Amino Acids (2003) 25: 207–218

DOI 10.1007/s00726-003-0011-2

Free radical-mediated oxidation of free amino acids and amino

acid residues in proteins

Opening Review Article

E. R. Stadtman and R. L. Levine

Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, U.S.A.

Received March 19, 2003

Accepted May 7, 2003
Published online July 29, 2003; # Springer-Verlag 2003

Summary. We summarize here results of studies designed to elucidate species (ROS) that: (a) are present as pollutants in the
basic mechanisms of reactive oxygen (ROS)-mediated oxidation of proteins
and free amino acids. These studies have shown that oxidation of proteins
atmosphere; (b) are generated as by-products of normal
can lead to hydroxylation of aromatic groups and aliphatic amino acid side metabolic processes; and (c) are formed during exposure
chains, nitration of aromatic amino acid residues, nitrosylation of sulfhydryl to X-,
-, or U.V.-irradiation. The mechanisms of these
groups, sulfoxidation of methionine residues, chlorination of aromatic
ROS-mediated oxidations reactions were elucidated by
groups and primary amino groups, and to conversion of some amino acid
residues to carbonyl derivatives. Oxidation can lead also to cleavage of the Garrison et al. (1962), Garrison (1987), Swallow (1960),
polypeptide chain and to formation of cross-linked protein aggregates. and Shuessler and Schilling (1984) who exposed solutions
Furthermore, functional groups of proteins can react with oxidation pro- of amino acids, peptides, and proteins to ionizing radia-
ducts of polyunsaturated fatty acids and with carbohydrate derivatives
(glycation=glycoxidation) to produce inactive derivatives. Highly specific tion under conditions where
OH or a mixture of
OH and
methods have been developed for the detection and assay of the various O2
 are formed. Results of these studies demonstrated
kinds of protein modifications. Because the generation of carbonyl deriva- that the
OH-dependent abstraction of a hydrogen atom
tives occurs by many different mechanisms, the level of carbonyl groups in
proteins is widely used as a marker of oxidative protein damage. The level
from the -carbon of amino acids and the protein poly-
of oxidized proteins increases with aging and in a number of age-related peptide backbone and also from the aliphatic side chains
diseases. However, the accumulation of oxidized protein is a complex of hydrophobic amino acid residues of proteins are initial
function of the rates of ROS formation, antioxidant levels, and the ability
sites of attack. As illustrated in Fig. 1, abstraction of the
to proteolytically eliminate oxidized forms of proteins. Thus, the accumula-
tion of oxidized proteins is also dependent upon genetic factors and indi- hydrogen atom leads to formation of a carbon-centered
vidual life styles. It is noteworthy that surface-exposed methionine and radical (reaction c), which in the presence of oxygen is
cysteine residues of proteins are particularly sensitive to oxidation by almost rapidly converted to the peroxyl radical (reaction d). This
all forms of ROS; however, unlike other kinds of oxidation the oxidation of
these sulfur-containing amino acid residues is reversible. It is thus evident peroxyl radical is readily converted to the alkyl peroxide
that the cyclic oxidation and reduction of the sulfur-containing amino acids by reaction with the protonated form of the superoxide
may serve as an important antioxidant mechanism, and also that these radical (reaction e) or by abstraction of a hydrogen atom
reversible oxidations may provide an important mechanism for the regula-
tion of some enzyme functions.
from another molecule (reaction f ). Further reactions with
HO2
can lead to formation of the alkoxyl radical (reac-
Keywords: Protein carbonyls – Oxygen free radicals – Methionine tion h) and its conversion to the hydroxy derivative (reac-
oxidation=reduction – Oxidized protein proteolysis
tion j). Although the reaction sequence illustrated in Fig. 1
was established by using ionizing radiation for the gen-
eration of
OH and HO2
, it is likely that cleavage of
Introduction
H2O2 by iron or copper (reaction b, Fig. 1) is a major
Free amino acids and amino acid residues in proteins are source of
OH under physiological conditions. Further-
highly susceptible to oxidation by one or more reactive more, as shown in Fig. 1, Fe(II) is also able to replace
208 E. R. Stadtman and R. L. Levine

tions C and D, Fig. 2). In addition, based on the finding

that the number of peptide fragments formed in the radi-
olysis of proteins is approximately equal to the number of
prolyl residues, Schuessler and Schilling (1984) proposed
that oxidation of prolyl residues might lead to protein
fragmentation. This was subsequently verified by the stud-
ies of Uchida et al. (1990) showing that the oxidation of
proline residues of proteins can lead to peptide bond clea-
vage by a mechanism that involves oxidation of the pro-
line residues to the 2-pyrrolidone derivative (reaction B,
Fig. 2). It is noteworthy that acid hydrolysis of 2-pyrroli-
done yields 4-aminobutyric acid. Thus, the presence of 4-
aminobutyric acid in protein hydrolysates might be a mea-
Fig. 1. Free radical-mediated oxidation of the protein polypeptide sure of cleavage by the prolyl oxidation pathway.
backbone

Site-specific metal-catalyzed oxidation

HO2
in the reactions leading to formation of the alkyl
In addition to the reactions summarized in Figs. 1 and 2,
peroxide, alkoxyl radical, and the hydroxy derivative
the side-chains of amino acid residues of some proteins
(reactions g, i, and k ). Significantly, all reactions depicted
are readily oxidized by metal ion-catalyzed oxidation
in Fig. 1, following the hydrogen abstraction by
OH, are
(MCO) systems (Levine, 1983; Rivett et al., 1985; Fucci
dependent upon the addition of O2 to the carbon-centered
et al., 1983; Stadtman, 1990; Amici et al., 1989). Oxida-
radical (reaction d). In the absence of O2, two carbon-
tion of the side-chains of lysine, arginine, proline, and
centered radicals can react with one another to produce
threonine residues has been shown to yield carbonyl deri-
carbon–carbon cross-linked derivatives (reaction l, Fig. 1).
vatives and histidine residues are converted to 2-oxo-his-
tidine (Table 1). In studies with E. coli glutamine synthe-
tase, it was found that amino acid residues situated at
Peptide bond cleavage
metal binding sites on the enzyme are uniquely sensitive
In addition to the reactions illustrated in Fig. 1, the oxida- to metal-catalyzed oxidation by a site-specific mechanism
tion of proteins by ROS can lead also to the cleavage of (Farber and Levine, 1986; Climent and Levine, 1991;
peptide bonds. As discussed by Garrison (1987), the alk- Sahakian et al., 1991). The case in which a lysine residue
oxyl radicals and alkylperoxide derivatives of proteins is assumed to be the target for Fe(II)-catalyzed oxidation
can undergo cleavage by either the -amidation or di- is illustrated in Fig. 3. According to this mechanism, the
amide pathways (Fig. 2). In the -amidation pathway, chelate complex formed by the binding of Fe(II) to the
the C-terminal amino acid of the fragment derived from amino group of lysine can react with hydrogen peroxide
the N-terminal region of the protein will exist as the to generate a hydroxyl radical that will preferentially
amide derivative and the N-terminal amino acid of the attack the lysine moiety leading to its conversion to a 2-
fragment derived from the C-terminal portion of the pro- amino-adipic-semialdehyde residue. Similar reactions of
tein will exist as an -keto-acyl derivative. In contrast, the the Fe(II) with other amino acid targets lead to the gen-
C-terminal amino acid of the fragment derived from the eration of carbonyl derivatives (Table 1). This site-specific
N-terminal portion of the protein via the diamide pathway mechanism is supported by the demonstration that the
will exist as the diamide derivative and the N-terminal metal-catalyzed reactions are inhibited by catalase but
amino acid residue of the peptide fragment derived from not by
OH scavengers, presumably because the scaven-
the C-terminal region of the protein will exist as the iso- gers cannot compete with the ‘‘caged’’ reaction of
OH
cyanate derivative. with amino acids at the metal binding site. The impor-
In other studies reviewed by Garrison (1987), it was tance of this site-specific mechanism of amino acid resi-
demonstrated that the oxidation of glutamyl and aspartyl due oxidation is supported by recent studies showing that
residues of proteins can also lead to peptide bond clea- the oxidation of proline, lysine, and arginine residues to
vage in which the N-terminal amino acid of the C-term- aldehyde derivatives accounts for all of the protein car-
inal fragment will exist as the N-pyruvyl derivative (reac- bonyl groups generated in the oxidation of glutamine
 Protein oxidation 209

Fig. 2. Free radical-mediated cleavage of the polypeptide chain. A Cleavage by the diamide and -amidation pathways. B Cleavage by oxidation of
prolyl residues. C Cleavage by oxidation of glutamyl residues. D Cleavage by oxidation of aspartyl residue
210 E. R. Stadtman and R. L. Levine

Table 1. Oxygen free radical-mediated oxidation of protein amino acid residue side chains

Amino acid Products Reference

Arginine Glutamic-semialdehyde Amici et al. (1989); Requena et al. (2001)

Lysine 2-Amino-adipic-semialdehyde Amici et al. (1989); Berlett et al. a; Requena et al. (2001)
Proline Glutamic-semialdehyde Amici et al. (1989)
2-pyrrolidone Uchida et al. (1990)
4-,5-hydroxyproline Poston (1988); Creeth (1983)
pyroglutamic acid
Cysteine Cysteine disulfides, Sulfenic acid Garrison (1987); Swallow (1960)
Threonine 2-amino-3-keto butyric acid Taborsky (1973)
Leucine 3-,4-,5-hydroxyleucine Garrison (1987); Kopoldova and Liebster (1963)
Histidine 2-oxo-histidine Uchida and Kawakishi (1993)

a
Berlett, BS, Miller DG, Szweda L, Stadtman ER, unpublished data

Knecht et al., 1995) and also by Michael- addition reac-

tions of lysine, cysteine, or histidine residues with , -
unsaturated aldehydes formed during the peroxidation of
poly-unsaturated fatty acids (Schuenstein and Esterbauer,
1979; Uchida and Stadtman, 1993; Friguet et al., 1994;
Nadkarni and Sayre, 1995; Sayre et al., 1993; Bruenner
et al., 1994 and 1995). Because the presence of protein
carbonyl derivatives in cells reflects damage induced by
multiple forms of ROS, a number of analytical procedures
have been developed to determine the protein carbonyl
content of biological materials (Lenz et al., 1989; Levine

Fig. 3. Site-specific metal-catalyzed oxidation of protein amino acid

residues

synthetase by MCO systems in vitro and for 50–60 per-

cent of the reactive carbonyl groups detected in proteins
present in rat liver extracts (Requena et al., 2001, and this
volume).

Protein carbonylation
As illustrated in Figs. 2 and 3 and in Table 1, direct reac-
tion of proteins with ROS can lead to formation of protein
derivatives or peptide fragments possessing highly reac-
tive carbonyl groups (ketones, aldehydes). But, as illu-
strated in Fig. 4, proteins containing reactive carbonyl
groups can also be generated by secondary reactions of
primary amino groups of lysine residues of proteins with
reducing sugars or their oxidation products (glycation=
glycoxidation reactions) (Cerami, 1987; Lee and Cerami,
1990; Wolf and Dean, 1987; Monnier et al., 1990 and
Fig. 4. Generation of carbonyl derivatives of proteins. A By glycation=
1995; Grandhee and Monnier, 1991; Kristal and Yu, glycoxidation of lysine amino groups. B By reactions of --unsaturated
1992; Mullarkey et al., 1990; Verzijl et al., 2000; Wells- aldehydes with lysine, cysteine, or histidine residues of proteins
 Protein oxidation 211

et al., 1990 and 2000; Shacter et al., 1994; Keller et al., Because protein carbonyl levels are appreciably greater
1993; Buss et al., 1997; Smith et al., 1998; Robinson et al., than other oxidative modifications and because some
1999). These methods have become widely used mea- workers (Cao and Cutler,1995) have had difficulties with
sures of oxidative stress-induced cellular damage. Based protein carbonyl assays, it was argued (Dean et al., 1993)
on such measurements, it is well established that the accu- that other kinds of amino acid modifications are better
mulation of oxidized proteins is associated with aging, markers of protein oxidative damage. Difficulties encoun-
ischemia-reperfusion injury, and a number of age-related tered in the assay of protein carbonyl levels may reflect
diseases including diabetes, Alzheimer’s disease, amyo- lack of attention to experimental details (Levine, 2002).
trophic lateral sclerosis, cataractogenesis, atherosclerosis, However, variations in the levels of protein carbonyl con-
and many others. For reviews, see Stadtman and Berlett tents among different species or among individuals of the
(1997 and 1998) and Levine (Levine, 2002). same species may reflect differences in their resistance to

Fig. 5. Oxidation of aromatic amino acid residues

212 E. R. Stadtman and R. L. Levine

oxidative stress. Such variation is highlighted by results of for regulation of cellular metabolism (Levine et al., 2000).
studies in this laboratory showing that there was an age- Methionine is readily oxidized to methionine sulfoxide by
dependent increase in the protein carbonyl content of liver many different reactive oxygen and nitrogen species
proteins of one batch of rats, but not in a second batch of (Lavine, 1947; Vogt, 1995). The oxidation of surface-
the same strain of rats obtained 10 years later from the exposed methionines can thus serve to protect other func-
same breeder, who maintained that the dietary and hous- tional essential residues from oxidative damage (Reddy
ing conditions were unchanged. The observed differences et al., 1994; Levine et al., 1996). Methionine sulfoxide
were not due to differences in analytical procedures since reductases have the potential to reduce the residue back to
re-assay of samples from the first batch of animals that methionine, increasing the scavenging efficiency of the
had been kept at  80 C conditions yielded results simi- system. The anti-oxidant function of methionines in pro-
lar to those originally observed. The obvious conclusion is teins need not be limited to the protein itself. An elegant
that during the 10-year interval between the first and the example comes from Stocker and colleagues who estab-
second study, the rats had either adapted to an abnormal lished that high density lipoproteins reduce cholesteryl
environment or a strain of rats had been inadvertently ester hydroperoxides to alcohols, with the concomitant
selected for that was more resistant to the stresses respon- oxidation of two methionine residues to the sulfoxides
sible for the age-related changes observed in the earlier (Garner et al., 1998). Since the oxidized apolipoprotein
study. Significantly, according to the breeder, the life-span was reduced by methionine sulfoxide reductase (Sigalov
of the second batch of animals was 30% greater than that and Stern, 1998), the apolipoprotein could function cata-
of the first batch. In any case, the results of these studies lytically in the reduction of the hydroperoxides.
emphasize the need for caution in the interpretations of Further, the cyclic oxidation and reduction of methio-
apparently conflicting results obtained in two different nine is a reversible covalent modification. Such reversible
laboratories, or even two different studies within the same modifications have long been recognized to provide the
laboratory. mechanistic basis for most systems of cellular regulation,
with phosphorylation–dephosphorylation being especially
pervasive. Thus, interconversion of methionine and me-
Aromatic amino acid oxidation
thionine sulfoxide can also function to regulate the bio-
The aromatic amino acid residues of proteins are prime logical activity of proteins, through alteration in catalytic
targets for oxidation by various forms of ROS. As illu- efficiency and through modulation of the surface hydro-
strated in Fig. 5, phenylalanine residues are oxidized phobicity of the protein (Ciorba et al., 1997; Levine et al.,
to ortho- and meta-tyrosine derivatives (Maskos et al., 1996).
1992; Wells-Knecht et al., 1993; Balakrishnan and Reddy,
1970); tyrosine residues are converted to the 3,4-dihy-
Protein–protein cross linkage
droxy (dopa) derivative (Fletcher and Okada, 1961; Davies
et al., 1987; Maskos et al., 1992; Dean et al., 1993), As illustrated in Fig. 6, oxidative modification of proteins
and also to bi-tyrosine cross-linked derivatives (Boguta can also give rise to intra- or inter-protein cross-linked
and Dancewiez, 1981; Guilivi and Davies, 1993; Wells- derivatives by several different mechanisms, includ-
Knecht et al., 1993; Huggins et al., 1993; Heinecke et al., ing: (a) direct interaction of two carbon-centered radicals
1993; Jacob et al., 1996; Leuwenburgh et al., 1977). Trypto- (Garrison, 1987); (b) interaction of two tyrosine radicals
phan residues are converted to either the 2-, 4-, 5-, 6-, or 7- (Fig. 5); (c) oxidation of cysteine sulfhydryl groups
hydroxy derivatives, and also to N-formylkynurenine and (Garrison, 1987; Zhou and Gafni, 1991; Brodie and Reed,
kynurenine (Armstrong and Swallow, 1969; Winchester 1990; Takahashi and Goto, 1990); (d) interactions of the
and Lynn, 1970; Kikugawa et al., 1994, Maskos et al., carbonyl groups of oxidized proteins with the primary
1992b). amino groups of lysine residues in the same or a different
protein; (e) reactions of both aldehyde groups of malon-
dialdehyde with two different lysine residues in the same
Methionine oxidation
or two different protein molecules; (f) interactions of
The roles of methionine residues in proteins have not been glycation=glycoxidation derived protein carbonyls with
well defined, but consideration of available studies leads either a lysine or an arginine residue of the same or a
to the conclusion that methionine, like cysteine, can func- different protein molecule (Grandhee and Monnier,
tion as an antioxidant and as a key component of a system 1991; Verzyl et al., 2000); Wells-Knecht et al., 1995);
 Protein oxidation 213

(2001) and Squadrito and Pryor (1998). Based on these

studies, it has been established that aromatic amino acid,
cysteine, and methionine residues of proteins are particu-
larly sensitive to modification by one or another forms of
reactive nitrogen species (RNS). Their ability to do so is
strongly dependent upon pH and the availability of CO2
(Tien et al., 1999). Results of in vitro experiments have
shown that NPC is primarily responsible for the nitration
of tyrosine residues of proteins (Tien et al., 1999; Gow
et al., 1996), whereas peroxynitrite is largely responsible
for the oxidation of methionine residues to methionine
sulfoxide (Tien et al., 1999) and the nitrosation of protein
sulfhydryl groups to form RSNO derivatives (Viner et al.,
1999; Stubauer et al., 1999; Rubbo et al., 1994). There is
still uncertainty about detailed mechanisms of these reac-
tions. For discussions, see Radi et al. (2001) and Wink
and Mitchell (1998).
The cyclic phosphorylation=dephosphorylation or
adenylylation=deadenylylation of the hydroxyl group of
unique tyrosine residues of some enzymes and cell signal-
ing molecules represents an important mechanism for cel-
lular regulation of their activities. Therefore, the effect of
tyrosine nitration on the activity of such proteins has
received considerable attention. In the case of E. coli
glutamine synthetase, it was shown that nitration of either
Fig. 6. Formation of protein–protein cross-linked derivatives one of two different tyrosine residues inhibited the ability
of the enzyme to be adenylylated; moreover, nitration
converted the enzyme to a form with regulatory charac-
teristics that were similar to those obtained by adenylyla-
(g) interaction of a primary amino group of a lysine resi-
tion of the enzyme (Berlett et al., 1996). In a similar study,
due with protein aldehydes obtained via Michael-addition
it was demonstrated that nitration of the tyrosine residue
reactions with the lipid peroxidation products (viz. 4-
of the tyrosine residues of a penta-decameric peptide cor-
hydroxy-2-nonenal) (Uchida and Stadtman, 1993; Friguet
responding to the active site of a substrate for the lck-
et al., 1994).
kinase inhibited the ability of the kinase to phosphorylate
the peptide (Kong et al., 1996 ). In view of the fact that
the nitration reactions are non-reversible, these studies
Protein modification by reactive
demonstrate that nitration of tyrosine residues of regula-
nitrogen species
tory proteins may seriously compromise cyclic cascades
Nitric oxide (NO
) generated from arginine by the action involved in cell signaling and in the regulation of meta-
of nitric oxide synthetases plays an important role in the bolic enzyme activities. To the contrary, peroxynitrite-
regulation of various physiological functions (Ignarro dependent nitrosation of sulfhydryl groups of cell signal-
et al., 1989; Moncada et al., 1991; Culotta and Koshland, ing proteins has been shown to facilitate their biological
1992; Wink and Mitchell, 1998). But it also reacts with functions (Mohr et al., 1987; Li et al., 1997; Stamier et al.,
O2
 to form peroxynitrite (ONOO  ) (Pyror and Squadrito, 1998).
1995; Beckman et al., 1990; Radi et al., 1991), which
under physiological conditions can react with CO2 to form
Chlorination reactions
nitrosoperoxocarboxylate [(NPC), ONOOCO2  ] (Lymar
and Hurst, 1995 and 1996). Much of our knowledge of When stimulated to undergo oxidative burst, neutrophils
NO
chemistry comes from studies carried out in a num- produce O2
 and also release myeloperoxidase, which
ber of different laboratories. For review, see Radi et al. catalyzes conversion of H2O2 and Cl  , the highly reactive
214 E. R. Stadtman and R. L. Levine

Fig. 7. Oxidation of proteins by hypochlorous

acid

HOCl (Harrison and Schultz, 1976; Michaelis et al., be formed by so many different mechanisms and because
1992). As is illustrated in Fig. 7, HOCl has been shown protein carbonyl levels are orders of magnitude greater
to oxidize methionine residues to Met(O), to chlori- than any other type of amino acid oxidation (Dean et al.,
nate tyrosine residues, to form chloramine derivatives 1997), the carbonyl level is probably the best overall
of amino groups of lysine residues, to oxidize sulfhy- marker of oxidative stress-induced cellular damage. For
dryl groups to sulfenic acid derivatives, and to oxidize review, see Levine (2002). Even so, because various forms
lysine amino groups to carbonyl derivatives. In addition, of ROS differ in their abilities to oxidize a given amino
HOCl has been shown to convert lysine amino groups of acid residue, further studies to identify the amino acid
proteins to nitrogen-centered radicals (Hawkins and modifications associated with a given physiological dis-
Davies, 1998). The further observation that reaction of order may lead to a better understanding of the ROS
HOCl with O2
 leads to formation of
OH provides involved.
another mechanism for the initiation of protein oxidation
as described in Fig. 1. Because
OH is also formed during
the oxidation of ferrocyanide to ferricyanide by HOCl, it Accumulation of oxidized proteins
was proposed that the oxidation of Fe(II) to Fe(III) may
The steady-state level of oxidatively modified proteins
provide another mechanism for the generation of
OH
is dependent on a multitude of factors that influence
(Candeis et al., 1994).
the rates of ROS generation, the ability of cells to scav-
enge ROS, and also the levels and activities of the 20 S
proteasome and other proteases that catalyze the de-
Other markers of protein oxidation
gradation of oxidized proteins. For reviews see: Oliver
In addition to carbonyl levels, methods are now available et al. (1981, 1987); Rivett et al. (1985); Stadtman (1988,
for the assay of other types of ROS-mediated protein 1988); Rivett (1986); Levine (1989); Davies (1985, 1986,
damage. These include methods for the estimation of di- 1987); Wolf et al. (1986); Dean et al. (1984). It is note-
tyrosine (Giulivi and Davies, 1993; Henicke et al., 1993; worthy that oxidative modifications of some proteins,
Huggins et al., 1993; Jacob et al., 1996; Leeuwenburgh especially those leading to generation of cross-linkages,
et al., 1997), 3-nitro-tyrosine (Hensley et al., 1997; Uppu lead to derivatives that are not only resistant to degrada-
et al., 1998), chlorine derivatives (Kettle, 1996), and var- tion by the proteasome, but inhibit the ability of the
ious mono- or di-hydroxy phenylalanine(DOPA) deriva- proteasome to degrade oxidized forms of other proteins
tives (Armstrong and Dean, 1995; Hensley et al., 1997; (Rivett, 1986; Friguet et al., 1994; Grant et al., 1992;
Huggins et al., 1993). In view of the multiplicity of factors Davies, 1987). Observations that an age-related decrease
involved and complexity of the interrelationships between in proteasome activity is sometimes accompanied by an
ROS generation, antioxidant activities, biological targets, age-related increase in the levels of oxidized proteins
and especially differences in the susceptibility of indivi- (Starke-Reed and Oliver, 1991; Carney et al., 1991;
duals to ROS damage, no single marker of oxidative stress Stadtman et al., 1991) suggests that the two events may
is all inclusive. Nevertheless, because carbonyl groups can be related.
 Protein oxidation 215

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