the of biological vol. 262, no. 20, issue of july 15, pp ... · solutions of bsa (0.33 mg of...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1987 by The American Society of Biological Chemists, Inc.

Vol. 262, No. 20, Issue of July 15, pp. 9902-9907 1987 Printed in (j.S.A.

Protein Damage and Degradation by Oxygen Radicals 11. MODIFICATION OF AMINO ACIDS*

(Received for publication, December 30, 1986)

Kelvin J. A. Davies, Marta E. DelsignoreS, and Sharon W. Lin From the Institute for Toxicology and the Department of Biochemistry, The University of Southern California, Los Angeles, California 90033 ~

Exposure of proteins to the hydroxyl radical (‘OH) or to the combination of ‘OH plus the superoxide anion radical (‘OH + 0;) causes gross structural modification. Such modified proteins can undergo spontaneous fragmentation or can exhibit substantial increases in proteolytic susceptibility. In the present study, with the representative protein bovine serum albumin (BSA), we report that alterations to primary structure underlie such gross structural modifications. All amino acids in BSA were susceptible to modification by both ‘OH and ‘OH + 0; (+02), although tryptophan, tyrosine, histidine, and cysteine were particularly sensitive. At a radical/BSA molar ratio (nmol of radicals/ nmol of BSA) of 10, we observed an average 9-10% destruction of amino acids; whereas at a ratio of 100, the average loss was 45%. Decreasing tryptophan fluorescence provided a useful index of amino acid loss and exhibited a clear dose dependence with ‘OH or with ‘OH + 0; (+02). Linear production of the biphenol bityrosine was observed with ‘OH treatment. In contrast, ‘OH + 0; (+02) induced only a limited bityrosine production rate which reached an early plateau. Stud- ies with various chemical scavengers (t-butyl alcohol, isopropyl alcohol, mannitol, urate) and gasses (N20, N2, 02, air) revealed that ‘OH is the primary radical responsible for all amino acid modifications, but that 0; and O2 can further transform the products of ‘OH reactions. Thus, 0,/02 can potentiate *OH-dependent destruction of many amino acids (e.g. tryptophan) while inhibiting production of bityrosine by reacting with tyrosyl (phenoxyl) radicals. No amino acid loss or bityrosine production occurred with exposure to 0; (+02) alone. Amino acid modifications caused both by ‘OH alone and by *OH + 0; (+02) progressively affected the overall electrical charge of BSA. In a pH range of 3.7-6.2, some 16 new isoelectric focusing bands were induced by ‘OH, and some eight new bands were induced by ‘OH + 0; (+02). The alterations to primary structure observed provide the key to an understanding of the link between oxidative modification and increased proteolytic susceptibility.

In the preceding paper (I), it was reported that oxygen radicals can oxidatively modify proteins and enhance their

* This work was supported by Grant ES 03598 from the National Institutes of Health (to K. J. A. D.). Part of this work has been published in preliminary form (Davies, K. J. A., and Delsignore, M. E. (1984) Fed. Proc. 43,1858 (Abstr. 2579)). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in zccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Present address: Colgate-Palmolive Co., 909 River Rd., Piscata- way, NJ 08854.

degradation by intracellular proteolytic systems. A growing body of literature now suggests that oxidative modification and proteolysis may be closely linked (1-19). The mechanisms by which oxidatively modified proteins are recognized and degraded, however, are poorly understood. To learn how intracellular proteolytic systems recognize and degrade oxidatively modified proteins, it is first necessary to carefully characterize the modifications.

Previous studies of free radical damage to proteins have indicated that a wide variety of reactions can occur (20-28). In the preceding paper (l), some of these reactions were found to coincide with increased proteolytic susceptibility, although no causal relationships have been established thus far. To improve our understanding of potential “signals” for proteolysis, we have now performed dose-response studies of the modification of protein primary structure by oxygen radicals. Alterations to primary structure undoubtedly underlie the increased proteolysis which we and others have observed (1- 19). Modifications to secondary and tertiary structure are considered in the subsequent paper (2), and the links between modification and proteolytic susceptibility are explored in the fourth paper of this series (3).

We decided to concentrate our efforts on bovine serum albumin (BSA)’ for a variety of reasons. In the preceding paper ( l ) , BSA was shown to be modified by oxygen radical exposure and to exhibit either fragmentation’ or increased proteolytic susceptibility. BSA appeared to be a good model for the 17 proteins previously studied (1) and has the added advantage that it is free of prosthetic groups and other com- plicating factors. The primary, secondary, and tertiary structure of BSA has been quite well characterized in the literature (29, 30), and several reports of BSA modification by oxygen radicals are available for comparison (31-35). Finally, BSA represents a major class of animal proteins and is readily available in a purified form. As in the previous report ( l ) , we have exposed BSA to the hydroxyl radical (‘OH), to superoxide (O;), and to the combination of ’OH + 0; (+02), which may best mimic biological exposure conditions.

EXPERIMENTAL PROCEDURES

Materials-The experiments reported in this paper were performed with a fatty acid- and globulin-free BSA (M, 66,200) from Sigma (A 0281). In preliminary experiments with other BSA preparations (not shown), Sigma product A 4378 and Miles Scientific (Naperville, IL) products 81-001-2,81-028-2, and 81-018-1 gave very similar results.

The abbreviations used are: BSA, bovine serum albumin; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid.

The term “protein fragmentation” refers to the direct breakdown of proteins by oxygen radicals. Such processes have been found to involve both main chain scission and side chain scission by mechanisms which differ from peptide hydrolysis. In contrast, the term “protein degradation” refers to peptide bond hydrolysis by proteolytic enzymes.

9902

Protein Damage and Degradation by Oxygen Radicals 9903

Exposure of BSA to Oxygen Radicals-BSA was exposed to 'OH alone, to 02 alone, or to 'OH + 0 2 using 'Wo radiation. Exposure to 'OH alone was achieved by irradiation under 100% N,O. Exposure to 0; alone involved irradiation under 100% 0, in the presence of 0.01 M sodium formate. Exposure to 'OH + 0; was achieved by irradiation under 100% 0,. Radiation was conducted with 5.0 pM solutions of BSA (0.33 mg of protein/ml) in double distilled and deionized water (no buffer). These procedures are described in detail in the preceding paper (1). The dose rate was 634 f 5 rads/min as measured by Fricke and Hart dosimetry (36). Radiation times were varied in order to achieve oxygen radical exposures of 1-120 nmol of radicals/nmol of BSA (total doses of 0.8-100 kilorads).

Amino Acid Analysis-BSA was hydrolyzed with 6 N HCl for 20 h (110 "C) prior to analysis. Native amino acids were quantified with both a Beckman 6300 and a Durrum D-500 amino acid analyzer. Results are presented as averages of the two instruments. Each run was calibrated with a standard mixture of all amino acids (10 nM each) and an internal standard of 0-thienylalanine. The detection limit was approximately 0.2 nM for each amino acid.

Fluorescence Measurements-Tryptophan destruction, bityrosine production, and fluorescamine reactivity were measured with an Aminco-Bowman fluorometer. Signal intensity was calibrated against a 0.1 mg/ml quinine sulfate solution in sulfuric acid as described in the preceding paper (1). To measure tryptophan loss and bityrosine production, 1.6 ml of BSA (0.53 mg of protein) was added to 0.4 ml of 0.1 M HEPES buffer (pH 7.0). Fluorescence emission at 340-350 nm (280 nm excitation) was used as a reflection of tryptophan content (37). Fluorescence loss was directly related to tryptophan oxidation in solutions of free tryptophan exposed to 'OH. Since fluorescence measurements are more complex with proteins, however, we did not attempt to convert fluorescence intensities to tryptophan residues. Bityrosine content was estimated at 325 nm excitation and 410-420 nm emission in comparison with authentic bityramine and bityrosine (38).

Free amino groups were assayed by reaction with fluorescarnine (39). Ten microliters of BSA (3.3 pg of protein) was added to 2.49 ml of 0.05 M sodium phosphate buffer (pH 8.0). While vortexing, 0.5 ml of fluorescamine (0.4 mg/ml in acetone) was added. The resulting fluorescence was measured at 390 nm excitation and 475 nm emission in comparison with standards.

Charge Changes-Isoelectric focusing gels were used to detect those alterations in primary structure which affected overall electrical charge. Ultrathin isoelectric focusing gels (0.2 mm) were used with GelBond backing film (40, 41). Ampholytes (Bio-Rad) were chosen such that the effective pH range was 3.7-6.2.

RESULTS

Generalized Loss of Amino Acids-BSA exposure to 'OH alone or to 'OH + 0; (+02) produced similar (dose-dependent) losses of most amino acids (Table I). The exception to this rule was half cystine which appeared to increase with exposure to 'OH and to decrease with exposure to 'OH + 0; (+02). Although published rate constants for reaction of free amino acids (42) were of some predictive value, it is clear that all amino acids must be considered. Presumably, the primary, secondary, and tertiary structure of a protein greatly influences the reactivity of each amino acid. The amino acid losses reported in Table I would be expected to have a dramatic effect on protein structure and function.

Studies with the 'OH scavenger mannitol indicated that all amino acid modifications (except some related to half-cystine) were initiated by 'OH. Amino acid modifications reported in Table I were inhibited more than 90% by inclusion of 1 mM mannitol during exposure to 'OH alone or to 'OH + 0: (+O,). This inhibition was observed at oxygen radical/BSA molar ratios of both 10 and 100 for all amino acids except half-cystine, which was only protected by 60-75%. Increasing the mannitol concentration to 10 or 100 mM had little effect. Since mannitol does not react with O;, these data indicate that 0; is largely unreactive with amino acids except cystine/ cysteine.

Exposure to 0; alone produced no detectable alterations to

TABLE I Modification of the amino acid composition of bovine serum albumin

bv axwen radicals ~

Amino acid composition of BSA

Amino Un- 10 nrnol radicals/nmol 100 nmol radicals/nmol acids treated BSA" BSA"

Ala Arg Asxb 1/2cys Glx GlY His Ile Leu LY s Met Phe Pro

Thr Ser

TYr Val

-

47 23 50 35 84 19 17 14 64 59 4

28 28 23 32 20 39

'OH 'OH+O; 'OH 'OH + 0; mol amino acidlmol BSA 43 43 29 30 22 22 14 15 43 44 24 31 59 9 212 7 71 76 42 50 17 18 11 14 13 14 6 8 13 13 8 8 61 59 39 39 54 56 33 37 3 3 2 2

25 25 16 15 26 26 15 13 21 21 13 14 30 30 18 19 16 18 11 10 37 36 25 24

Average % change -10.4 ? 1.5 -8.5 f 0.9 -45.7 f 2.6 -44.7 f 3.3 Values are total oxygen radical yields, i.e. all ' OH or 50% ' OH + Asx, aspartic acid + asparagine; Glx, glutamic acid + glutamine.

50% 0; (+Oz).

any amino acid except half-cystine, which increased approximately %fold at an O;/BSA molar ratio of 120 (data not shown). Since 0; alone did not significantly damage amino acids, it is interesting that 'OH and 'OH + 0; (+02) had similar effects (Table I). Exposure to 'OH + 0; (+02) involved only half the 'OH yield of exposure to 'OH alone since the solvated electron (e,) was converted to 0; by reaction with oxygen (1). Dismutation of 0 2 produces H202; but in separate experiments, no amino acid alterations were produced by H20z alone at BSA/H202 molar ratios of up to 150 unless transition metals were added (data not shown).

Loss of Tryptophan-Although BSA contains several tyrosine and phenylalanine residues and only 2 tryptophans, more than 99% of the fluorescence observed at 340-350 nm emission (280 nm excitation) can be attributed to tryptophan (37, 43). Exposure of BSA to 'OH alone or to 'OH + 0; (+Oz) resulted in the rapid loss of native tryptophan fluorescence (Fig. 1). The shape of the tryptophan destruction curves indicates that modified tryptophans compete with native tryptophan residues for the available oxygen radicals. Exposure to 0; alone produced no significant tryptophan loss (Fig. I), in agreement with results for other amino acids referred to above.

&Butyl alcohol is an efficient scavenger of 'OH, but does not react with H ' (44-46): H' represents some 8% of the radiolytic yield under 100% NzO. When irradiated under N,O in the presence of 1.0 M t-butyl alcohol, little tryptophan loss was observed at first (Fig. 2). With increasing radiation, tryptophan fluorescence slowly decreased, probably due to reactions with the t-butyl alcohol radical. During radiation under 100% nitrogen, isopropyl alcohol scavenges both 'OH and H ' leaving only e, unreacted (44-46). The combination of 100% Nz plus 1.0 M isopropyl alcohol (Fig. 2) produced very similar results to N20 plus t-butyl alcohol, indicating that tryptophan destruction is largely due to ' OH.

Mannitol (42) and uric acid (47-49) are frequently used in the laboratory as scavengers of 'OH. Uric acid may also be an important (water-soluble) biological antioxidant (47-49).

9904 Protein Damage and Degradation by Oxygen Radicals

: 3. 3- 0 5e m I 7 2.

; 2. e (1 1-

m \

U II

1. u) 0) b 0. 3

LL 4

0 20 4 0 60 80 100 120

nmol Oxygen Radicals/nmol 8SA

FIG. 1. Tryptophan destruction by oxygen radicals. Tryp- tophan fluorescence of BSA was monitored by fluorescence emission between 340 and 350 nm, with excitation at 280 nm, as described under “Experimental Procedures.” BSA was exposed to ‘OH, to 02 + 02, or to ‘OH + 0; + 02. Oxygen radical/BSA molar ratios are total yields, i.e. all ’ OH, all 02, or 50% .OH + 50% 0;. Fluorescence intensity values are means of three independent determinations for which standard errors were less than 10%.

\ 0 m E 2.0 (Y

5 1.5 v)

L - 5 1.0 L L

0 5 10 15 20 25 30 nmol Rodicals/nmol BSA

FIG. 2. Inhibition of tryptophan modification by isopropyl alcohol and t-butyl alcohol. BSA was exposed to ’OH (A) or to . OH + 0; + O2 (0) as described in the legend to Fig. 1. Alternatively, BSA was irradiated under 100% N20 in the presence of 1 M t-butyl alcohol (0) or under 100% N1 in the presence of 1 M isopropyl alcohol (0). Values are means of three independent determinations for which standard errors were less than 10%.

Both mannitol and urate gave significant protection against tryptophan loss (Fig. 3).

Bityrosine Production-. OH induced a linear production of bityrosine at all doses tested (Fig. 4). In contrast, exposure to ‘OH + 0; (+02) induced a more limited response which reached an early plateau. Little or no bityrosine was produced by exposure to 0; alone (Fig. 4). Isopropyl alcohol strongly inhibited bityrosine production (Fig. 4). These results are consistent with the concept that ’OH initiates bityrosine production. Meaningful inhibitory experiments could not be conducted with t-butyl alcohol due to production of an inter- fering fluorescent product. Mannitol was an effective inhibitor of bityrosine production during exposure to ‘OH or to ‘OH + 0 2 + O2 (Fig. 5 ) . Urate gave little or no protection against bityrosine produced by *OH alone. Urate did, however, par- tially inhibit bityrosine produced by exposure to ‘OH + 0 5 + O2 (Fig. 5).

Our results are consistent with earlier reports (38, 50, 51)

0 5 10 15 20 25 30 nmol *OH/nmol BSA

4. 0 Exposure to .OH+O,-

- 0 5 10 I5 20 25 30 nmol -OH+02-/nmol BSA

FIG. 3. Inhibition of tryptophan modification by mannitol and uric acid. Results shown in A represent tryptophan loss for BSA exposed to ‘OH alone (A), whereas B shows the results of exposure to ‘OH + 0; + O2 (0). The additions (in both A and B) are as follows: 0,l mM mannitol; A, 10 mM mannitol; 0,50 1M urate; 0, 0.5 mM urate. Oxygen radical/BSA ratios are total yields, i.e. all ’OH or 50% ‘OH + 50% 0;. Values are means of three independent determinations (performed as described in the legend to Fig. 1) for which standard errors were less than 10%.

/

nmol Oxygen Radicals/nmol BSA

FIG. 4. Bityrosine production by oxygen radicals. The bityrosine content of BSA was assessed by fluorescence emission between 410 and 420 nm, with excitation at 325 nm, as described under “Experimental Procedures.” BSA was exposed to ‘OH alone (A), to 0; + O2 (0), or to ’OH + 0; + O2 (0). Oxygen radical/BSA ratios are total yields, i.e. all ‘OH, all O;, or 50% ‘OH + 50% 0;. Also shown are the results of irradiation under 100% NS in the presence of 1 M isopropyl alcohol (0). Values are means of three independent determinations for which standard errors were less than 10%.

that tyrosyl (phenoxyl) radicals are produced as a result of hydrogen abstraction by . OH. Tyrosyl radicals may then react with other tyrosyl radicals or with tyrosine molecules to form several stable biphenolic compounds. The 2,2‘-biphenol, bityrosine, appears to be the major product (38). Both 0; and O2 may compete for reaction with tyrosyl radicals, thus ex- plaining their inhibitory effects on bityrosine production (38, 52). The inability of urate to act as an efficient inhibitor of bityrosine production may be related to the observation that proteins can act as “electron wires” with tyrosine as an “electron sink” (23, 53). Thus, a bulk-phase antioxidant may need to be used in relatively high concentration if it is to be effective (urate has only limited solubility).

Effects of Various Buffering Agents-Although our studies were performed on BSA in double distilled and deionized water, other researchers have often used buffers to stabilize the proteins they were investigating. Several buffers which

Protein Damage and Degradation by Oxygen Radicals 9905

A /i 0.510r/ B Exposure to .OH+Oz-

I . . . . . I 0 5 10 15 20 25 30 0 5 10 15 20 25 30

nmol -OH/nmol BSA nmol *OH+Oz-/nmol BSA

FIG. 5. Inhibition of bityrosine production by mannitol and uric acid. Results shown in A represent bityrosine production for BSA exposed to ’OH alone (A), whereas R shows the results of exposure to ‘OH + 0, + O2 (0). The additions (in A and E ) were as follows: 0, 1 mM mannitol; A, 10 mM mannitol; 0, 50 p M urate; 0, 0.5 mM urate. Oxygen radical/BSA ratios are total yields, i.e. all ‘OH or 50% ’OH + 50% 0;. Values are means of three independent determinations for which standard errors were less than 10%.

TABLE I1 Effects of variow buffering agents or tryptophan loss

and bityrosine production BSA was exposed to ‘OH alone or to ‘OH + 0, + O2 or was

untreated. A molar ratio of 30 nmol of oxygen radicals/nmol of BSA was used. BSA was dissolved either in Hz0 or in 100 mM buffer; all BSA solutions were pH 7.0. Tryptophan measurements are absolute fluorescence intensities at 280 nm excitation and 340-350 nm emission. Bityrosine measurements are absolute fluorescence intensities at 325 nm excitation and 410-420 nm emission. Detailed procedures are given under “Experimental Procedures.” Values are means of three independent determinations for which standard errors were less than 10%

Tryptophan Treatmentbuffer

Bityrosine

‘OH ‘OH+O; ‘OH ‘ O H + O ;

Untreated (H20) 3.52 3.51 0.06 0.06

Radical-treated Hz0 1.35 1.11 0.63 0.42 Tris 2.90 2.09 0.08 0.15 HEPES 3.11 3.51 0.14 0.08 Carbonate 0.79 0.91 4.55 1.15 Phosphate 1.41 1.51 0.60 0.33

~

are commonly used for protein studies had marked effects on BSA modification, as reflected by tryptophan damage and bityrosine production (Table 11). Tris and HEPES provided significant protection against tryptophan loss and bityrosine production. Carbonate enhanced tryptophan loss and greatly increased 410-420 nm fluorescence. This increase probably reflects production of a fluorescent carbonate product rather than true bityrosine. Phosphate buffer provided results which were more similar to those obtained with water. These data indicate that Tris, HEPES, and carbonate should not be used for studies of free radical modification of proteins. Phosphate buffer may be a reasonably suitable alternative, but one must be wary of iron contamination in all phosphate salts.

Charge Changes-Dramatic charge changes in BSA were induced by exposure either to ‘OH alone or to ’OH + 0; (+02). These changes are reflected in the isoelectric focusing gels shown in Fig. 6. Oxygen radical exposure produced a series of new bands with PI values more basic than that of native BSA. Charge changes were evident even at a radical/ BSA molar ratio of 1 and progressively increased with higher

’ 0 1 2.5 5 7.5 10 12.5 15 20 25 50 75 100

nmol Oxygen Radicals / nmol BSA

FIG. 6. Charge changes induced by oxygen radicals. BSA was exposed either to ‘OH alone ( A ) or to ’OH + 0; + O2 ( B ) . Isoelectric focusing gels (pH 3.7-6.2) were prepared and run as described under “Experimental Procedures.” Gels were treated with silver stain. Oxygen radical/RSA ratios are total yields, i.e. all ‘OH or 50% ‘OH + 50% 0,. The gels shown are representative of several experiments.

a. 4 ~

0 25 50 75 100 125 nmol Oxygen Rodlcols/nmol 05A

FIG. 7. Effect of oxygen radical treatment on pH of BSA solutions. BSA was exposed to ‘OH or to ‘OH + 02 + 02 in the absence of buffer. The pH of BSA solutions was rechecked immedi- ately following oxygen radical exposure. Oxygen radical/BSA ratios are total yields, i.e. all ‘OH or 50% ‘OH + 50% 0;. Values are means of three independent determinations for which standard errors were less than 10%.

ratios. Alteration of the BSA band was more rapid in samples exposed to ‘OH alone than in samples exposed to ’OH + 0; (+02). The general pattern of new band formation was also different for the two exposure conditions, and the most basic bands were only seen with exposure to ‘OH alone. Decreased positive charge was also evident from 10-2076 reductions in fluorescamine reactivity with free amino groups (see Ref. 2). Loss of staining intensity, at radical/BSA molar ratios greater than 25 (see Fig. 6), was found to result from decreased adherence to the surface of the ultrathin gels rather than to decreased reaction with the silver stain reagent.

When exposed to ‘OH alone, unbuffered BSA solutions showed no change in pH. In contrast, exposure to ‘OH + 0; (+02) induced significant pH changes (Fig. 7). The shape of the pH curve is similar to that which would be obtained by titration of a weak acid with a pK of approximately 6.2. Thus, the plateau in the pH curve may reflect the buffering capacity of histidine (pK 6.0). As histidine is gradually modified (Table I) , its buffering capacity would be lost, resulting in a sharp drop in pH as seen between ( ‘OH + O;)/BSA molar ratios of 90-120 in Fig. 7. One source of protons for the pH drop during ‘OH + 0; (+02) exposure is the dissociation of HO; to 0; and H’ (1). Furthermore, BSA undergoes fragmentation when exposed to ‘OH + 0; (1,2), and this process may also release protons.

9906 Protein Damage and Degradation by Oxygen Radicals

DISCUSSION

Following exposure to ‘OH or to ’OH + 0; (+02), the proteolytic susceptibility of BSA is greatly increased (1, 3). Although alterations to secondary and tertiary structure may provide the actual signals for proteolysis (1-3), the present data indicate that modifications to primary structure underlie all other changes.

All amino acids in the protein were susceptible to modification by ‘OH or by ’OH + 0; (+02). In contrast, 0; alone appeared only to reduce cystine residues. In agreement with known rate constants for reaction of free amino acids with ‘OH (42), tryptophan, histidine, and cysteine were more vulnerable than most other residues. Tyrosine should also be added to the list of sensitive amino acids since the results of Table I include tyrosine formed by reaction of ’OH with phenylalanine. Tyrosine is formed as a simple addition product of ‘OH + phenylalanine (42), and this “inflates” the remaining tyrosine values of Table I. Although apparent, the selectivity of .OH for tryptophan, tyrosine, histidine, and cysteine was much less than might be expected from reactions with free amino acids (42). Thus, it is clear that primary, secondary, and tertiary protein structure can greatly influence reactivity with oxygen radicals.

Bityrosine production appears to be quite a useful “marker” for protein modification by ‘OH. In related studies (to be published elsewhere), we have found that xanthine oxidase, horseradish peroxidase, tyrosinase, and H202 + Fez+ can also react with BSA (and several other proteins) to produce bityrosine.

Studies with scavengers and inhibitors (t-butyl alcohol, isopropyl alcohol, mannitol, urate) indicate that all modifications to primary structure are initiated by ’OH. Oxygen or 0; appears to exacerbate many effects of ‘OH. Theoretical considerations suggest that several amino acid residues may react with ‘OH to produce radicals which may decay harm- lessly. Oxygen or O;, however, may react with such species to produce permanently modified residues. We suggest that such considerations can explain the similar amino acid losses (without regard to the products formed) observed with exposure to ‘OH alone and to ‘OH + 0; (+02). Charge change results are also consistent with this interpretation and further demonstrate that different products are formed by ’OH than by ‘OH + 0; (+02). This point is explored in detail in the following paper (2).

Our studies of bityrosine formation also demonstrate that 0; can actually inhibit protein modification. This process probably involves re-reduction of tyrosyl (phenoxyl) radicals by 0; (38). Such reduction reactions can prevent the formation of bityrosine and may also inhibit production of other biphenols. In the following paper (a), we report that exposure to ‘OH (in the absence of O2 or 0;) can produce covalently bound BSA aggregates. Intermolecular (between two BSA molecules) bityrosine formation is certainly one mechanism for protein aggregation, although other cross-links can also be formed (20-28). Thus, it is important to note that 0; and 02, which inhibit bityrosine formation, also inhibit formation of BSA aggregates (2).

Although 0; alone had little effect on BSA, our results should not be construed to show that 0; is harmless. First, 0; did alter the half-cystine results of Table I. This was probably due to reduction of cystine disulfide bridges to sulfhydryl groups (cysteine residues). Although such reactions may not be harmful to cystine/cysteine residues, they can cause denaturation (unfolding) and loss of function. Second, proteins which have heme, iron-sulfur, copper, or other reac-

tive prosthetic groups may undergo more serious and perma- nent damage when exposed to 0;.

Glutamine synthetase undergoes oxidative modification of a histidine residue when exposed to mixed-function oxidases or to ascorbate + iron (8-10, 16). Such treatments also cause inactivation and promote the degradation of the enzyme in bacteria (8, 9, 16). Proteases have been isolated from liver and Escherichia coli which selectively degrade the oxidatively modified enzyme (10, 16). The oxidative modification of glutamine synthetase by ascorbate + iron appears to be a far more selective process than is reported here and may involve a site-directed mechanism. Nevertheless, in preliminary studies: metalloproteases from E. coli and liver which degrade modified glutamine synthetase also degraded . OH-modified BSA several times faster than untreated BSA.

Oxidative modification of hemoglobin was previously linked with proteolytic degradation, both in intact red blood cells (4, 6, 18, 19) and in extracts of erythrocytes and reticulocytes (5 , 7,18,19). Oxygen radicals and other activated oxygen species generated by xanthine oxidase, ascorbate + iron, or H20z + iron were effective initiators, as were mixtures of ‘OH and 0; generated by 6oCo radiation (in the presence of 02). As with the studies of glutamine synthetase described above, we were not able to identify specific initiating species or mechanisms.

The present results demonstrate that ’OH can modify essentially any aspect of protein primary structure. Oxygen and 0; can act to transform the initial products of .OH reaction, resulting either in increased damage (e.g. tryptophan loss) or in “repair” (e .g . prevention of bityrosine formation). In the following paper (2), we report that such oxidative modifications to primary structure underlie the alteration of secondary and tertiary structure. In the preceding paper (l), we noted that oxidative modification of primary, secondary, and tertiary structure commonly coincides with increased proteolytic susceptibility. The fourth and final paper (3) of this series shows that oxidative modification of protein structure can be directly linked with increased proteolytic susceptibility. Whereas oxidative modification and proteolysis are undoubtedly more complicated in vivo (e.g. by multiple radicals/oxidants, prosthetic groups, transition metals, etc.), our model studies provide a detailed rationale upon which to build.

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the of biological vol. 262, no. 20, issue of july 15, pp ... · solutions of bsa (0.33 mg of...

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