Formation of o-Tyrosine and Dityrosine in Proteins during Radiolytic and Metal-catalyzed Oxidation *

To evaluate their usefulness as chemical indicators of cumulative oxidative damage to proteins, we studied the kinetics and extent of formation of ortho-tyrosine (0-Tyr), dityrosine (DT), and dityrosine-like fluorescence (Ex = 3 17 nm, E,,, = 407 nm) in the model proteins RNase and lysozyme exposed to radiolytic and metalcatalyzed (H20z/Cu2+) oxidation (MCO). Although there were protein-dependent differences, o-Tyr, DT, and fluorescence increased coordinately during oxidation of the proteins in both oxidation systems. The contribution of DT to total dityrosine-like fluorescence in oxidized proteins varied from 2-10070, depending on the protein, type of oxidation, and extent of oxidative damage. In proteins exposed to MCO, DT typically accounted for >50% of the fluorescence at DT wavelengths. These studies indicate that o-Tyr and DT should be useful chemical markers of cumulative exposure of proteins to MCO in vitro and in vivo.

Oxygen radicals are formed ubiquitously in biological systems by both enzymatic and metal-catalyzed oxidation (MCO)' reactions (1). These radicals may react with and modify neighboring molecules, and there is evidence that oxidative modification of proteins may be physiologically important, serving as a "marking" step for initiating protein degradation (2-4). There is increasing evidence that oxidative stress, resulting from either excessive oxygen radical production or compromised anti-oxidant defenses, may be involved in the natural aging of tissue proteins as well as the pathogenesis of atherosclerosis, ischemic-reperfusion injury, inflammatory diseases, and diabetes (5)(6)(7)(8). Oxidation of proteins by microsomal oxidases and MCO treatment is known to produce carbonyl modifications of amino acids, and proteinbound carbonyl groups are observed to increase in tissue proteins with age and in cultured cells with donor age and population doubling number (2-4). Increased levels of protein carbonyl groups have also been reported in fibroblasts cultured from progeric patients and following ischemic-reperfu-sion injury (2). However, only trace levels of carbonyl compounds are detectable in tissue proteins, undoubtedly because of the reactivity of aldehydes and ketones with nucleophiles under physiological conditions. Increases in levels of these compounds with age and pathological conditions are also limited, generally less than %fold, consistent with their reactivity and possible role as markers for the catabolism of proteins. For these reasons, carbonyl compounds may be useful as indicators of steady-state oxidative stress to protein, but may not be good candidates for assessing cumulative damage to long-lived proteins by oxidation reactions.
In contrast to the lability of carbonyl compounds, some products of oxidation of Phe and Tyr are stable, even to acid hydrolysis of proteins. These include dityrosine (DT) and ortho-tyrosine (0-Tyr) which are formed during radiolytic oxidation of proteins (9)(10)(11)(12) and, during limited radiolysis, increase gradually in protein as a function of absorbed dose (Reaction 1). While DT may also be formed in proteins by enzymatic oxidation (13), there is no information on formation of o-Tyr under these conditions, and, in general, there is little information on the formation of o-Tyr and DT in proteins exposed to MCO treatment or comparative formation of these oxidation products during radiolytic and MCO. In the present work, we set out to develop sensitive and specific assays for these amino acid oxidation products and to evaluate their usefulness as markers of cumulative oxidative modification of protein. RNase and lysozyme were chosen as model proteins for the studies since they are chemically and structurally well characterized. Additionally, they provide a valuable contrast since RNase lacks Trp while lysozyme is relatively rich in this amino acid (6 mol of Trp/mol); thus, stable products of oxidation of Trp might also be identifiable for further study. Since DT is a fluorescent compound and oxidation of protein is accompanied by an increase in DT-like, alkaline blue-green fluorescence (E, = 317 nm, E , = 407), we also addressed the contribution of DT to the total DT-like fluorescence in oxidized proteins. The results presented below indicate that o-Tyr and DT are sensitive indicators of a protein's cumulative exposure to both radiolytic oxidation and MCO and suggest that these compounds should be useful as indicators of progressive oxidative damage to proteins in vitro and in vivo. In the accompanying paper (14), we describe results of measurement of these compounds, as well as DTlike fluorescence, in human lens protein as a function of age.

EXPERIMENTAL PROCEDURES
Materials-Unless otherwise indicated, reagents were of the highest quality obtainable from either Sigma or Aldrich Chemical Co. RNase (type XII-A), lysozyme (grade I), horseradish peroxidase (type 11), bovine liver catalase (catalog C-30), and chymotrypsin (type I-S) were purchased from Sigma, and d5-L-phenylalanine (98% pure) from Cambridge Isotope Laboratories (Woburn, MA). Preparation of Standards-0-Tyr, deuterated at four positions on the phenyl ring (d4-o-Tyr), was prepared by oxidation of d,-L-Phe with H202 in the presence of Cu2+. Briefly, 25 mg of deuterated Phe was dissolved in 25 ml of deionized water, and the solution was made 100 mM in Hz02 and 1 mM in &SO4. The mixture was adjusted to pH 9.0 with 0.1 N NaOH and incubated overnight at 37 "C. The reaction was quenched by adjusting to pH 6.0 with 1 N HCl. Selected ion monitoring-gas chromatography/mass spectrometry (SIM-GC/ MS) was used to detect d4-o-Tyr (see below), and its concentration was determined by amino acid analysis using authentic o-Tyr as standard. The d,-o-Tyr was used as internal standard for GC/MS assays without further purification.
DT was synthesized as described by Ushijima et al. (15) by oxidation of L-Tyr with horseradish peroxidase and H202 and was purified by reversed-phase HPLC (RP-HPLC) using conditions described below for analysis of the DT content of oxidized proteins. The concentration of DT in standard solutions was determined by the trinitrobenzenesulfonic acid assay, as described by Spadaro et al. (16), using Tyr as standard (2 mol of Tyr/mol of DT).
Oxidation of Proteins-RNase A and lysozyme were oxidized by both radiolytic and MCO reactions. For radiolytic reactions, protein (10 mg) was dissolved in deionized water (0.5 mg/ml) and exposed to oxygen radicals produced by 18 MV x-rays generated by a linear accelerator (Varian Clinac 20, Varian Instruments). The absorbed dose rate was 5 & 0.05 kilorads/min as measured by Fricke-Hart dosimetry (17); exposure time was varied from 0 to 15 min, yielding total doses of 0-75 kilorads. The radiation doses (15-75 kilorads) were based on those used by  in studies on effects of radiolytic oxidation (0.5-100 kilorads) on susceptibility of proteins to proteolytic degradation. For MCO, protein (10 mg) was dissolved in deionized water (0.5 mg/ml) and incubated at room temperature with H202 (0-10 mM) in the presence of 0.1 mM CuS04 as catalyst. After 4 h, the MCO reactions were quenched by the addition of 1 mM diethylenetriaminepentaacetic acid and 170 units of catalase. Control MCO samples were treated identically with oxidized samples, except that H202 was omitted from the incubations.
The concentrations of H202 used in the MCO experiments (0.5-10 mM) were also in the range typically used by other investigators (4,21). Data shown in the figures and tables are typical of 3-4 independent experiments.
Protein Hydrolysis-Following oxidation reactions, control and treated samples were dried in U~C U O using a Savant Speed-Vac concentrator (Savant Instruments, Farmingdale, NY) and then resuspended at 5 mg of protein/ml of deionized water. Proteins (2 mg) were hydrolyzed in uucuo in 2 ml of 6 N HCI for 24 h at 110 "C. The hydrolysates were dried in U~C U O to remove acid, then resuspended in 200 pl of deionized water, and separate aliquots were taken for amino acid analysis and determination of a-Tyr and DT. When known amounts of authentic o-Tyr or DT were mixed with native proteins, 90-95% of added amino acid was recovered (see below), indicating that the conditions used for protein hydrolysis did not generate or cause significant losses of these oxidation products. Similarly, addition of Phe and Tyr in amounts equivalent to that in protein did not increase the amounts of o-Tyr or DT, respectively, in the hydrolysates of native proteins.
Measurement of o-Tyr in Oxidized Proteim-The o-Tyr content of hydrolyzed proteins was determined following addition of the deuterated internal standard and conversion of amino acids to their N,Oacetyl isopropyl ester derivatives. Briefly, 250 ng of d4-o-Tyr was added to the hydrolysate (-1 mg of protein), and the sample was dried in uacuo. For preparation of the isopropyl esters, the sample was dissolved in 1 ml of 1 N isopropanolic HCl and heated for 30 min at 110 "C. Solvent was evaporated under a stream of nitrogen, and the product was redissolved in 0.8 ml of pyridine. Acetic anhydride (0.2 ml) was then added, and the mixture was incubated at room temperature for 30 min to obtain the N,O-acetyl derivatives. After removal of the reagents under a stream of nitrogen, the sample was redissolved in 100 p1 of ethyl acetate. To correct for variations in protein content, the o-Tyr measured in each sample was normalized to the Val content of an aliquot of the original hydrolysate, determined by amino acid analysis; the Val content of the proteins was unchanged during the oxidation reactions. Amino acid analyses were performed on a Waters HPLC amino acid analyzer system using a cation exchange column, as described previously (22).  (Fig.  2, below).
The DT content of proteins was determined by measuring DT fluorescence at alkaline pH after mixing the column effluent (1 ml/ min flow rate) with an equal volume of 25 mM potassium phosphate, pH 11.5. The final pH was 11.2, and the eluate was monitored for fluorescence at E, = 317 nm, E, = 407 nm using a Shimadzu RF5000U spectrofluorophotometer (Shimadzu Corp., Tokyo, Japan) for RNase and a Gilford Fluoro IV Spectrofluorometer (Gilford, Oberlin, OH) for lysozyme. Peak areas were integrated using a Hewlett-Packard 3390A recording integrator, and the assay was standardized by external standardization using a calibration curve generated with synthetic DT. To correct for variations in protein content, DT concentration was normalized to the Val content of each sample measured by amino acid analysis as above. Final DT concentrations were expressed per mol of Tyr in the orginal protein using the molar ratio of Va1:Tyr in the protein.
Measurement of Protein Fluorescence-For measurement of total protein fluorescence at DT wavelengths, protein (1 mg/ml) was digested with 50 pg of chymotrypsin for 24 h at 37 "C in 50 mM phosphate buffer, pH 8.5. An aliquot of the digested protein, typically

O-Tyrosine and Dityrosine in
Oxidized Proteins 12343  (23). Proteins were detected by staining with Coomassie Brilliant Blue R-250.

Effects of Oxidation on Protein Structure and Amino Acid
Composition-The effects of radiolytic and MCO treatment on the integrity of RNase and lysozyme were evaluated by SDS-PAGE. Oxidation of both proteins was accompanied by a dose-dependent decrease in the intensity of the monomer band, and an increase in staining in the low molecular weight region of the gels, but discrete products were not detected (data now shown). Amino acid analyses of the oxidized proteins indicated that, among the amino acids measured (i.e. excluding Pro, Trp, and Cys), only His, Phe, and Tyr were partially destroyed ( Table I).
Formation of o-Tyr during Oxidation of Proteins-0-Tyr was not detectable in the native proteins, but accumulated in radiolyzed and MCO-treated RNase and lysozyme in a dosedependent manner (Fig. 1) (Table I).
Formation of D T during Oxidation of Proteins-DT was also not detectable in native RNase or lysozyme, but was readily identified by RP-HPLC in hydrolysates of the oxidized proteins (Fig. 2). Identification of the DT peak was confirmed by its co-elution with authentic D T in mixing experiments (not shown), its fluorescence spectrum and decrease in fluo-  rescence at acid pH, and its mass spectrum (the latter using methods described in the accompanying paper (14)). The chromatograms in Fig. 2, A and C, indicate that in addition to DT a number of DT-like fluorescent products, presently uncharacterized, are formed during radiolytic oxidation of the proteins. D T was, in fact, only a minor contributor to the total alkaline blue-green fluorescence generated during radiolytic oxidation, and the number of products and their relative yield from the two proteins were quite different. In contrast, on MCO treatment of the proteins (Fig. 2, B and D ) , D T accounted for the majority of fluorescence in both proteins and nearly quantitatively for the DT-like fluorescence in RNase, which lacks Trp. The yield of DT was dependent on dose for both proteins in both oxidation systems, but was 20-50-fold greater in the MCO system (Fig. 3). For MCO-treated proteins, D T accounted for up to 2% of the total Tyr content of the protein (Fig. 3) and up to 10% of the Tyr destruction (Table I). Thus, DT, like o-Tyr, appears to be a sensitive indicator of exposure of these proteins to MCO treatment.
Increase in Alkaline Blue-Green Fluorescence during Oxidation of Proteins- Fig. 4 compares the dose and concentration dependence of generation of fluorescence at DT wavelengths in RNase and lysozyme during exposure to radiolysis and MCO treatment. Lysozyme yielded higher levels of fluorescence than RNase in both oxidation systems, while MCO treatment yielded higher absolute levels of fluorescence for both proteins. The higher total fluorescence in lysozyme compared to RNase may result from the presence of Trp and its oxidation products in lysozyme. Fig. 5  cence accumulating in the proteins during oxidation. Since acid hydrolysis yielded a 4-10-fold increase in total fluorescence in the hydrolysate compared to the oxidized protein, measurements of total fluorescence were carried out on chymotrypsin-digested proteins. This treatment was performed in order to eliminate effects of protein structure on fluorescence, although the total fluorescence of the protein was not significantly affected (<lo% change) by the proteolytic treatment. The results in Fig. 5 support the general observation in Fig. 2 that DT accounted for only a fraction of the total DTlike fluoresence formed during radiolytic oxidation of the proteins, whereas it was the major fluorophore in protein oxidized by MCO treatment, accounting for essentially 100% of the fluorescence in MCO-treated RNase.
Spectrum of Alkaline Fluorescence Generated during Onidation of Proteins-The three-dimensional fluorescence plots shown in Fig. 6 illustrate that although there is some variability in the visible wavelength fluorescence maxima of oxidized proteins, in general, the maxima for oxidized proteins are similar to those of DT (E, = 317, E,,, = 407). Trp fluorescence is particularly apparent in native lyzozyme (Fig. 6 B ) , and its near absence from the spectrum of the oxidized protein (Fig. 6, D and F ) is consistent with rapid destruction of Trp during oxidation. Although the fluorescent maxima of MCO and radiolyzed RNase and lysozyme are similar, the shapes of the spectra of irradiated protein are more complex than their MCO counterparts. The similarity of the fluorescent maxima of oxidized RNase and lysozyme indicate that fluorescent products originating from the oxidation of Trp did not significantly alter the overall fluorescent maxima of lysozyme compared to RNase.

DISCUSSION
The long term goal of this research is to identify useful biomarkers of oxidative damage to proteins in vivo. To address this question we have developed assays for two chemical indicators of radiolytic and metal-catalyzed oxidation of protein, o-Tyr, and DT, which are produced by the oxidative modification of Phe and Tyr, respectively. These studies show that o-Tyr, DT, and DT-like fluorescence increase gradually in proteins during exposure to both radiolytic and MCO treatment, and that DT is a major "alkaline blue" fluorophore in proteins oxidized by MCO treatment, even in lysozyme which is rich in Trp residues. Comparison of the SDS-PAGE analyses (data not shown) and the o-Tyr and D T analyses ( Figs. 1 and 3) indicates that both o-Tyr and DT are detectable in protein prior to extensive cleavage and fragmentation of the protein. Overall, these findings indicate that both o-Tyr and DT should be useful as markers of oxidative damage to protein since neither is present in the native protein, and both accumulate in protein in a dose-dependent manner during exposure to oxidative stress.
Although the amounts of o-Tyr formed in RNase and lysozyme were comparable during radiolytic and MCO treatment, the yield of DT was 20-50 times higher in MCO-treated RNase and lysozyme than in the radiolyzed proteins. The difference in DT yield does not appear to result from greater oxidative damage to protein by MCO treatment, since SDS-PAGE (data not shown) and amino acid analysis (Table I  by MCO treatment of protein is a site-specific process, occurring in regions where transition metals are complexed to the protein. The increased D T formation during Cu2+/H2O2 treatment of RNase and lysozyme suggests that Tyr residues may be adjacent to these metal binding sites. Visible wavelength, alkaline blue fluorescence also increased steadily in RNase and lysozyme during exposure to oxidative stress, and this commonly termed DT-like fluorescence did have excitation and emission maxima similar to those of DT. Earlier reports in the literature (25)(26)(27) have implied that DT is a significant contributor to the nontryptophan fluorescence that develops during protein oxidation, and we observed that DT was the major non-tryptophan fluorophore produced during MCO of proteins, accounting for 100% and 50% of the total protein fluorescence in RNase and lysozyme (Fig. 5 B ) , respectively. In contrast, DT was a minor contributor to protein fluorescence formed during radiolytic oxidation of RNase and lysozyme, accounting for only 16% and 2% of the fluorescence in the proteins at a dose of 75 kilorads. The contribution of DT to total fluorescence was greater for RNase, which lacks Trp, than for lysozyme which contains 6 mol of Trp/mol of protein (Fig. 5).
The above results have shown that both o-Tyr and DT are markers of metal-catalyzed oxidative damage to protein. In addition, D T has been shown to account for a large fraction of the fluorescence that accumulates in protein upon exposure to Cu2'/H202. These findings, coupled with the physiological significance of MCO reactions, indicated that o-Tyr and DT should be useful as biomarkers of oxidative damage to tissue proteins and that DT may account for a sizeable fraction of the non-tryptophan fluorescence that accumulates in longlived proteins exposed to MCO in vivo. In the accompanying paper (14), we used these biomarkers to assess the role of MCO reactions in the aging of lens proteins.