic fluids employs salicylate as a competitive scav1
To whom correspondence should be addressed. Fax: (212) 3481310. 232
enger: Salicylate is hydroxylated to generate 2,3- and 2,5-dihydroxybenzoate as relatively stable end products, which are conveniently measured by HPLC with electrochemical detection (1, 2). In order to directly evaluate tissue damage, relatively stable and specific cellular oxidation products are required. A popular method for assessing protein damage by oxidants, such as •OH, is via the formation of protein carbonyl groups (3). This method measures mainly damage to lysine, arginine, proline, and threonine residues. The carbonyl moieties can be generated not only by •OH, but also by other reactive oxidizing species as well (4). We were interested in developing methodology that would be more specific as an indicator for •OH damage to proteins. To this end, we investigated the hydroxylation of L-tyrosine in proteins to form L-Dopa (3,4dihydroxyphenylalanine).2 Although hydroxylation of the aromatic ring in tyrosine forms two products, namely, 2,4-Dopa and 3,4-Dopa, the catechol (orthodihydroxybenzene) grouping in 3,4-Dopa provides a special advantage: It permits the use of alumina (Al2O3) to bind catechols and, hence, isolate the Dopa from potentially interfering contaminants. Therefore, the problem of obtaining an index of •OH attack on proteins reduces to the measurement of Dopa by standard alumina purification and HPLC technology used to study brain catecholamines. Since Dopa is not normally a constituent of mammalian proteins, the formation of Dopa in proteins can be taken as an index of oxidative damage by •OH. Formation of Dopa and other protein products (dimers, organic peroxides, etc.) have previously been observed in pulse radiolysis experiments or under conditions of a Fenton reaction, both of which generate •OH (5, 6). On the other hand, other investigators reported loss of protein tyrosine 2 Abbreviations used: Dopa, 3,4-dihydroxyphenylalanine; TCA, trichloracetic acid; PCA, perchloric acid; DTPA, diethylenetriaminepentaacetate.
0003-2697/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
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IN PROTEINS
without formation of Dopa or failure to hydroxylate phenylalanine or tyrosine in solution (7, 8). In these studies, we confirmed that a Fenton •OHgenerating system is capable of generating Dopa in tyrosine-containing peptides and proteins. Dopa is isolated by hydrolysis of peptide bonds with concentrated HCl vapor, followed by adsorption onto and elution from alumina. These experiments lay the groundwork for further studies of damage to proteins in inflammatory or degenerative disorders (e.g., multiple sclerosis, Parkinson’s disease). Because a prominent site of damage in multiple sclerosis (an inflammatory demyelinating disease) is myelin basic protein, for which protein fragments are excreted in the urine (9, 10), we specifically studied myelin basic protein as a model protein. The method, however, is flexible, requiring only the presence of L-tyrosine in proteins and, therefore, it may prove valuable in a general way for studying a variety of tissues under conditions of oxidative stress. METHODS
Hydroxylation of Substrates Two systems were used to hydroxylate tyrosine (11). One was a Fenton-type reaction between an iron-chelate (Fe21-DTPA) and H2O2, which generates hydroxyl radicals, and the other was the iron/EDTA/ascorbate system, which generates H2O2 and consists of a Fenton-type reaction between ferrous EDTA and H2O2, with recycling of ferric ions to the ferrous state by ascorbate. In general, the yield of Dopa was greater with the iron/EDTA/ascorbate system, which was progressive with time; similar observations had been made previously during the study of hydroxylation of dopamine (3,4-dihydroxyphenylethylamine) to 6-hydroxydopamine (12). Therefore, the iron/EDTA/ascorbate system was preferred. The iron/EDTA/ascorbate system (13) consisted of 50 mM sodium phosphate buffer (pH 7.2), containing 240 mM EDTA and 1 mM ascorbic acid. Tyrosine or the dipeptides were generally present at 100 –500 mM. The reaction was initiated by the addition of Fe21 to a final concentration of 200 mM; the final reaction volume was 1.0 ml. For the preparation of reaction mixtures, it is important that Fe21 be added last (see Discussion). The samples were incubated in wells of a 24-well tissue culture plate (Falcon, 18 mm diameter; Becton–Dickinson, Lincoln Park, NJ) at room temperature; magnetic stirring was used to improve oxygenation (except for initial experiments with tyrosine). Control samples lacked iron, EDTA, and ascorbate; if only the iron was omitted, a low level of hydroxylation occurred, which was attributed to trace amounts of iron present in the phosphate buffer and the EDTA. In experiments with tyrosine, for direct HPLC assay (without isolation of Dopa with alumina), 10- or 50-ml aliquots were quenched by dilution either 100- or 10-
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fold, respectively, into 0.4 M PCA in plastic microcentrifuge tubes; the tubes were capped, mixed, and stored on ice for analyses by HPLC. The contents of the tubes were transferred to glass micro-insert vials used for the automated HPLC sample injector. For evaluation of the hydrolysis and alumina isolation procedures, 10 ml of sample was added to hydrolysis tubes, as described below. For studies with chymotrypsin, protein was added to a concentration of 2.86 mg/ml, and following exposure to iron/EDTA/ascorbate, 0.35 ml (1 mg protein) was taken for analysis. Trichloroacetic acid (TCA, 117 ml, 40% w/v) was added (10% final), and the samples were mixed and centrifuged at 8000g for 15 min. The supernatant was removed and the pellet was taken for assay. Myelin basic protein was hydroxylated at a concentration of 1.43 mg/ml and then 0.35 ml (0.5 mg) with added 0.5 mg chymotrypsin (as a carrier protein) was processed as above. For brain homogenates, rats were decapitated under CO2 anesthesia and 30 mg of cerebral cortex was homogenized in 0.9 ml of buffer, which contained ascorbate and EDTA; iron was added to 200 mM to initiate the reaction. After 60 min, 0.35 ml was processed with TCA. The protein pellet was subsequently washed by resuspension (vortex), followed by recentrifugation three times with fresh TCA in order to remove soluble contaminants, including any endogenous Dopa. Hydrolysis of Dipeptides and Proteins Hydrolysis of dipeptides was conducted in the presence of 1 mg chymotrypsin as a carrier and protective protein (see Results). To 100 ml chymotrypsin (10 mg/ ml) in an amber 0.7-ml glass minivial was added 100 ml of 20% (w/v) TCA; samples were lightly vortexed and centrifuged in carrier 2-ml plastic microcentrifuge tubes at 8000g for 15 min in a microcentrifuge operated in a cold room. The TCA was subsequently aspirated and discarded. To the protein pellet was added 10 ml of sample and 10 ml of 12 M HCl, which contained 10% (v/v) phenol. For studies with chymotrypsin, 10 ml of 6 M HCl containing 5% (v/v) phenol was added to the pelleted protein. For studies with myelin basic protein, chymotrypsin (0.5 mg) was used as a carrier protein for precipitation with TCA. The vials were flushed with argon, sealed with a crimped cap with a Teflon liner, and heated in a sand bath at 145°C; the sand bath was covered with aluminum foil to maintain the hydrolysis temperature. After 60 min, the vials were removed and allowed to cool at room temperature for 5 min. Isolation of Dopa with Alumina Just prior to incubation with alumina, 500 ml of 0.1 M Tris buffer (pH 8.5) was added to the sample, which was mixed by repeated aspiration and release with a
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Pasteur pipet and transferred to a 1.5-ml microcentrifuge tube; a second 500-ml aliquot of Tris was used to rinse the original tube. The samples were still acidic at this point. Ascorbic acid (25 ml) and DTPA (100 ml) were added to final concentrations of 5 and 2.4 mM, respectively; DTPA (metal chelator) and ascorbate (antioxidant) protected the sensitive catechol group in Dopa from oxidation during subsequent binding to alumina at pH 8.5. Alumina (Al2O3) was prepared by washing with HCl solution (14). This procedure removes fine particles that are not easily sedimented; although small in relative weight, the fine particles contribute a disproportionately large surface area for binding and they can induce major losses in Dopa when the supernatant fluid is discarded. The alumina (about 100 g) was washed in a 2-L round-bottom flask, first by swirling with 1 L of distilled water, allowing the heavier particles to settle, and generously discarding the relatively turbid supernatant; this was done twice. The process was then repeated 3– 4 times with 1 M HCl until the remaining particles settled quickly and the supernatant was essentially clear. The alumina particles were subsequently rinsed twice with 1 M HCl in methanol, followed by six washes with distilled water. The final water rinse had a pH between 4 and 5 as judged with pH paper. After two final rinses with pure methanol, the alumina was transferred with methanol to a sintered glass funnel and air dried with a protective cover. To the tube containing the hydrolyzed experimental sample (in Tris containing DTPA and ascorbic acid) was added 50 mg of acid-washed alumina and either 80 or 160 ml of 2 M Tris base, depending on whether 10 ml 6 M HCl (protein) or 20 ml (soluble peptides) was used, which brought the pH to 8.5. As soon as Tris base was added to a tube, it was capped and inverted five times to initiate binding of Dopa to alumina. The binding process was completed by repeated inversion of all tubes for 5 min. Subsequently, the tubes were brought to speed in a microfuge for 10 –15 s, and the supernatant was aspirated and discarded. The alumina was then washed twice with 1.0-ml portions of distilled water by repeated inversion for 30 s, followed by centrifugation and removal of the supernatant. Then, 1.0 ml of 0.4 M perchloric acid (PCA) was added to elute the Dopa. Tubes were repeatedly inverted for 2.5 min and centrifuged (10 –15 s), and the supernatant was taken for HPLC analysis. High-Performance Liquid Chromatography Dopa and Dopa dipeptides were measured by HPLC with electrochemical detection. The detector was a glassy carbon electrode set at 0.65 V vs a Ag/AgCl reference electrode. A C-18 reverse-phase column
(Beckman Ultrasphere IP, 5-mm beads, 4.6 mm 3 15 cm) was used. The mobile phase consisted of 50 mM phosphate buffer containing 1 mM EDTA, adjusted to pH 2.9 (15), and operated at a flow rate of 0.25–1.0 ml/min. Injections of sample (20 ml) were made with a refrigerated autosampler (Waters, Milford, MA). External standards of L-Dopa (0.1 mg/ml) were used for calibration. Reagents The sources of chemicals were as follows: hydrolysis grade 6 M HCl (GFS Chemicals, Powell, OH); 12 M HCl (HCl reagent, ACS grade), disodium EDTA, ferrous ammonium sulfate •6H2O, and L-ascorbic acid (Fisher, Fair Lawn, NJ); chymotrypsin A4 (achymotrypsin, bovine pancreas; Boehringer Mannheim, Indianapolis, IN); myelin basic protein (bovine brain), glycyl-tyrosine, tyrosyl-glycine, and tyrosyltyrosine (Sigma, St. Louis, MO). Solutions of Fe21, prepared in distilled water, were stable to oxidation. Gly-tyr was prepared in water; tyr-gly and tyr-tyr were dissolved in 1 M HCl, neutralized with 1 N NaOH, and diluted into water. RESULTS
Methodological Aspects The analytic procedure consisted of vapor-phase hydrolysis of dipeptides or proteins with concentrated HCl (to release Dopa) followed by HPLC analyses. To protect the sensitive catechol group of Dopa from oxidation by oxygen, samples were flushed with argon prior to heating (140 –145°C) with HCl. Investigation of procedural details showed that L-Dopa was unstable in concentrated HCl (Fig. 1, curve B) compared to 0.4 M PCA as a comparison medium (curve A). However, addition of phenol (5% w/v, 0.6 M) to the HCl sample protected the L-Dopa (curve C). The cause of L-Dopa instability is unknown, but it may be due to the presence of chlorine atoms or chlorine molecules in the concentrated HCl. Phenol, added in relatively high concentration, acts as a competitive substrate (or scavenger) for the L-Dopa-destroying species. Figure 1 shows that a relatively high concentration of phenol (5% w/v) provided complete protection (curve C), while a much lower concentration (0.01% w/v) provided partial protection (curve D). In separate experiments, conducted under hydrolysis conditions (145°C), L-Dopa disappeared completely after 10 min in the absence of phenol. Lastly, in experiments concerning hydroxylation of protein tyrosine residues in chymotrypsin, we noted that the presence of chymotrypsin provided protection for added L-Dopa in the absence of added phenol. Bovine serum albumin, on the other hand, could not substitute for chymotrypsin over 60 min of hydrolysis.
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FIG. 1. Instability of L-Dopa (6 mg/ml) in 6 M HCl at room temperature. Samples were prepared in either 6 M HCl or 0.4 M PCA (control). For analyses, the samples were diluted to 0.1 mg L-Dopa/ml in cold 0.4 M PCA. Curve A, 0.4 M PCA; curve B, 6 M HCl; curve C, 6 M HCl with 5% (w/v) phenol; curve D, 6 M HCl with 0.01% (w/v) phenol.
Based on this experience, chymotrypsin was routinely added to samples to additionally protect Dopa during hydrolysis. Chymotrypsin also served to help pellet myelin basic protein. In addition, TCA rather than PCA was used to pellet the protein. PCA induced some loss of L-Dopa during hydrolysis; this effect may be related to the mild oxidizing properties of perchlorate, which would be amplified at elevated temperature. Hydroxylation of Tyrosine Figure 2 shows experiments carried out with tyrosine. Exposure to iron/EDTA/ascorbate resulted in hydroxylation of tyrosine to Dopa. The accumulation of Dopa was dependent upon both the concentration of tyrosine and the time of incubation. The conversion of tyrosine to Dopa was approximately 10% at 60 min for each concentration of tyrosine. A likely explanation for the lack of linearity with time is that the initial rate reflects the rapid oxidation of ferrous ions by dissolved molecular oxygen to generate H2O2, which supports a Fenton reaction, followed by a slower rate dependent on the diffusion of oxygen into the system. In support of this assumption, separate experiments conducted with magnetic stirring for better aeration produced higher levels of Dopa. For example, with 0.5 mM tyrosine, levels of Dopa without and with stirring, respectively, were 31 and 56 mM at 30 min and 41 and 60 mM at 60 min. In order to test for losses in Dopa during the hydrolysis and alumina isolation procedures used to study
FIG. 2. Formation of Dopa from tyrosine (0.10, 0.25, and 0.50 mM) induced by the iron/EDTA/ascorbate system. The reaction was conducted in 50 mM phosphate buffer at pH 7.2, in the presence of 200 mM Fe21, 240 mM EDTA, and 1.0 mM ascorbic acid. Samples were diluted 10-fold into cold 0.4 M PCA for HPLC analyses. Results are the mean and SE for 4 –12 samples. Where error bars are not visible, they are encompassed by the data point.
dipeptides and proteins, aliquots from tyrosine hydroxylation experiments were analyzed either directly or after the hydrolysis and purification procedures. The results shown in Table 1 tested the procedure over a range of 8.7 to 44.8 mM Dopa (the amounts formed during hydroxylation of pure tyrosine). Recoveries of Dopa were quite good, and ranged from 78.6 to 90.1% (mean 5 83.4% 6 1.7% SE). The small average loss of Dopa (16.7%) consists of losses both during hydrolysis and during isolation of product with alumina.
TABLE 1
Recovery of Dopa after Standard Hydrolysis and Alumina Isolation Procedures Sample (mM tyrosine)
Time (min)
% Recovery a
0.10
30 60 30 60 30 60
90.1 6 2.5 80.7 6 4.1 86.7 6 1.3 78.6 6 0.8 84.2 6 0.9 79.9 6 0.9
0.25 0.50
a Tyrosine was hydroxylated to form Dopa by exposure to iron/ EDTA/ascorbate. Aliquots were quenched with 0.4 M PCA (controls) or they were hydrolyzed with 6 M HCl and purified with alumina (recovery samples). Results are the means of four independent samples (6 SE).
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FIG. 3. Formation of glycyl-Dopa from glycyl-tyrosine (0.10, 0.25, and 0.50 mM) by iron/EDTA/ascorbate. Experiments were conducted as described in the legend to Fig. 2, except that glycyl-tyrosine was used in place of tyrosine. Samples were hydrolyzed in 6 M HCl at 145°C to release Dopa for HPLC analyses.
Hydroxylation of Tyrosine-Containing Dipeptides Figure 3 shows similar experiments carried out with glycyl-tyrosine (gly-tyr). Again the accumulation of Dopa was dependent upon both the concentration of substrate and the time of incubation. As with tyrosine, an initial faster rate was replaced by a slower rate over time. Direct comparison of the data in Figs. 2 and 3 is complicated by the use of stirring to improve oxygenation in Fig. 3. Separate experiments, with and without stirring, showed that without stirring, the yield of Dopa from dipeptides was decreased by 30% at 60 min (similar to the effect seen with tyrosine). When correction is made for the effect of stirring, the amount of Dopa formed with dipeptides was approximately half that seen with tyrosine.
In additional experiments, the concentration of glytyr was raised to 1 and 5 mM, which resulted in higher concentrations of Dopa. With 1 mM gly-tyr, the yields of Dopa were 31.3 6 0.8 mM and 40.2 6 1.6 mM at 30 and 60 min, respectively; the corresponding yields with 5 mM substrate were 50.0 6 1.6 mM and 64 6 1.6 mM, respectively. Table 2 shows data for experiments with two additional tyrosine dipeptides as substrates: tyr-gly and tyr-tyr. The accumulation of Dopa increased with time and concentration of substrate. Similar amounts of Dopa were formed with all three substrates, although the yield with gly-tyr was somewhat greater (P , 0.01). Representative HPLC chromatograms for experiments with gly-tyr and tyr-gly are shown in Fig. 4. After hydroxylation, each of the dipeptides gave rise to an HPLC peak (A and E), which grew with time (B and F). The new peaks, presumably, gly-Dopa (peak 1) and Dopa-gly (peak 2), exhibited somewhat different retention times (legend to Fig. 4). No Dopa was present in the unhydrolyzed samples (A, B, E, and F). Small peaks in the area of DOPA in B, E, and F have a different retention time from DOPA. However, Dopa (peak 3) was present after hydrolysis (C, D, G, and H). Peaks 1, 2, and 3 were absent from control samples (not shown). The broad peak after the initial baseline offset (A) is ascorbic acid. The peak diminishes in size between 30 and 60 min (A vs B and E vs F), reflecting loss of ascorbate. The larger ascorbic acid peaks seen after purification with alumina (C, D, G, and H) reflect the ascorbic acid added during the purification procedure with alumina. Hydroxylation of Proteins Experiments were carried out with two proteins, chymotrypsin and myelin basic protein. Results are shown in Table 3. a-Chymotrypsin has 4 tyrosine residues (16), representing 1.7% of the total of 241 amino acid residues. Human and bovine myelin basic protein have 4 tyrosines out of a total of 170 amino acids (2.4%
TABLE 2
Hydroxylation of Tyrosine in Three Dipeptides by Iron/EDTA/Ascorbate (with Stirring) Dopa formed (mM)a Data at 30 min
Data at 60 min
Dipeptide
0.10 mM
0.25 mM
0.50 mM
0.10 mM
0.25 mM
0.50 mM
Glycyl-tyrosine Tyrosyl-glycine Tyrosyl-tyrosine
6.5 6 0.2 4.3 6 0.3 5.1 6 0.3
12.7 6 0.5 11.0 6 0.2 11.9 6 0.6
20.6 6 0.7 18.2 6 0.4 17.2 6 0.6
8.2 6 0.3 6.9 6 0.2 7.6 6 0.2
18.2 6 0.2 16.2 6 0.4 15.7 6 0.5
28.7 6 0.2 25.9 6 0.8 24.4 6 2.2
a Results are the means 6 SE (n 5 5– 6 per group) in three independent experiments. Dopa levels with glycyl-tyrosine were significantly higher than with the other two dipeptides (P , 0.01, ANOVA followed by Tukey–Kramer multiple comparison test), except for 0.25 mM tyrosyl-tyrosine at 30 min, and both tyrosyl-tyrosine and tyrosyl-glycine at 0.50 mM and 60 min.
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FIG. 4. Chromatograms from experiments with 0.5 mM glycyl-tyrosine (A–D) and 0.5 mM tyrosyl-glycine (E–H) exposed to iron/EDTA/ ascorbate. After hydroxylation, new peaks appeared (A and E, at 30 min, no hydrolysis), which grew with time (B and F, 60 min). The new peaks have somewhat different retention times, depending upon the dipeptide studied (peak 1, 28 min, gly-Dopa; and peak 2, 32.5 min, Dopa-gly). The corresponding chromatograms after hydrolysis and purification with alumina are shown in C and D (30 and 60 min) and G and H. Dopa (peak 3) had a retention time of 15 min.
tyrosine) (17). The formation of Dopa in chymotrypsin (MW 24,500) indicated conversion of 0.34% of the available tyrosines in 1 h or 1.4% of the protein molecules (assuming an average of one conversion per chymotrypsin molecule). For myelin basic protein (MW 18,500), the yield was about 0.077% based on tyrosine (or 0.3% of protein molecules) in 1 h. A similar calculation cannot be performed for rat brain because the mean protein tyrosine content is not known. Chromatograms of chymotrypsin hydrolysates are shown in Fig. 5. After hydroxylation and hydrolysis,
chymotrypsin showed a series of peaks on HPLC, including Dopa (A). After purification with alumina (with added ascorbic acid), prominent Dopa and ascorbic acid peaks are seen (B). Chymotrypsin that had not been exposed to iron/EDTA/ascorbate showed a tiny peak in the area of Dopa (C).
TABLE 3
Formation of Dopa Residues in Chymotrypsin, Myelin Basic Protein, and Rat Brain Homogenate (Cortex) pmol Dopa/mg protein Protein
30 min
60 min
Chymotrypsin Myelin basic protein Rat brain
368 6 20 (n 5 6) 124 6 14 (n 5 7)
548 6 35 (n 5 4) 167 6 16 (n 5 7) 137 6 10 (n 5 4)
Note. Following the exposure to iron/EDTA/ascorbate (with stirring), the protein was precipitated with TCA and hydrolyzed, followed by purification with alumina. Results are the means 6 SE.
FIG. 5. HPLC of chymotrypsin hydrolysates. (A) Unpurified (no alumina) hydrolysate after exposure to iron/EDTA/ascorbate for 30 min. Retention times: peak 1, ascorbic acid, 8.5–9.4 min; peak 2, Dopa, 14.3 min. (B) Same sample after purification with alumina; Dopa, 14.4 min. (C) Control sample not exposed to iron/EDTA/ascorbate; Dopa-like peak, 14.4 min.
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COHEN, YAKUSHIN, AND DEMBIEC-COHEN
DISCUSSION
Ring hydroxylation of aromatic compounds can be used to monitor •OH production in model systems in vitro (1, 18). For in vivo application, methods based on the hydroxylation of salicylate or the conversion of free phenylalanine to o-tyrosine have been utilized to monitor the production of •OH, including application in a variety of clinically relevant circumstances (19 –21). Similarly, the presence of Dopa in proteins can serve as an index of oxidative damage to proteins by •OH. Our experiments were conducted mainly with an iron/EDTA/ascorbate system. It is known that the spontaneous oxidation of Fe21-EDTA chelate by molecular oxygen in phosphate buffer at neutral pH is very rapid; the reaction is complete in less than 1 min (22). The speed of this initial reaction makes it imperative to add the ferrous salt last (i.e., in the presence of substrate). H2O2 produced in the initial burst (Eq. [1]) reacts with remaining Fe21-EDTA in a Fenton-type reaction to produce •OH (Eq. [2]). This accounts for the initial burst in formation of Dopa from free tyrosine or tyrosine-containing dipeptides (Figs. 2 and 3). Subsequently, the reaction is sustained at a slower pace by the reduction of Fe31-EDTA to Fe21-EDTA by ascorbate (AH2, Eq. [3]) and by diffusion of oxygen into the solution. The limiting nature of oxygen diffusion is evident from the higher yield of Dopa when stirring is employed. 2Fe21-EDTA 1 O2 1 2H1 3 2Fe31-EDTA 1 H2O2 [1] Fe21-EDTA 1 H2O2 3 Fe31-EDTA 1 •OH 1 OH2 [2] 2Fe31-EDTA 1 AH2 3 2Fe21-EDTA 1 A 1 2H1
[3]
The initial product of •OH attack on tyrosine is an hydroxycyclohexadienyl radical (•Tyr-OH, Eq. [4]). Formation of stable product (Dopa) requires oxidation (loss of an electron), which is achieved by either Fe31EDTA or molecular oxygen (Eqs. [5] and [6]). Although two hydroxylated products are formed, namely, 2,4Dopa and 3,4-Dopa, in this study we focused on the formation of 3,4-Dopa, which was isolated by binding to alumina. The overall yield of product in the iron/EDTA/ ascorbate system is somewhat suppressed because ascorbate (1 mM) acts as a competitive scavenger for • OH. Other investigators have observed hydroxylation of phenylalanine or tyrosine by pulse or g-radiolysis and in Fenton-type oxidations (23, 24). Although Fenton-type oxidations can also produce ferryl radicals (FeO)21 as reactive intermediates, Maskos et al. (23) suggest that the similar distribution of isomeric hydroxylated products formed from phenylalanine (viz, o-, m-, and p-tyrosine) indicate that the same hydroxy-
cyclohexadienyl radicals (or hydroxyl radical adducts) are formed by both g-radiolysis and Fenton reactions. Tyr 1 •OH 3 •Tyr-OH
[4]
Tyr-OH 1 Fe31-EDTA 3 Dopa 1 Fe21-EDTA 1 H1 [5]
•
•
1 Tyr-OH 1 O2 3 Dopa 1 •O2 2 1 H
[6]
Recovery of Dopa through the hydrolysis and alumina procedures was 78.6–90.1% (mean 83.4%). High recoveries in the described procedure require acid washing of the alumina to remove turbidity and fine particles (see under Methods). Isolation of Dopa with alumina not only eliminates potentially interfering peaks (Fig. 5), but also serves to remove contaminants which may affect column chromatography, particularly for the analysis of tissue extracts. In addition, replacement of air by argon, and the presence of phenol during the vapor state hydrolysis procedure were beneficial; phenol and argon (or nitrogen) have been used in a routine way by others investigators (6, 25) to protect sensitive products during hydrolysis. However, it should be emphasized that phenol is absolutely required to prevent the very rapid loss of L-Dopa in HCl under hydrolysis conditions. In addition, we found that chymotrypsin exerted a protective effect on Dopa formed from tyrosine or dipeptides. Therefore, hydrolysis of soluble reaction products of dipeptides was routinely performed in the presence of pelleted chymotrypsin. Chymotrypsin was also added just prior to protein precipitation in experiments with myelin basic protein; chymotrypsin was not needed for a crude brain homogenate, which possessed endogenous protective proteins. In our studies, external standards were used in order to evaluate absolute recovery in the procedure. Therefore, dilutional effects of fluid remaining in the tube (elution from alumina), systematic losses on handling, and chromatographic effects, such as peak spread, are all included in the overall recovery. For routine use, we recommend use of an internal standard to correct for losses; a convenient standard might be alpha-methyl-Dopa. Conversion of tyrosine to Dopa was also observed with three dipeptides: gly-tyr, tyr-gly, and tyr-tyr. No Dopa was seen before hydrolysis (Figs. 4A, 4B, 4E, and 4F); however, it was seen after hydrolysis (Figs. 4C, 4D, 4G, and 4H). Therefore, Dopa does not arise from scission of the peptide bonds by iron/EDTA/ascorbate, followed by hydroxylation of free tyrosine. The new peaks appearing in Fig. 4 are obviously the corresponding dipeptides gly-Dopa and Dopa-gly, which give rise to free Dopa via hydrolysis. The yields of Dopa with gly-tyr and tyr-gly were similar, although the yield with gly-tyr was moderately higher than tyr-gly at several concentrations and time points (Table 2). A surprising aspect was that the yield for tyr-tyr fell into
L-DOPA
IN PROTEINS
a similar range. An increased yield was anticipated because the available tyrosines are effectively doubled in tyr-tyr, and the overall yield increases with concentration of substrate (Fig. 3). A possible explanation is that the first order rate constant for •OH reacting with tyrosine is k 5 0.6 –2.2 3 1010M21 s21, while that for glycine is only k 5 0.6 –1.0 3 107M21 s21 (26, 27), providing a large competitive advantage to tyrosine. This might mean that attack by •OH on tyr-tyr would not give very different results from that for tyr-gly or gly-tyr. A second possibility is that attack by •OH on glycine residues produces an unstable radical that rearranges electronically to yield hydroxylated tyrosine as a stable end product. Exposure of chymotrypsin, myelin basic protein, or a crude brain homogenate to iron/EDTA/ascorbate produced protein Dopa residues (Table 3). Absolute yields of Dopa with proteins should be affected by several factors: Protein folding and three-dimensional structure will make amino acids either more or less accessible to attack by iron/EDTA/ascorbate, depending upon their positioning. In addition, aromatic amino acids, such as phenylalanine, tryptophan, and histidine, and the thiol group in cysteine, are good competitive substrates for •OH attack (k 5 0.5–1.4 3 1010M21 s21) (27). Lastly, •OH attack on proteins produces a variety of products (e.g., dimers, fragmentation, peroxides, isomeric products). Given these limitations, the overall yield of Dopa in proteins is remarkably high and indicates that protein tyrosine is a good target for oxidative attack (hydroxylation) by •OH. Protein L-Dopa occurs naturally as a posttranslational change in nonmammalian organisms, such as molluscs, annelids, and coelenterates, in insoluble sclerotized structures (25). Dopa residues may be essential for the unusual properties of the water-resistant adhesive produced by mussels (28). Mammalian tyrosinase is also capable of transforming protein tyrosine to L-Dopa (25, 29). Therefore, the presence of protein Dopa as an index of •OH-induced damage should be approached with caution in the study of skin or melanoma. The enzyme tyrosine hydroxylase, present in catecholamine neurons and the adrenal gland, also needs to be investigated for its potential to generate protein L-Dopa. The current study provides an evaluation of methodology for hydrolysis, isolation, and measurement of protein Dopa as an index of oxidative damage. The methodology appears suitable to detect and monitor protein damage by •OH in tissues. ACKNOWLEDGMENTS This work was supported by Grant NS-23017 from the U.S. Public Health Service and by grants from the Multiple Sclerosis Foundation and the National Multiple Sclerosis Society.
239
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