Protein Oxidation in Aging,
Disease, and Oxidative Stress*

Barbara S. Berlett and Earl R. Stadtman‡

From the Laboratory of Biochemistry, NHLBI, National
Institutes of Health, Bethesda, Maryland 20892

The demonstration that oxidatively modified forms of proteins

accumulate during aging, oxidative stress, and in some pathologi-
cal conditions has focused attention on physiological and non-
physiological mechanisms for the generation of reactive oxygen
species (ROS)

1

and on the modification of biological molecules by

various kinds of ROS. Basic principles that govern the oxidation of
proteins by ROS were established in the pioneering studies of
Swallow (1), Garrison (2, 3), and Scheussler and Schilling (4) who
characterized reaction products formed when proteins were ex-
posed to ionizing radiation under conditions where only zOH, O

2

., or

a mixture of both was made available. Results of these studies
demonstrated that the modification of proteins is initiated mainly
by reactions with zOH; however, the course of the oxidation process
is determined by the availability of O

2

and O

2

. or its protonated

form (HOz

2

). Collectively, these ROS can lead to oxidation of amino

acid residue side chains, formation of protein-protein cross-link-
ages, and oxidation of the protein backbone resulting in protein
fragmentation. In the meantime, it has been shown that other
forms of ROS may yield similar products and that transition metal
ions can substitute for zOH and O

2

. in some of the reactions.

2

Oxidation of the Protein Backbone

As is illustrated in Fig. 1, oxidative attack of the polypeptide

backbone is initiated by the zOH-dependent abstraction of the

a-hy-

drogen atom of an amino acid residue to form a carbon-centered
radical (Fig. 1, Reaction c). The zOH needed for this reaction may be
obtained by radiolysis of water or by metal-catalyzed cleavage of
H

2

O

2

(Reactions a and b). The carbon-centered radical thus formed

reacts rapidly with O

2

to form an alkylperoxyl radical intermediate

(Reaction d), which can give rise to the alkylperoxide (Reaction f),
followed by formation of an alkoxyl radical (Reaction h), which may
be converted to a hydroxyl protein derivative (Reaction j). Signifi-
cantly, many of the steps in this pathway that are mediated by
interactions with HOz

2

can be catalyzed also by Fe

2

1

(Reactions e, g,

and i)

2

or by Cu

1

(not shown). The alkyl, alkylperoxyl, and alkoxyl

radical intermediates in this pathway may undergo side reactions
with other amino acid residues in the same or a different protein
molecule to generate a new carbon-centered radical (Reaction 1)
capable of undergoing reactions similar to those illustrated in Fig. 1.

R

1

Cz

P

P

or R

1

OOz or R

1

Oz

1 R

2

C

P

P

H 3 R

2

Cz

P

P

1 R

1

H or R

1

OOH or R

1

OH

R

EACTION

1

Moreover, in the absence of oxygen, when Reaction d in Fig. 1 is
prevented, the carbon-centered radical may react with another
carbon-centered radical to form a protein-protein cross-linked de-
rivative (Reaction 2).

R

1

Cz

P

P

1 R

2

Cz

P

P

3 R

1

CCR

2

R

EACTION

2

Protein Fragmentation

The generation of alkoxyl radicals (Fig. 1, Reactions h and g) sets

the stage for cleavage of the peptide bond by either the diamide or
a-amidation pathways. Upon cleavage by the diamide pathway (Fig.
2, Pathway a), the peptide fragment derived from the N-terminal
portion of the protein possesses a diamide structure at the C-terminal
end, whereas the peptide derived from the C-terminal portion of the
protein possesses an isocyanate structure at the N-terminal end. In
contrast, upon cleavage by the

a-amidation pathway (Fig. 2, Pathway

b), the peptide fragment obtained from the N-terminal portion of the
protein possesses an amide group at the C-terminal end, whereas the
N-terminal amino acid residue of the fragment derived from the
C-terminal portion of the protein exists as an N-

a-ketoacyl deriv-

ative. Upon acid hydrolysis, the peptide fragments obtained by the
diamide pathway will yield CO

2

, NH

3

, and a free carboxylic acid,

whereas hydrolysis of the fragment obtained by the

a-amidation

pathway yields NH

3

and a free

a-ketocarboxylic acid.

Peptide bond cleavage can occur also as a result of ROS attack of

glutamyl, aspartyl, and prolyl side chains. As described by Garri-
son (2), zOH-dependent abstraction of a hydrogen atom from the
g-carbon atom of a glutamyl residue, followed by reactions analo-
gous to Reactions d, f, and h in Fig. 1, will lead eventually to
peptide bond cleavage by a mechanism in which oxalic acid is
formed and the N-terminal amino acid of the peptide derived from
the C-terminal portion of the protein will exist as an N-pyruvyl
derivative (Reaction 3).

Based on the observation that the number of peptides formed

during radiolysis of proteins is approximately equal to the number
of prolyl residues, Schuessler and Schilling (4) proposed that oxi-
dation of prolyl residues would lead to peptide bond cleavage. This
was verified by studies of Uchida et al. (5) showing that oxidation
of proline residues leads to the formation of 2-pyrrolidone and
concomitant peptide bond cleavage (Reaction 4). Because acid hy-
drolysis of 2-pyrrolidone yields 4-aminobutyric acid, the presence of
4-aminobutyric acid in protein hydrolysates is presumptive evi-
dence for peptide bond cleavage by the proline oxidation pathway.

Oxidation of Amino Acid Side Chains

All amino acid residues of proteins are susceptible to oxidation

by zOH. However, the products formed in the oxidation of some
residues have not been fully characterized. Table I lists some of the
products formed during the oxidation of the residues that are most
susceptible to oxidation.

Oxidation of Sulfur-containing Amino Acid Residues—Cysteine

and methionine residues are particularly sensitive to oxidation by
almost all forms of ROS. Under even mild conditions cysteine
residues are converted to disulfides and methionine residues are
converted to methionine sulfoxide (MeSOX) residues. Most biolog-
ical systems contain disulfide reductases and MeSOX reductases
that can convert the oxidized forms of cysteine and methionine
residues back to their unmodified forms. These are the only oxida-
tive modifications of proteins that can be repaired. Based on the
observation that preferential oxidation of several exposed methio-
nine residues in some proteins has little effect on their biological
function, it was proposed that the cyclic oxidation-reduction of
methionine residues serves as a “built-in” ROS scavenger system to
protect such proteins from more extensive irreversible oxidative
modifications (6). This proposition is supported by results of recent
studies showing that a “knock-out” strain of yeast lacking MeSOX
reductase is more sensitive to H

2

O

2

toxicity than the wild-type

strain and that, when grown in the presence of H

2

O

2

, the protein

and free amino acid pool of the mutant strain contain higher levels

* This minireview will be reprinted in the 1997 Minireview Compendium,

which will be available in December, 1997. This is the fourth article of five in
the “Oxidative Modification of Macromolecules Minireview Series.”

‡ To whom correspondence should be addressed: Laboratory of Biochem-

istry, Bldg. 3, Rm. 222, NHLBI, National Institutes of Health, Bethesda, MD
20892. Tel.: [phone]; Fax: [phone].

1

The abbreviations used are: ROS, reactive oxygen species; MeSOX, me-

thionine sulfoxide reductase; PN, peroxynitrite; GS, glutamine synthetase.

2

For review, see Donald C. Borg and Karen M. Schaich (1988) in Oxygen

Radicals and Tissue Injury (Halliwell, B., ed) pp. 20 –26, Proceedings of an
Upjohn Symposium, Federation of American Societies for Experimental Bi-
ology, Bethesda, MD.

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of MeSOX than are present in the wild type strain.

3

Aromatic Amino Acid Residues—Aromatic amino acid residues

are among the preferred targets for ROS attack. As shown in Table
I, tryptophan residues are readily oxidized to formylkynurenine
and kynurenine and to various hydroxy derivatives; phenylalanine
and tyrosine residues yield a number of hydroxy derivatives; his-
tidine residues are converted to 2-oxohistidine, asparagine, and
aspartic acid residues.

Reactions with Peroxynitrite—With the discovery that nitric ox-

ide is a normal product of arginine metabolism and that it reacts
rapidly with O

2

. to form peroxynitrite (Reaction 5), the biological

effects of peroxynitrite (PN) have been extensively studied.

O

2

. 1 NOz 3 ONOO

2

R

EACTION

5

Methionine and cysteine residues of proteins are particularly

vulnerable to oxidation by PN, and tyrosine and tryptophan resi-
dues are selective targets for PN-dependent nitration. The nitra-
tion of tyrosine residues may be of singular importance since ni-
tration precludes the ability of tyrosine residues to undergo cyclic

interconversion between phosphorylated and unphosphorylated
forms (7) or between nucleotidylated and unmodified forms (8).
Accordingly, nitration would compromise one of the most important
mechanisms of cellular regulation of key enzyme activities and of
signal transduction networks (7). This possibility is underscored by
the demonstration that nitration of tyrosine residues in model
substrates prevents the phosphorylation of these residues by pro-
tein tyrosine kinases (9, 10) and by the demonstration that nitra-
tion of tyrosine residues in Escherichia coli glutamine synthetase
(GS) converts the enzyme to a form with regulatory properties
similar to those obtained by in vivo enzyme-catalyzed adenylyla-
tion of a single tyrosine residue in each subunit of the enzyme (11).
The enzyme-catalyzed cyclic adenylylation and deadenylylation of
GS is the basis of an exquisite mechanism for the feedback regu-
lation of GS activity by diverse end products of glutamine metab-
olism (8). In contrast, the nitration of tyrosine residues is an
irreversible process and therefore locks the enzyme into a rela-
tively inactive configuration.

The ability of PN to nitrate tyrosine residues and oxidize methi-

onine residues of proteins is dependent upon the availability of
CO

2

. In the absence of CO

2

, PN is in equilibrium with an activated

form (PN*) of unknown structure that reacts rapidly with methio-
nine residues to form MeSOX (12), but in the presence of CO

2

, PN

is almost instantly converted to a derivative (possibly O

5NOOCO

2

2

or O

2

NOCO

2

2

) that can nitrate aromatic compounds (13–16). Accord-

ingly, the nitration of tyrosine and the oxidation of methionine resi-
dues of proteins are mutually exclusive processes that are differen-
tially regulated by the availability of CO

2

, as illustrated in Scheme 1.

Curiously, in the case of GS, there is little or no nitration of

tyrosine residues in the complete absence of CO

2

, and no oxidation

of methionine residues occurs in the presence of physiological con-
centrations of CO

2

(i.e. 5% CO

2

, pH 7.4). Even so, the PN-depend-

ent oxidation of methionine residues in the absence of CO

2

and the

nitration of tyrosine residues in the presence of CO

2

both convert

GS to a form with regulatory properties similar to those obtained
by enzyme-catalyzed adenylylation of a single tyrosine in each
subunit of the enzyme (17).

Generation of Protein Carbonyl Derivatives

As already noted, oxidative cleavage of proteins by either the

a-amidation pathway (Fig. 2) or by oxidation of glutamyl side
chains (Reaction 3) leads to formation of a peptide in which the
N-terminal amino acid is blocked by an

a-ketoacyl derivative. How-

ever, as shown in Table I, direct oxidation of lysine, arginine,
proline, and threonine residues may also yield carbonyl deriva-
tives. In addition, carbonyl groups may be introduced into proteins
by reactions with aldehydes (4-hydroxy-2-nonenal, malondialde-
hyde) produced during lipid peroxidation (Fig. 3A) (18 –20) or with
reactive carbonyl derivatives (ketoamines, ketoaldehydes, deoxyo-
sones) generated as a consequence of the reaction of reducing
sugars or their oxidation products with lysine residues of proteins
(21–23) (glycation and glycoxidation reactions) (Fig. 3B). The pres-
ence of carbonyl groups in proteins has therefore been used as a
marker of ROS-mediated protein oxidation, and several sensitive
methods for the detection and quantitation of protein carbonyl
groups have been developed (24). As judged by the presence of
carbonyl groups, it has been established that protein oxidation is
associated with aging, oxidative stress, and a number of diseases.

Oxidative Stress-induced Protein Oxidation—Elevated levels of

oxidized protein are present in animals and cell cultures following
their exposure to various conditions of oxidative stress. Thus, ex-
posure of animals or cell cultures to either hyperoxia, forced exer-
cise, ischemia-reperfusion, rapid correction of hyponatremia, para-
quat toxicity, magnesium deficiency, ozone, neutrophil activation,
cigarette smoking, x-radiation, chronic alcohol treatment, or mixed
function oxidation systems leads to an increase in the level of
oxidized protein.

4

Protein Oxidation and Aging—Aging is associated with the ac-

cumulation of inactive or less active, more heat-labile forms of
numerous enzymes (25, 26). The possibility that these age-related
changes are due, at least in part, to oxidative modification is

3

J. Moskovitz, B. S. Berlett, M. J. Poston, R. L. Levine, and E. R. Stadt-

man, unpublished results.

4

For review, see E. R. Stadtman and B. S. Berlett (1997) Chem. Res.

Toxicol. 10, 485– 494.

F

IG

. 1. Oxygen free radical-mediated oxidation of proteins.

F

IG

. 2. Peptide bond cleavage by the (a) diamide and (b)

a-amida-

tion pathways.

R

EACTION

3

R

EACTION

4

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indicated by the facts. (a) In vitro exposure of enzymes to ROS
elicits changes in catalytic activity, heat stability, and proteolytic
susceptibility similar to those that occur during aging (27–30). (b)
Brief exposure of animals to oxidative stress leads to alterations in
enzymes similar to that associated with aging (31, 32). (c) Old
animals are more susceptible than young animals to protein dam-
age during oxidative stress, e.g. x-radiation, H

2

O

2

(33, 34). (d)

There is an age-related increase in the carbonyl content of protein
in human brain (35), gerbil brain (36), eye lens (37), rat hepatocytes
(32), whole body protein of flies (38), and human red blood cells
(28). (e) The carbonyl content of protein in cultured human fibro-
blasts increases exponentially as a function of the age of the fibro-
blast donor (28). (f) There is an inverse relationship between regi-
mens that lead to an increase in life span and regimens that lead to
an increase in protein carbonyl content and vice versa (39). For
example, diet (caloric) restriction of rats (39) and mice (40) leads to
an increase in life span and to a decrease in the level of protein
carbonyls. When compared at the same chronological age, strains of
short lived houseflies contain higher levels of oxidized proteins
than their longer lived cohorts (41).

Protein Oxidation and Disease—Accumulation of oxidized pro-

tein (protein carbonyls) is associated with a number of diseases,
including amyotrophic lateral sclerosis, Alzheimer’s disease, respi-
ratory distress syndrome, muscular dystrophy, cataractogenesis,
rheumatoid arthritis, progeria, and Werner’s syndrome.

4

Although

the level of carbonyl has not been directly determined, there is
reason to believe that oxidative modification of proteins is impli-
cated also in atherosclerosis, diabetes, Parkinson’s disease, essen-
tial hypertension, cystic fibrosis, and ulcerative colitis.

4

Accumulation of Oxidized Protein

The intracellular level of oxidized protein reflects the balance

between the rate of protein oxidation and the rate of oxidized
protein degradation. This balance is a complex function of numer-
ous factors that lead to the generation of ROS, on the one hand, and
of multiple factors that determine the concentrations and/or activ-
ities of the proteases that degrade oxidatively damaged protein, on
the other. As illustrated in Fig. 4, many different physiological and
environmental processes lead to the formation of ROS. Collectively,
these processes can promote the generation of a battery of ROS,
including a number of free radicals (zOH, O

2

., Rz, ROOz, ROz, NOz,

RSz, ROSz, RSOOz, and RSSR.), various non-radical oxygen deriv-
atives (H

2

O

2

, ROOH,

1

O

2

, O

3

, HOCl, ONOO

2

, O

5NOCO

2

2

,

O

2

NOCO

2

2

, N

2

O

2

, NO

2

1

, and highly reactive lipid- or carbohydrate-

derived carbonyl compounds, viz. 4-hydroxy-2-nonenal, malondial-
dehyde ketoamines, ketoaldehydes, and deoxyosones. Any one of
these ROS is capable of promoting the modification of proteins.
However, as shown in Fig. 4, their abilities to do so are dependent
upon the concentrations of a myriad of enzymic and non-enzymic
factors (antioxidants) that can either inhibit the formation of ROS
or facilitate their conversion to inactive derivatives. For example,
the O

2

. formed by several pro-oxidant systems shown in Fig. 4 is

readily converted to H

2

O

2

by the action of superoxide dismutase.

This H

2

O

2

together with H

2

O

2

produced by various oxidases and

metal-catalyzed oxidation systems is readily degraded by catalase,
glutathione peroxidase, thiol-specific antioxidant enzymes, and
other peroxidases. However, if in the course of metabolism the
concentrations of these antioxidant activities become insufficient to
decompose all of the H

2

O

2

formed, the H

2

O

2

may undergo metal

ion-catalyzed cleavage by the Fenton reaction to generate the even
more toxic zOH. This reaction is dependent upon the availability of
iron and copper, which is determined by the concentrations of
metal-binding proteins (ferritin, transferrin, lactoferrin, and ceru-
loplasmin), and of multiple factors (iron-responsive elements, etc.)
that control the intracellular concentrations of these proteins, as
well as factors that influence the binding and/or the release of
metal ions from these binding proteins. The level of ROS is also a
function of the concentrations of vitamins (A, C, and E) and of me-
tabolites (uric acid, bilirubin, etc.) that are capable of either scaveng-
ing free radicals directly or of facilitating the regeneration of metab-
olites that do so. Finally, metal ion chelators can either suppress or
enhance the rates of ROS generation by forming complexes with iron
or copper that inhibit their ability to catalyze ROS formation or alter
their redox potentials and therefore their ability to undergo cyclic
interconversion between oxidized and reduced states. Furthermore,
other divalent cations (Mg

2

1

, Mn

2

1

, and Zn

2

1

) may compete with

Fe(II) or Cu(I) for binding to metal binding sites on proteins and
thereby prevent site-specific generation of zOH, which is likely the
most important mechanism of protein damage (42). In addition,
Mn(II) is able to inhibit the reduction of Fe(III) to Fe(II) (43) and thus

T

ABLE

I

Amino acids most susceptible to oxidation

Amino acids

Oxidation products

Cysteine

Disulfides, cysteic acid

Methionine

Methionine sulfoxide, methionine sulfone

Tryptophan

2-, 4-, 5-, 6-, and 7-Hydroxytryptophan, nitrotryptophan, kynurenine, 3-hydroxykynurinine, formylkynurinine

Phenylalanine

2,3-Dihydroxyphenylalanine, 2-, 3-, and 4-hydroxyphenylalanine

Tyrosine

3,4-Dihydroxyphenylalanine, tyrosine-tyrosine cross-linkages, Tyr-O-Tyr, cross-linked nitrotyrosine

Histidine

2-Oxohistidine, asparagine, aspartic acid

Arginine

Glutamic semialdehyde

Lysine

a-Aminoadipic semialdehyde

Proline

2-Pyrrolidone, 4- and 5-hydroxyproline pyroglutamic acid, glutamic semialdehyde

Threonine

2-Amino-3-ketobutyric acid

Glutamyl

Oxalic acid, pyruvic acid

S

CHEME

1

F

IG

. 3. Formation of protein carbonyls by glycation, glycoxidation,

and by reactions with peroxidation products of polyunsaturated
fatty acids (PUFA). A, reactions of sugars with protein lysyl amino groups
(P-NH

2

). B, Michael addition of 4-hydroxy-2-nonenal to protein lysine (P-

NH

2

), histidine (P-His), or cysteine (PSH) residues. C, reaction of protein

amino groups (PNH

2

) with the lipid peroxidation product, malondialdehyde.

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prevent its ability to promote formation of zOH by the Fenton reaction
as well as the generation of other forms of ROS as illustrated in
Fig. 1.

As noted above, the accumulation of oxidized protein reflects not

only the rate of protein oxidation but also the rate of oxidized
protein degradation, which (as shown in Fig. 4) is also dependent
upon many variables, including the concentrations of proteases
that preferentially degrade oxidized proteins and numerous factors
(metal ions, inhibitors, activators, and regulatory proteins) that
affect their proteolytic activities. For example, oxidized forms of
some proteins (e.g. cross-linked proteins (44 – 46)) and proteins
modified by glycation (47) or lipid peroxidation products (44) are
not only resistant to proteolysis but, in fact, can inhibit the ability
of proteases to degrade the oxidized forms of other proteins (44, 48).

The foregoing discussion and some of the points illustrated in

Fig. 4 are by no means comprehensive. They are intended to call
attention to the extraordinary complexity of ROS biochemistry.
Numerous other factors not discussed are certainly important in
determining the steady-state level of oxidative damage under vary-
ing physiological and environmental conditions. It is our belief that
during aging there is a progressive accumulation of errors at the
level of DNA that affect any one or more of the factors that govern
the dynamics of protein oxidation and oxidized protein degrada-
tion. This leads to a shift in the balance between these processes in
favor of oxidized protein accumulation and attendant loss of bio-
logical function. Two observations are consistent with this hypoth-
esis. (a) The level of oxidized protein in cultured fibroblasts is a
function of the age of the fibroblast donor and is independent of the
cell passage number. (b) Chronic injection of the free radical scav-
enger tert-butyl-

a-phenylnitrone leads to a reversal of some age-

related changes in the gerbil brain, but when the tert-butyl-

a-

phenylnitrone treatment is discontinued the age-related changes
reappear (36). Both observations indicate that the level of oxidative
damage is determined by the genetic make-up of the cell, which
changes with age. According to this proposition, the aged pheno-
type could be expressed (a) by a single point mutation that could
impair a biological activity that occupies a central role in biological
functions, such an alteration of helicase as occurs in Werner’s
syndrome (49), or (b) by the accumulation over time of numerous
errors leading to deficiencies in the synthesis and/or activities of a
multiplicity of the factors that govern the balance between protein
oxidation and degradation. From this perspective, aging could be
looked upon as a degenerative process (disease?) that might include
aberrations that contribute to the development of other pathologies,
such as Alzheimer’s disease, amyotrophic lateral sclerosis, diabetes,
etc., in which the accumulation of oxidatively modified protein has
been demonstrated. Perhaps in these diseases one or more of the

specific processes summarized in Fig. 4 are exaggerated, leading to
unique manifestations that are characteristic of the disorder.

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F

IG

. 4. Accumulation of oxidized protein is dependent upon the

balance between pro-oxidant, antioxidant, and proteolytic activi-
ties. MSR, methionine sulfoxide reductase; GPx, glutathione peroxidase;
CAT, catalase; RSH-Px, thiol-specific peroxidase; NOS, nitric oxide synthe-
tase; SOD, superoxide dismutase; GST, glutathione transferase.

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