Tài liệu Báo cáo khoa học: Inhibition of glyceraldehyde-3-phosphate dehydrogenase by peptide and protein peroxides generated by singlet oxygen attack docx

Inhibition of glyceraldehyde-3-phosphate dehydrogenase by peptide
and protein peroxides generated by singlet oxygen attack
Philip E. Morgan
1
, Roger T. Dean
2
and Michael J. Davies
1
1
EPR and
2
Cell Biology Groups, The Heart Research Institute, Sydney, New South Wales, Australia
Reaction of certain peptides and proteins with singlet oxygen
(generated by visible light in the presence of rose bengal dye)
yields long-lived peptide and protein peroxides. Incubation
of these peroxides with glyceraldehyde-3-phosphate dehy-
drogenase, in the absence of added metal ions, results in loss
of enzymatic activity. Comparative studies with a range of
peroxides have shown t hat this inhibition is concentration,
peroxide, a nd time dependent, with H
2
O
2
less efficie nt than
some peptide peroxides. Enzyme inhibition correlates with
loss of both the peroxide and e nzyme thiol residues, with a
stoichiometry of two thiols lost per peroxide c onsumed.
Blocking the thiol residues prevents reaction with the per-
oxide. This stoichiometry, the lack of metal-ion dependence,
and the absence of electron paramagnetic resonance (EPR)-
detectable species, is consistent with a molecular (nonradi-

cal) reaction between the active-site thiol of t he enzyme and
the peroxide. A number of low-molecular-mass compounds
including thiols and ascorbate, but not Trolox C, can pre-
vent inh ibition by removing the initial peroxide, or s pecies
derived from it. In contrast, g lutathione reductase and lac-
tate dehydrogenase are poorly inhibited by these peroxides
in the absence of added Fe
2+
–EDTA. The presence of this
metal-ion complex enhanced the inhibition observed with
these enzymes consistent with the o ccurrence of radical-
mediated reactions. Overall, these studies demonstrate that
singlet oxygen-mediated damage to a n i nitial target protein
can r esult in selective subsequent damage to other proteins,
as evidenced by loss of enzymatic activity, via the formation
and subsequent reactions of protein peroxides. These reac-
tions may be important in the development o f cellular d ys-
function as a result of photo-oxidation.
Keywords: protein oxidation; protein peroxides; protein
radicals; s inglet oxygen; photo-oxidation.
Singlet oxygen (molecular o xyge n in its
1
D
g
state;
1
O
2
)is
generated by a number of enzymatic and chemical reactions,

by UV exposure, and by visible light in the presence of a
number of exogenous or endogenous cellular sensitisers.
1
O
2
generation has been reported in myeloperoxidase- and
eosinophil peroxidase-catalysed reactions [1–3], and by
some activated cell types including neutrophils [4], eosino-
phils [3,5], and macrophages [6]. As a result of the wide-
spread exposure of humans to UV and visible light,
1
O
2
has
been sugge sted t o p lay a key role in the development of a
number of human pathologies including cataract, sunburn,
some skin cancers and aging [7–12].
1
O
2
reacts with a range of biological molecules including
DNA [13,14], cholesterol [15,16], lipids [15,17,18], and
amino acids and proteins [12,19,20]. Proteins are major
biological targets as a result of their abundance and high
rate constants for reaction [21], with damage occurring
primarily at Trp, Met, Cys, His and Tyr side-chains
[12,19,20]. Reaction with Trp, H is and T yr residues h as
been shown t o yield peroxides, although the structure of
some of these materials remain s to be fully established
(reviewed in [12,19,20]). Previous studies have identified the

C-3 site on the indole ring of Trp as a major site of peroxide
formation [22], and our recent studies have demonstrated
that the major peroxide generated with Tyr residues is a
ring-derived, C-1, dieneone hydroperoxide (A. Wright,
W. A. Bubb, C. L. Hawkins & M. J. Davies, unpublished
results). Further species are also formed with free Tyr [23].
Both endo- and hydro-peroxides have been reported with
His [24].
1
O
2
-mediated oxidation of proteins also yields
peroxides, with Tyr, Trp and His residues likely targets [25].
All of these peroxides are unstable in solution, with
decomposition enhanced by reducing agents, UV light and
metal ions ([25]; A. W right, W. A. Bubb, C. L. Hawkins &
M. J. Davies, unpublished results). Reaction with some
metal ions generates radical species ([25]; A. Wright, C. L.
Hawkins & M. J. Davies, unpublished r esults).
Previous studies with protein peroxides generated by
high-energy radiation (e.g. c-sources, X-rays), meta l ion/
peroxide systems, thermal sources of peroxyl radicals,
peroxynitrite, and activated white cells [26,27], have shown
that these species play a key role in the propagation of
oxidative chain reactions within proteins [12,28]. These
species can oxidize other biomolecules, including lipids,
Correspondence to M. J. Davies, EPR Group, The Heart Research
Institute, 145 Missenden Road, Camperdown, Sydney, New South
Wales 2050, Australia. Fax: + 61 29550 3302,
E-mail:

Abbreviations: EPR, electron paramagnetic resonance; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase; GR, glutathione reduc-
tase; GSH, reduced glutathione; LDH, lactate dehydrogenase; 2MPG,
N-(2-mercaptopropionyl)glycine; N-Ac-Trp-OMe, N-acetyl trypto-
phan methyl ester; N-Ac-Trp-OMe-OOH, peroxides formed on
N-acetyl tryptophan methyl ester by reaction with
1
O
2
;NEM,
N-ethylmaleimide;
1
O
2
, molecular oxygen in its first excited singlet
(
1
D
g
) state; PBN, N-t-butyl-a-phenylnitrone.
Enzymes: glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12);
glutathione reductase (EC 1.6.4.2); lactate dehydrogenase
(EC 1.1.1.27) .
Note: a website is available at www.hri.org.au
(Received 13 N ovember 2001, revised 12 February 2002, accepted 20
February 2002)
Eur. J. Biochem. 269, 1916–1925 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02845.x
antioxidants and DNA [27,29–31], with some of these
reactions involving peroxide-derived radicals [30–33]. The
reactions of protein peroxides with other proteins have not

been investigated in depth, and would be expected to be
distinct from the reactions of low-molecular-mass alkyl a nd
lipid peroxides. A preliminary report has appeared on the
inhibition of glutathione reductase by radiation-generated
protein peroxides [34], and evidence presented for inhibition
of enzymes, by radiation-generated species, in erythrocytes
[35]. Such results cannot be extrapolated to
1
O
2
-generated
species, owing to their different sites and chemistries ([25–
27]; A. Wright, C. L. Hawkins & M. J. Davies, unpublished
results).
In this study we have examined the inhibition of two thiol-
dependent enzymes [glyceraldehyde-3-phosphate dehydro-
genase (GAPDH) and glutathione reductase (GR)], and one
nonthiol-dependent enzyme (lactate dehydrogenase, LDH),
by
1
O
2
-generated peroxides f ormed on p eptides and proteins.
These data have been compared with those obtained u sing
H
2
O
2
. The role of radical vs. nonradical processes has been
investigated, as has the prevention of such damage.

MATERIALS AND METHODS
Amino acids, peptides and antioxidants were commercial
samples of high purity. BSA (fraction V, > 98%), lysozyme
(chicken egg white, % 95 %), RNase A (bovine pancreas,
essentially protease and salt free), GR [bakers yeast, in 3.6
M
(NH
4
)
2
SO
4
, pH 7 .0], GAPDH (rabbit muscle, lyophilized
powder) and LDH [rabbit muscle, in 3.2
M
(NH
4
)
2
SO
4
,
pH 6.0, or lyophilized powder] were from Sigma. GAPDH
[rabbit muscle, in 3.2
M
(NH
4
)
2
SO

4
+0.1m
M
EDTA,
pH 7.5], NADPH (tetrasodium salt), NAD
+
(free acid),
NADH (disodium salt) were f rom B oehringer Mannheim.
These preparations did not contain materials that interfered
with peroxide formation or e nzyme activity measurements.
The water used was passed through a four-stage Milli Q
system equipped with a 0.2-lm-pore-size final filter. Solu-
tions of Fe
2+
–EDTA (1 : 1 complex) were prepared using
de-oxygenated water and maintained under oxygen-free N
2
.
All concentrations given are final values.
Peroxides were generated on BSA, lysozyme, RNase A
(each 50 mgÆmL
)1
) and the peptides N-acetyl tryptophan
methyl ester (N-Ac-Trp-OMe), Gly-His-Gly and Gly-Tyr-
Gly (each 2.5 m
M
), by photolysis with visible light (from a
Kodak S-AV 2050 slide projector) through a 345-nm cut-off
filter in the presence of 10 l
M

rose bengal dye ([25];
A. Wright, C. L . Hawkins & M. J. Davies, unpublished
results). Solutions were kept on ice during photolysis
(30 min BSA, 60 min for lysozyme and peptides, 120 min
for RNase A), and were continually aerated. After cessation
of photolysis, c atalase (Sigma, bovine liver, 5 lgÆmL
)1
for
BSA, 50 lgÆmL
)1
for lysozyme and RNase A, 250 lgÆmL
)1
for p eptides) was added, unless stated otherwise, to remove
H
2
O
2
and the samples incubated for 30 min at room
temperature before freezing ()80 °C) in aliquots.
Peroxide concentrations were determined by a modified
FOX (FeSO
4
/xylenol orange) assay, using H
2
O
2
standards
[36]. This assay gives s imilar values to iodometric analysis
(A. Wright, C. L. Hawkins & M. J. Davies, unpublished
results). The effects of reductants on peroxides were

determined by incubation of the samples with an approx-
imately 20-fold excess of NaBH
4
over protein concentration
for 30 m in at room temperature. Control samples were
incubated under identical conditions. I mmediately post-
incubation, samples were separated on a PD-10 column
(Pharmacia), the protein fractions collected, and residual
peroxides determined after correction for sample dilution
(assessed by A
280
values).
Thiol groups on GAPDH (2 mgÆmL
)1
) were blo cked by
incubation for 30 min at room temperature with a 10-fold
excess (over protein thiol concentration) of N-ethylmalei-
mide (NEM) followed by separation of the treated protein
from excess reagent by PD-10 chromatography, with water
elution. Control samples were incubated in the absence of
NEM. Protein concentration after PD-10 chromatography
was determined by the BCA assay, using BSA standards
(Pierce).
Free thiol concentrations were assessed by incubation of
0.1 m gÆmL
)1
GAPDH with 500 l
M
5,5¢-dithiobis(2-nitro-
benzoic acid) (in 100 m

M
phosphate buffer, pH 7.4) for
30 min at room temperature, with quantification of the
released 5-thionitrobenzoic acid (TNB) anion measured
using its absorbance at 412 nm and e 13 600
M
)1
Æcm
)1
[37].
Electron paramagnetic resonance (EPR) samples were
prepared by addition of peroxide (200 l
M
) to the enzyme in
the presence of the spin trap N-t-butyl-a-phenylnitrone
(PBN) (9.4 m
M
in 50 m
M
phosphate buffer, pH 7.4). Fe
2+
–
EDTA (100 l
M
, 1 : 1 complex) was added where stated.
Samples were incubated for 5 min at 20 °Cbeforeexami-
nation in a standard, fl attened, aqueous solution cell (WG-
813-SQ; Wilmad, B uena, NJ, USA) using a Bruker EMX
X-band spectrometer equipped with 100 kHz modulation
and a cylindrical ER4103TM cavity. Typical spectrometer

settings were: gain 1.0 · 10
6
, modulation amplitude 0.1 mT,
time constant 163.8 ms, sweep time 81.9 s, centre field
348.0 mT, field sweep width 8.0 mT, microwave pow er
25.0 m W, frequency 9 .7 GHz, with four acquisitions aver-
aged.
GR ac tivity was examined at 37 °Cin50m
M
phosphate
buffer, pH 7.4, containing 0.025 UÆmL
)1
GR, 20 l
M
Fe
2+
–
EDTA (where indicated), peroxide (20–200 l
M
), and
antioxidant (10-fold excess over peroxide concentration).
Aliquots were removed as indicated, and residual activity
assayed by the sequential addition of oxidized glutath ione
(1 m
M
) and NADPH (0.1 m
M
), with consumption of the
latter measured at 340 nm and 37 °C over t he period from
1.2 to 3.0 min after the addition of NADPH. LDH activity

was assessed in a similar manner, except using 0.05 UÆmL
)1
enzyme, with pyruvic acid (1 m
M
)andNADH(0.1m
M
)
added to the aliquots, and the loss of the latter monitored at
340 nm. GAPDH activity was assessed in 50 m
M
pyro-
phosphate buffer, pH 7.4, with 0.15 U mL
)1
enzyme and
15–100 l
M
peroxides. Aliquots were removed as indicated,
glyceraldehyde-3-phosphate (1 m
M
), NAD
+
(0.5 m
M
), and
sodium arsenate (25 m
M
, in water) added, and NADH
formation monitored at 340 nm. All experiments were
performed in duplicate or greater.
Enzyme inhibition induced by the peptide and protein

peroxides was compared to controls containing the c orres-
ponding nonoxidized substrate. Statistical analyses com-
paring multiple enzyme activities were performed using a
one-way
ANOVA
and Dunnett’s posthoc test. Other analyses
comparing multiple conditions were performed using a
one-way
ANOVA
and Newman–Keuls posthoc test. Where
Ó FEBS 2002 Enzyme inhibition by
1
O
2
-mediated protein peroxides (Eur. J. Biochem. 269) 1917
only one condition was compared to its corresponding
control a Student’s pooled two-sample t-test was used. In
all cases significance was assumed if P < 0.05.
RESULTS
Formation of peptide and protein peroxides
Photolysis of solutions containing rose bengal and N-Ac-
Trp-OMe, Gly-His-Gly, Gly-Tyr-Gly, lysozyme or RNase
A with visible light (k > 345 nm) in the presence of O
2
resulted in the generation of peroxides as detected by a
modified FOX assay [25,36]. Treatment of such samples
with catalase, after the cessation of illumination, resulted,
in most cases, in a very rapid initial decrease in peroxide
concentration, and a subsequent slow decay (Fig. 1). The
absolute levels of peroxide lost in the rapid phase after

addition of catalase, was substrate dependent (Fig. 1). The
fast initial loss is ascribed to the removal of H
2
O
2
generated during the photolysis (e.g [38]). The subsequent
slow decay is a ssigned to thermal decomposition of
peptide or protein peroxides ([25]; reviewed in [12,19,22]).
In the case of lysozyme (Fig. 1C) only thermal decompo-
sition of protein peroxides is evident, as no H
2
O
2
appears
to be formed during the photolysis. The presence of
peroxide groups on the proteins tested was confirmed by
the coelution of the FOX assay-positive material with the
protein containing fractions from size-exclusion chroma-
tography columns (data not shown). High concentrations
of catalase (£ 250 lgÆmL
)1
), and a 30-min preincubation
period, were employed in all subsequent experiments to
ensure complete, and rapid, removal of H
2
O
2
before the
peroxides were u sed in other experiments. Omission of the
rose bengal, photolysis in the absence of O

2
, or incubation
of complete samples in the absence of light, resulted in
peroxide concentrations of < 5 l
M
(data not shown).
Stability of peptide and protein peroxides
The decay of the peroxides generated on N-Ac-Trp-OMe,
Gly-Tyr-Gly, Gly-His-Gly, lysozyme and RNase A was
studied over time at 37 °C (Fig. 2). Addition of GAPDH, to
these incubations resulted in a significantly (P < 0.01)
enhanced rate of decay of the peroxides formed o n N-Ac-
Trp-OMe, Gly-Tyr-Gly, and Gly-His-Gly consistent with
reaction of these peroxides with this enzyme (Fig. 2 ).
Similar experiments with lysozyme - and RNase A -derived
peroxides did not yield statistically significant data (data not
shown). Experiments with LDH i n the place o f GAPDH,
and the same peroxides, did not result in a statistically
enhanced rate of decay of N-Ac-Trp-OMe and Gly-Tyr-Gly
peroxides (P > 0.05). An enhanced rate of decomposition
was detected with Gly-His-Gly peroxide (%30% after
30 min compared to 12% in controls, P < 0.01), though
this was much less marked than that observed with
GAPDH (data not shown). Analogous experiments were
not carried out with GR due to the quantity of material
required.
Treatment of and RNase A peroxides with NaBH
4
(approx. 20-fold molar excess relative to protein concentra-
tion) for 3 0 min at room temperature, and subsequent

separation of the treated protein from excess reductant by
PD-10 chromatography, resulted in the loss of > 97% of
the initial peroxides. This is in accord with previous studies
[25,26]. Treatment with Fe
2+
–EDTA, reduced glutathione
(GSH), ebselen and other thiols a lso rapidly removes such
peroxides (data not shown; A. Wright, C. L. Hawkins &
M. J. Davies, unpublished results; P. E. Morgan, R. T.
Dean & M. J. Davies, unpublished data). Similar studies
were not carried out with peptide-derived peroxides as
excess reductant, w hich interferes with the peroxide assay,
Fig. 1. Formation of H
2
O
2
and peptide and protein peroxides after
photo-oxidation with
1
O
2
generated by rose bengal i n the presence of
visible light and oxygen. (a) RNase A (50 mgÆmL
)1
); (b) N-Ac-Trp-
OMe (2.5 m
M
); and (c) lysozyme (50 mgÆmL
)1
)werephotolysedwith

visible light for 60 min in the presence of rose b engal (10 l
M
)with
continuous gassing with air at 4 °C. Immediately after the cessation of
photolysis catalase w as added to part of the sample (50 lgÆmL
)1
for
proteins, 250 lgÆmL
)1
for N-Ac-Trp-OMe) and peroxide levels
assayed at the indicated times using a modified FOX assay. (¤)
Catalase added; (h) no catalase added. Initial peroxide concentrations
were in the range of 420–520 l
M
for RNase A, 540–660 l
M
for
N-Ac-Trp-OMe, and 130–180 l
M
for lysozyme. Data are means
± S D; where no error bar is visible it is obscured by the symbol.
Statistical analysis between conditions was by a Student’s pooled two-
sample t-test, ** P < 0.01.
1918 P. E. Morgan et al. (Eur. J. Biochem. 269) Ó FEBS 2002
could not be readily removed from the reaction mixture,
and lysozyme was unable to be analysed as the protein
precipitated on addition of NaBH
4
.
Interaction of GAPDH thiols with peptide peroxides

The interaction of free thiol groups on GAPDH with N-Ac-
Trp-OMe peroxides was assessed by measurement of the
change in GAPDH thiol concentration on incubation with
this peroxide. Figure 3A shows that as the peroxide
concentration decreased, a concomitant, time-dependent,
decrease in thiol concentration was observed . The concen-
tration of thiols lost (24.9 ± 1.2 l
M
at 30 min), is approxi-
mately double that of the peroxides lost under identical
conditions (11.2 ± 1.1 l
M
at 30 min, Fig. 2A), consistent
with a stoichiometry of two thiol groups lost per peroxide
molecule consumed.
Further evidence for an interaction of the thiol groups on
GAPDH with N-Ac-Trp-OMe peroxides was obtained by
pretreatment of the GAPDH with NEM. This resulted in
Fig. 2. Thermal decay of peptide peroxides over time at 37 °Cinthe
absence or presence of added GAPDH. Peptide peroxide samples were
generated as described in Fig. 1 a nd the text. Im mediately after c es-
sation of photolysis catalase was added and the samples incubated for
30 min at room temperature. The residual peroxide levels after further
incubation at 37 °C, were m easured at the indicated times for either
untreated controls (d), or samples with added GAPDH (n;
1mgÆmL
)1
), using a modified FOX assay. (A) N-Ac-Trp-OMe per-
oxides; ( B) Gly-His-Gly peroxides; (C) Gly-Tyr-Gly peroxides. In all
cases the initial (postcatalase treatment) peroxide concentration in the

incubation mixtures was 20 l
M
. Data are means ± SD; where no error
bar is visible it is obscured by the symbol. Statistical analysis was by a
Student’s pooled two-sample t-test, ** P <0.01.
Fig. 3. (A) Loss of thiol groups present o n GAPDH on incubation with
N-Ac-Trp-OMe peroxides, and (B) Effect of blocking the free thiols
groups on GAPDH on loss of N-Ac-Trp-OMe peroxides. (A) GAPDH
(1 m gÆmL
)1
) was incubated with 20 l
M
N-Ac-Trp-OMe peroxides for
30 min at 37 °C, with the concentration of free GAPDH thiols mea-
sured by reaction with 5,5¢-dithiobis(2-nit robe nzoic a cid) at the indi-
cated times. (r) GAPDH in presence of N-Ac-Trp-OMe peroxides;
(h) GAPDH in the absence of added peroxide. Initially, 7 .3 ± 0.2 free
thiols p er GAPDH tetramer were detected. Data are means ± SD;
where no e rror bar is visible it is obscured by the symbol. Statistical
analysis was by a Student’s pooled two-sample t-test, ** P < 0.01.
(B) GAPDH was incubated for 30 min at room temp with, or without
NEM, followed by PD-10 column treatment to re-isolate the enzyme
and remove excess reagent (c ontrol, 42 ± 6% of thiols free; N EM-
treated, 20 ± 3% of thiols free). The re-isolated enzyme was then
incubated with 20 l
M
N-Ac-Trp-OMe peroxides at 37 °C, and the
residual peroxide levels measured at the indicated times using a modi-
fied FOX assay. (j) Perox ide loss observed in prese nce of control
(non-NEM treated) GAPDH ; (·) peroxide loss observed in presence of

NEM-treated GAPDH; ( d) peroxide l oss in absence of adde d GAP-
DH (cf. Fig. 2A). Data are means ± S D; where no error bar is visible
it is obscured by the symbol. Statistical analysis was by one-way
ANOVA with Newman–Keuls posthoc test; unlike letters indicate
statistically distinct results at the P <0.05level.
Ó FEBS 2002 Enzyme inhibition by
1
O
2
-mediated protein peroxides (Eur. J. Biochem. 269) 1919
the blocking of % 50% of the free thiols on the enzyme when
compared to controls. Complete blocking of all thiol groups
was not attempted a s the requirement for high concentra-
tions of NEM can result in other modifications [39].
Subsequent incubation of such NEM-treated GAPDH with
N-Ac-Trp-OMe peroxides resulted in a much slower, and
less dramatic, loss in peroxide concentration compared to
the non-NEM treated control (Fig. 3B), confirming that the
thiol groups on GAPDH play a role in the peroxide loss.
Similar experiments were not carried out with other
peroxides or enzymes.
Enzyme inhibition studies
Peroxides g enerated on N-Ac-Trp-OMe, Gly-Tyr-Gly,
Gly-His-Gly, lysozyme and RNase A were incubated with
GAPDH, GR, and LDH, in the presence and absence of
added Fe
2+
–EDTA and the residual enzymatic activity
determined (Fig. 4, Table 1). Lower concentrations of
these enzymes were employed in these studies, c ompared to

those reported above, to prevent substrate depletion.
Comparative studies were also carried out with H
2
O
2
.
The EDTA complex of Fe
2+
was employed to prevent
potential binding of Fe
2+
to the target enzymes; omission
of the EDTA resulted in less efficient enzyme inhib ition
(data not shown).
GAPDH w as rapidly inactivated, in a time-dependent
manner, by all t he peroxides tested, in both the absence and
presence of added Fe
2+
–EDTA. The rate of i nhibition of
GAPDH by N-Ac-Trp-OMe peroxides was concentration
dependent over the range tested (20–200 l
M
peroxide).
Incubation of GAPDH with nonphotolysed samples of the
peptide or proteins (with, o r without, 20 l
M
Fe
2+
–EDTA),
or with Fe

2+
–EDTA alone, resulted in slow loss of
enzyme activity, presumably owing to slow denaturation
(Fig. 4A).
GAPDH was readily inhibited by H
2
O
2
,whichwas
employed as a positive control, in either the presence, or
absence, of Fe
2+
–EDTA. Comparison of the data obtained
with H
2
O
2
and N-Ac-Trp-OMe peroxides showed that
fivefold higher concentrations of H
2
O
2
needed to be
employed to generate a similar r ate and extent of inhibition
(Fig. 4 B). Inhibition by protein-derived peroxides was
slower than that induced by the peptide peroxides at
Fig. 4. Inhibition of glyceraldehyde-3-phosphate dehydrogenase on incubation with H
2
O
2

, and peptide- and protein-peroxides, in the presence and
absence of added Fe
2+
–EDTA. GAPDH was incubated at 37 °C for 30 min with (a) N-Ac-T rp-OMe p eroxides (20 l
M
); (b) H
2
O
2
(100 l
M
); (c) Gly-
His-Gly peroxides (15 l
M
); (d) Gly-Tyr-Gly peroxides (20 l
M
); (e) RNase A peroxides (100 l
M
); and 120 m in with (f) lysozyme peroxides (20 l
M
).
Fe
2+
–EDTA (20 l
M
) was added w here indicated. Control samples co ntained equal concentrations of nonphotolysed materials, or wate r (in the
case of H
2
O
2

). Activity is expressed as a percentage of that of the nonphotolysed (nonperoxide containing) samples without added Fe
2+
–EDTA.
For further details see the Materials and methods. (·) Peroxide-containing samples in presence of added Fe
2+
–EDTA; (h) Peroxide-containing
samples in absence of added Fe
2+
–EDTA; (n) nonp hotolysed/non H
2
O
2
containing sample s in presence of added Fe
2+
–EDTA; (r) n onpho-
tolysed/non H
2
O
2
containing samples in ab sence of added Fe
2+
–EDTA. Statistical a nalyses (one-way ANOVA with Dunnett’s posthoc test)
compared all conditions t o the nonphoto lysed/non -H
2
O
2
control without added Fe
2+
–EDTA, ** P < 0.01. Where n o error bar is visible it is
obscured by the symbol.

1920 P. E. Morgan et al. (Eur. J. Biochem. 269) Ó FEBS 2002
identical peroxide concentrations demonstrating that per-
oxide size and/or electronic charge p lay a role in d etermin-
ing the rate of inhibition.
Identical studies using GAPDH with samples of RNase
A peroxides which had been pretreated with NaBH
4
to
remove peroxides (see above), gave similar extents of
inhibition to control, nonperoxide-containing RNase sam-
ples, confirming the requirement for peroxide groups for
enzyme inhibition.
GR was inhibited on incubation with N-Ac-Trp-OM e
peroxides, but only at the highest concentrations tested
(200 l
M
) (Table 1). This inhibition was not stimulated by
added Fe
2+
–EDTA. In contrast, rapid inhibition of GR by
H
2
O
2
was only observed in the presence of Fe
2+
–EDTA
(Table 1). LDH was not inhibited by the highest concen-
trations of peptide and protein peroxides (200 l
M

)tested,in
either the presence or absence of Fe
2+
–EDTA (data not
shown), whereas this enzyme was readily inhibited by H
2
O
2
in the presence, but not absence, of Fe
2+
–EDTA (Table 1).
As with GAPDH, a slow loss of enzyme activity was
observed, with both GR and LDH, in control samples; this
has been ascribed to slow thermal i nactivation.
Examination of the role of peroxide-derived radicals
in enzyme inhibition
To examine whether radical species were generated during
the inactivation of GAPDH and L DH by peroxides in the
absence of added metal ions, GAPDH (24 mgÆmL
)1
)and
LDH (6 m gÆmL
)1
) were incubated with the spin trap PBN
(9.4 m
M
)andN-Ac-Trp-OMe peroxides or H
2
O
2

(both
200 l
M
) for extended periods and examined by EPR
spectroscopy. No radical adducts were detected above those
detected in controls. Experiments were not carried out with
GR owing to the quantity of material required. Previous
studies have demonstrated the formation of radicals from
these peptide and protein peroxides in the presence of Fe
2+
–
EDTA ([25]; A. Wright, C. L. Hawkins & M. J. Davies,
unpublished results).
Prevention of enzyme inhibition induced by peptide
peroxides
A number of compounds protected GAPDH or GR against
inactivation when these materials were coincubated with the
enzymes and N-Ac-Trp-OMe p eroxides or H
2
O
2
(Table 2).
GSH, N-(2-mercaptopropionyl)glycine, ascorbic acid and
dithiothreitol (all 200 l
M
) all offered highly significant
protection against the inhibition of GAPDH induced by
20 l
M
N-Ac-Trp-OMe peroxides in the presence of 20 l

M
Fe
2+
–EDTA (Table 2, cf. Figure 4A). Methionine, at an
identical concentration, had a much less marked, although
still statistically significant, effect. Trolox C was i neffective.
All the compounds tested showed a significant protective
effect at 2 m
M
in the LDH/Fe
2+
–EDTA/H
2
O
2
system
(Table 2). In some cases, inclusion of these compounds in
control samples resulted in minor changes in enzyme
activity. Thus Trolox C caused a significant decrease in
GR activity (P < 0.05), whilst blank experiments with
added N-(2-mercaptopropionyl)glycine ( 2MPG) resulted in
a significant increase in GR activity compared to the
absence of this compound (P < 0.05). The latter effect is
attributed to re-activation of inactive enzyme present in the
sample.
DISCUSSION
Exposure of amino acids, peptides and proteins to radiation
(ionizing, UV, or visible light in the presence of a
photosensitiser) in the presence of O
2

, gives rise to peroxides
[25–27,38]. With
1
O
2
, peroxides are formed primarily on
Tyr, Trp and His side-chains [12,19,24,25,40]. Peroxides
formed on N-Ac-Trp-OMe are primarily located at the C-3
site on the indole ring, and those on Gly-His-Gly and Gly-
Tyr-Gly at ring positions on the His and Tyr side-chains,
respectively ([19,23,24,40]; A. Wright, C. L. Hawkins &
M. J. Davies, unpublished results). The location of such
peroxides on proteins has yet to be fully determined.
Recent s tudies have shown that protein peroxides a re also
Table 1. Inhibition of glutathione reductase by H
2
O
2
and N-Ac-Trp-OMe peroxides, and lactate dehydrogenase by H
2
O
2
, in the presence and absence
of added Fe
2+
–EDTA. Samples c ontaining glutathione reductase (0.025 UÆmL
)1
) were incubated at 37 °Cfor120minwithN-Ac-Trp-OMe
peroxides (200 l
M

)andH
2
O
2
(200 l
M
). Samples containing lactate dehydrogenase (0.05 UÆmL
)1
) were incubated at 37 °Cfor30minwithH
2
O
2
(200 l
M
). 20 l
M
Fe
2+
–EDTA was present where indicated. Control solutions contain ed equal concentration s of nonphotolysed N-Ac-Trp-OMe,
or water in the case of H
2
O
2
. Activity is expressed as a percentage of that of the nonphotolysed/non H
2
O
2
containing samples without added Fe
2+
–

EDTA. Statistical analyses (one-way
ANOVA
with Dunnett’s p osthoc test ) compared all conditions to the nonphotolysed N-Ac-Trp-OMe/H
2
O
control w ithout added Fe
2+
–EDTA; * P < 0.01.
Enzyme Added agents Initial control activity (%)
GR N-Ac-Trp-OMe (control) 87 ± 1
N-Ac-Trp-OMe + Fe
2+
–EDTA 84 ± 1
N-Ac-Trp-OMe + peroxides 68 ± 1*
N-Ac-Trp-OMe + peroxides + Fe
2+
–EDTA 62 ± 4*
GR H
2
O (control) 99 ± 1
H
2
O+Fe
2+
–EDTA 97 ± 4
H
2
O
2
98 ± 2

H
2
O
2
+Fe
2+
–EDTA 23 ± 5*
LDH H
2
O (control) 72 ± 7
H
2
O+Fe
2+
–EDTA 70 ± 6
H
2
O
2
69 ± 2
H
2
O
2
+Fe
2+
–EDTA 10 ± 1*
Ó FEBS 2002 Enzyme inhibition by
1
O

2
-mediated protein peroxides (Eur. J. Biochem. 269) 1921
generated in cells on exposure to
1
O
2
(A. Wright, C. L.
Hawkins & M. J. Davies, unpublished results) or peroxyl
radicals [41].
This study has shown, for the first time, that low
concentrations of
1
O
2
-generated peptide- and protein-per-
oxides can transmit damage from the initial site of oxidation
to other cellular targets, and hence bring about chain
oxidation reactions where oxidative damage to on e protein
can r esult i n damage to multiple targets. It has been shown
that these protein peroxides can inhibit GAPDH, which is a
key cellular glycolytic enzyme, and GR, which r ecycles
oxidized glutathion e and thereby maintains reducing equi-
valents within the cell. Other enzymes, such as LDH, are
unaffected, so such damage is selective. The long lifetime of
these protein peroxides may allow these species to diffuse
considerable distances from their site of formation, and
hence induce damage at remote sites. The concentrations of
peptide and protein peroxides that induce inhibition of
GAPDH are similar to those which we have recently
detected (% 20 l

M
) on proteins in viable rose-bengal loaded
THP-1 cells exposed to visible light (A. Wright, C. L.
Hawkins & M. J. Davies, unpublished r esults).
Previous studies (reviewed in [42]) have shown that
GAPDH is rapidly, and specifically, inhibited in m yocytes,
aortic endothelial and U937 (pro-monocyte) cells on
exposure to H
2
O
2
in the absence of added metal ions [43–
45]. This inactivation arises via direct reaction of H
2
O
2
with
a particularly reactive Cys residue (Cys149), which has a
pK
a
of 5.4 owing to interaction with His176, in the a ctive
site o f the e nzyme. This process gives a sulfenic acid (R-S-
OH), which can be repaired by dithiothreitol. The isolated
enzyme can also be inhibited by UV light [46], n itric oxide
[37], superoxide radicals [42], ozone [47] and tert-butyl
hydroperoxide [48]. Inhibition can also arise via radical
Table 2. Percentage of enzyme activity retained after incubation of GAPDH, GR and LDH with N-Ac-Trp-OMe peroxides or H
2
O
2

at 37 °Cinthe
absence, or presence, of a 10-fold excess (over peroxide concentration) of putative antioxidant. Samples containing GAPDH (0.15 UÆmL
)1
)orLDH
(0.05 UÆmL
)1
) were incubated for 30 min; those containing GR (0.025 UÆmL
)1
) for 120 min. A ll incubations contained 20 l
M
Fe
2+
–EDTA.
Control samples contained nonphoto lysed N-Ac-Trp-OMe or H
2
O as appropriate. Statistical analyses (one-way ANOVA with Dunnett’s posthoc
test) compared all conditions to the nonperoxide containing controls; * P <0.01,**P < 0.05.
Enzyme Added agents Initial control activity (%)
GAPDH + Fe
2+
–EDTA N-Ac-Trp-OMe (control) 86 ± 2
N-Ac-Trp-OMe peroxides 14 ± 2*
N-Ac-Trp-OMe peroxides + GSH 83 ± 1
N-Ac-Trp-OMe peroxides + 2MPG 80 ± 1
N-Ac-Trp-OMe peroxides + Methionine 25 ± 4*
N-Ac-Trp-OMe peroxides + Trolox C 13 ± 1*
N-Ac-Trp-OMe peroxides + Dithiothreitol 87 ± 1
N-Ac-Trp-OMe peroxides + Ascorbic Acid 73 ± 3*
GAPDH + Fe
2+

–EDTA H
2
O (control) 67 ± 5
H
2
O
2
7±3*
H
2
O
2
+ GSH 59 ± 2
H
2
O
2
+ 2MPG 69 ± 5
H
2
O
2
+ Methionine 2 ± 1*
H
2
O
2
+ Trolox C 4 ± 1*
H
2

O
2
+ Dithiothreitol 100 ± 3*
H
2
O
2
+ Ascorbic Acid 64 ± 4
GR + Fe
2+
–EDTA N-Ac-Trp-OMe (control) 88 ± 3
N-Ac-Trp-OMe peroxides 45 ± 1*
N-Ac-Trp-OMe peroxides + GSH 74 ± 4**
N-Ac-Trp-OMe peroxides + 2MPG 93 ± 2
N-Ac-Trp-OMe peroxides + Methionine 52 ± 2*
N-Ac-Trp-OMe peroxides + Trolox C 44 ± 10*
N-Ac-Trp-OMe peroxides + Dithiothreitol 93 ± 3
GR + Fe
2+
–EDTA H
2
O (control) 97 ± 4
H
2
O
2
23 ± 15*
H
2
O

2
+ GSH 88 ± 5
H
2
O
2
+ 2MPG 109 ± 7
H
2
O
2
+ Methionine 109 ± 6
H
2
O
2
+ Trolox C 80 ± 1
H
2
O
2
+ Dithiothreitol 55 ± 1*
LDH + Fe
2+
–EDTA H
2
O (control) 70 ± 6
H
2
O

2
10 ± 1*
H
2
O
2
+ GSH 99 ± 8
H
2
O
2
+ 2MPG 102 ± 10**
H
2
O
2
+ Methionine 95 ± 18
H
2
O
2
+ Trolox C 112 ± 9*
H
2
O
2
+ Dithiothreitol 91 ± 5
H
2
O

2
+ Ascorbic Acid 70 ± 6
1922 P. E. Morgan et al. (Eur. J. Biochem. 269) Ó FEBS 2002
reactions (e.g. involving HO
Æ
[46]. or O
2
– Æ
[42,49]) that
involve oxidation of Cys-149 to cysteic acid [47].
GR also contains an active site Cys r esidue [50]. GR is
less-readily inhibited than GAPDH by H
2
O
2
, and loss of
activity has been reported to require metal ions, be radical-
mediated, involve oxidation of other residues in addition to
the active site Cys (e.g. His467, Tyr114 and Trp residues
[50]), and result in the formation of carbonyl groups [50–52].
A preliminary report has appeared on the inhibition of GR
by radiation-generated peroxides [34]. LDH has been shown
to be inhibited by a number of oxidants, with this requiring
the p resence of metal ions [53], but is less sensitive to
inhibition than GAPDH [42]. A similar pattern appears to
hold with t he peptide and protein peroxides investigated in
the current study, with GAPDH being more sensitive than
GR and LDH, and inactivation of G APDH being metal-
ion independent, whereas inhibition of GR and LDH by
H

2
O
2
occurs most rapidly in the presence of metal ions.
It is proposed that the inhibition of GAPDH and GR by
these
1
O
2
-generated peptide and protein peroxides occurs
via direct (nonradical) oxidation of the active site Cys
residues (i.e. reaction 1, see below). This is s upported by the
observations, with GAPDH, that thiol group loss occurs
with similar kinetics to peroxide loss, and that blocking of
% 50% of the thiol groups on the enzyme inhibits peroxide
loss. Furthermore removal of the peroxide groups by
reduction p revents enzyme inhibitio n. Such a mechanism is
also supported by the inefficient inhibition of LDH by these
peroxides, in the absence of metal ions, as this enzyme does
not contain an active site thiol. The nonradical nature of the
GAPDH and GR inhibitio n in the absence of metal ions is
confirmed by the E PR studies where no r adicals were
detected; previous studies have shown that radicals formed
from these p eroxide can be detected using the methodology
employed when peroxide formation is stimulated with metal
ions [25,32,33]. Though it is possible that the inactivation of
GAPDH and GR occurs via oxidation of nonactive site
residues, that bring about loss of functional integrity, the
stoichiometry of inactivation ( i.e. the loss of two molecules
of thiol per molecule of peroxide consumed) suggests that

the inactivating reaction(s) are highly specific. This is
inconsistent with a radical-mediated process. This stoichi-
ometry detected with GAPDH is consistent with the
occurrence of both reaction 1 and subsequent reaction of
the s ulfenic acid formed with a second thiol to give a
disulfide bond (reaction 2). Previous studies have provided
direct evidence for the formation of intramolecular disulfide
bonds du ring oxidation of GAPDH between the active site
thiol Cys149 and a further thiol residue, Cys153, which is in
very close proximity to the former species [37,54,55]. No
direct evidence for the formation of such a disulfide has been
obtained in the current study, but such a mechanism seems
highly likely on the basis of the data obtained. In contrast to
this direct (nonradical) inactivation, inhibition of GR and
LDH by H
2
O
2
is believed to occur via radical-mediated
reactions, catalysed by the added Fe
2+
–EDTA.
Enzyme-SH þ Peptide-/Protein-OOH ! Enzyme-S-OH
þ Peptide-/Protein-OH ð1Þ
Enzyme-S-OH þ Enzyme-SH ! Enzyme-S-S-Enzyme
ð2Þ
Previous studies have shown that some Met residues also
react with hydroperoxides, to give the sulfoxide, with
concomitant reduction of the hydroperoxide to the alcohol
(e.g [56,57]). It is therefore possible that

1
O
2
-generated
peptide and protein peroxides may also inhibit enzymes
containing critical Met residues. These previous observa-
tions are consistent with the statistically significant protec-
tive effect offered by f ree Met in the i nhibition experiments
carried out with N-Ac-Trp-OMe peroxides and GAPDH
(cf. Table 2, P <0.01byone-way
ANOVA
with Dunnett’s
posthoc test, when the Met treated sample was compared to
photolysed N-Ac-Trp-OMe with added Fe
2+
–EDTA).
However the extent of protection afforded by Met was
much less marked that that seen with the thiol compounds
and ascorbate at equimolar concentrations, suggesting that
reaction of these peptide and protein peroxides with Met
residues is kinetically uncompetitive when compared with
reaction with activated (low pKa) Cys residues.
A previous study has shown that peroxides formed by
1
O
2
on peptides and proteins a re not removed by catalase
[25], and this has b een confirmed in the present study.
These peroxides are also likely to be poor substrates for the
glutathione peroxidase family, as a result of their steric

bulk, and the buried position of most Tyr, His and Trp
residues in proteins. This hypothesis is supported by a
previous report that showed that radiation-generated
protein peroxides are not removed rapidly by this enzyme,
though some amino-acid peroxides are [27,29]. Reaction
with low-molecular-mass reducing agents a nd antioxidants
is therefore likely t o be t he major r oute for the removal of,
or protection against, such peroxides in cells [26,29]. The
studies reported here show that thiols can ameliorate
inactivation of GAPDH induced by these
1
O
2
-generated
peptide and protein peroxides, presumably by acting as
sacrificial targets. This is in accord with the known rapid
depletion of GSH and other thiols (both low-molecular-
mass and protein-bound) in photo-oxidized cells, and that
maintenance of thiol levels offers protection [58–61].
Similarly, it has been shown that ascorbate and thiols
can readily remove radiation-generated peptide and p rotein
peroxides [26,27,29]. It has also been sh own that over-
expression, in human fibroblast cells, of the enzyme
thioredoxin, which maintains low-molecular-mass thiols
in a reduced form, protects cells against photo-oxidative
damage and cell death [62,63]. Whether the p rotection
offered by thiols is o wing to direct scavenging of
1
O
2

,
removal of peroxides (H
2
O
2
and/or protein), or repair of
reversibly damaged targets, such as the enzymes investi-
gated here, r emains to be established.
ACKNOWLEDGEMENTS
The authors a re grateful to the Australian Research Council and the
Juvenile Diabetes Foundation International for financial support, and
to Dr Clare Hawkins for helpful discussions.
REFERENCES
1. Rosen, H. & Michel, B.R. (1997) Redundant contribution of
myeloperoxidase-dependent systems to neutrophil-mediated kill-
ing of Escherichia coli. Infect. Immun. 65, 4173–4178.
2. Kiryu, C., Makiuchi, M., Miyazaki, J., Fujinaga, T. &
Kakinuma, K. ( 1999) P hysiological production of singlet mole-
Ó FEBS 2002 Enzyme inhibition by
1
O
2
-mediated protein peroxides (Eur. J. Biochem. 269) 1923
cular oxygen in the myeloperoxidase-H
2
O
2
-chloride system. FEBS
Lett. 443, 154–158.
3. Kanofsky, J.R., Hoogland, H., Wever, R. & Weiss, S.J. (1988)

Singlet o xygen production by human eosinophils. J. Biol. Chem.
20, 9692–9696.
4. Valenzeno, D.P. (1987) Photomodification of biological mem-
branes with emphasis on singlet oxygen mechanisms. Photochem.
Photobiol. 46, 147–160.
5. Teixeira, M.M., Cunha, F.Q., N oronha-Dutra, A. & Hothersall,
J. (1999) Production of singlet oxygen by eosinophils activated
in vitro by C5a and leukotriene B4. FEBS L ett. 453, 2 65–268.
6. Steinbeck, M.J., Khan, A.U. & Karnovsky, M.J. (1993) Extra-
cellular production o f singlet oxygen by s timulated macrophages
quantified using 9,10-diphenylanthracene and perylene in a poly-
styrene film. J. Biol. C hem. 268, 15649–15854.
7. Sohal, R.S. & Weindruch, R. (1996) Oxidative stress, caloric
restrict ion , and aging. Science 273, 59–63.
8. Black, H.S., deGruijl, F.R., Forbes, P.D., Cleaver, J.E., Anan-
thaswamy, H.N., deFabo, E.C., Ullrich, S.E. & Tyrrell, R.M.
(1997) Photocarcinogenesis: an overview. J. Photochem. Photobiol.
B. 40, 29–47.
9. Dean, R.T., Fu, S., Stocker, R. & Davies, M.J. (1997) Biochem-
istry and pathology of radical-mediated protein oxidation. Bio-
chem. J. 324, 1–18.
10. Ortwerth, B.J., Casserly, T.A. & O lesen, P.R. (1998) Singlet oxy-
gen production correlates with His and Trp destruction in
brunescent cataract water-insoluble p roteins. Exp. Eye Res. 67,
377–380.
11. Stadtman, E .R. & Berlett, B.S. (1998) Reactive oxygen-mediated
protein oxidation in aging and disease. Drug Metab. Rev. 30,225–
243.
12. Davies, M.J. & Truscott, R.J.W. (2001) Photo-oxidation o f pro-
tein and its role in cataractogenesis. J. Photochem. Photobiol. B.

63, 114–125.
13. Lutgerink, J.T., van der Akker, E., Smeets, I., Pachen, D., van
Dijk, P., Aubry, J M., Joenje, H., Lafleur, M.V.M. & Retel, J.
(1992) Interaction of singlet oxygen with DNA and biological
consequence s. Mutat. Res. 27 5, 377–386.
14. Cadet, J., Berger, M., Decarroz, C., Wagner, J.R., Van Lier, J.E.,
Ginot, Y.M. & Vigny, P. (1986) Photosensitised reactio ns of
nucleic acids. Biochemie 68 , 813–834.
15. Vever-Bizet, C., Dellinger, M., Brault, D., Rougee, M. & Ben-
sasson, R.V. (1989) Singlet molecular oxygen q uenching by satu-
rated and unsaturated fatty-acids and by cholesterol. Photochem.
Photobiol. 50, 321–325.
16. Girotti, A.W. (1992) Photosensitized oxidation of cholesterol in
biological systems: reaction pathways, cytotoxic effects an d defense
mechanisms. J. Photochem. Photobiol. B: Biol. 13 , 105–118.
17. Wagner,J.R.,Motchnik,P.A.,Stocker,R.,Sies,H.&Ames,B.N.
(1993) The oxidation of blood plasma and low density lipoprotein
components by chemically generated singlet oxygen. J. Biol.
Chem. 268, 18502–18506.
18. Matsuo, I., Yoshino, K. & Ohkido, M. (1983) Mechanism of skin
surface lipid peroxidation. Curr. Probl. Dermatol. 11, 135–143.
19. Straight, R.C. & Spikes, J.D. (1985) Photosensitized oxidation of
biomolecules. In Singlet O
2
(Frimer, A.A., ed.), pp. 91–143. CRC
Press, Boca Raton, FL, USA.
20. Bensasson, R.V., Land, E.J. & Truscott, T.G. (1993) Excited
States and Free Radicals in Biology and Medicine. Oxford Uni-
versity Press, Oxford, U K.
21. Wilkinson, F., Helman, W.P. & Ross, A.B. (1995) Rate constants

for the decay and reactions o f the lowest el ectronically excited state
of m olecular oxygen in solution. An expanded and revised com-
pilation. J. Phys. Chem. Ref. Data. 24, 663–1021.
22. Saito, I., Matsuura, T., Nakagawa, M. & Hino, T. (1977) Per-
oxidic intermediates in photosensitized oxygenation of tryptophan
derivatives. Acc. Chem. Res. 10 , 346–352.
23. Jin, F .M., Leitich, J. & von Sonntag, C. (1995) The photolysis
(k ¼ 254 nm) of tyrosine in aqueous solutions in the absence and
presence of oxygen – the reaction of tyrosine with singlet oxygen.
J. Photochem. Pho tobiol. A: Chem. 92, 147–153.
24. Tomita, M., Irie, M. & Ukita, T. (1969) Sensitized photooxidation
of histidine and its derivatives. Products and mechanism of the
reaction. Biochemist ry 8, 5149–5160.
25. Wright, A., Hawkins, C.L. & Davies, M.J. (2000) Singlet oxygen-
mediated protein oxidation: evidence for the formation of reactive
peroxides. Redox Report 5, 159–161.
26. Simpson, J.A., Narita, S., Gieseg, S., Gebicki, S., Gebicki, J.M. &
Dean, R.T. (1992) Long-lived reactive species o n free-radical-
damaged proteins. Biochem. J. 28 2 , 621–624.
27. Gebicki, J.M. (1997) Protein hydroperoxides as new reactive
oxygen species. Redox Report 3, 99–110.
28. Hawkins, C.L. & Davies, M.J. (2001) Generation and propagation
of radical reactions on proteins. Biochim. Biophys. Acta. 1504,
196–219.
29. Fu,S.,Gebicki,S.,Jessup,W.,Gebicki,J.M.&Dean,R.T.(1995)
Biological fate of amino acid, peptide and protein hydroperoxides.
Biochem. J. 311, 821–827.
30. Luxford, C., Morin, B., Dean, R.T. & Davies, M.J. (1999)
Histone H1- and other protein- and amino acid-hydroperoxides
can give rise to free radicals which oxidize DNA. Biochem. J. 344,

125–134.
31. Luxford, C., Dean, R.T. & Davies, M.J. (2000) Radicals derived
from h istone hydroperoxides damage nucleobases in RNA and
DNA. Chem. Res. T oxicol. 13, 665–672.
32. Davies, M.J., Fu, S. & Dean, R.T. (1995) Protein hydroper-
oxides can give rise to reactive free radicals. Biochem. J. 305,
643–649.
33. Davies, M.J. (1996) Protein and peptide alkoxyl radicals can give
rise to C-terminal decarboxylation and backbone cleavage. Arch.
Biochem. Biophys. 336, 163–172.
34. Gebicki, S., Dean, R.T. & Gebicki, J.M. (1996) Inactivation
of glutathione reductase by protein and amino acid peroxides.
In Oxidative Stress and Redox Regulation: Cellular Signalling,
AIDS, Cancer and Other Diseases. pp. 139. Institute Pasteur,
Paris, France.
35. Soszynski, M., Filipiak, A., Bartosz, G. & Gebicki, J.M. (1996)
Effect o f amino acid peroxides on the Erythrocyte. Free Radic.
Biol. Med. 20, 45–51.
36. Gay, C., Collins, J. & Gebicki, J.M. (1999) Hydroperoxide assay
with the ferric-xylenol orange comple x. Anal. Biochem. 273,149–
155.
37. Ishii, T., Sunami, O., Nakajima, H., Nishio, H., Takeuchi, T. &
Hata, F. (1999) Critical role of sulfenic acid formation of thiols in
the inactivation of glyceraldehyde-3-phosphate dehydrogenase by
nitric oxide. Biochem. Pharmacol. 58, 133–143.
38. Silvester, J.A., Timmins, G.S. & Davies, M.J. (1998) Protein
hydroperoxides and carbonyl groups generated by porphyrin-
induced photo-oxid ation of bovine serum albumin. Arch.
Biochem. Biophys. 350, 249–258.
39. Means, G.E. & Feeney, R.E. (1971) Chemical Modification of

Proteins. Holden-Day, San Francisco, CA, USA.
40. Michaeli, A. & Feitelson, J. (1994) Reactivity of singlet oxygen
towardaminoacidsandpeptides.Photochem. Photobiol. 59,284–
289.
41. Gieseg, S., Duggan, S. & Gebicki, J.M. (2000) Peroxidation of
proteins before lipids in U937 cells exposed t o peroxyl radicals.
Biochem. J. 350, 215–218.
42. Armstrong, D.A. & Buchanan, J.D. (1978) Reactions of O
À
2
Æ,
H
2
O
2
and other oxidants with sulfhydryl enzymes. Photochem.
Photobiol. 28, 743–755.
43. Janero, D.R., Hreniuk, D. & Sharif, H.M. (1994) Hydroperoxide-
induced oxidative stress impairs heart muscle cell carbohydrate
metabolism. Am. J. Physiol. 266, C179–C188.
1924 P. E. Morgan et al. (Eur. J. Biochem. 269) Ó FEBS 2002
44. Ciolino, H.P. & Levine, R.L. (1997) Modification of p roteins in
endothelial cell death during oxidative stress. Free Radic. Biol.
Med. 22, 1277–1282.
45. Colussi, C., Albertini, M.C., Coppola, S., Rovidati, S., Galli, F. &
Ghibelli, L . (2000) H
2
O
2
-induced block of glycolysis as an active

ADP-ribosylation reaction protecting cells from apoptosis.
FASEB J. 14, 2266–2276.
46. Hu, M.L. & Tappel, A.L. (1992) Potentiation of oxidative damage
to proteins by ultraviolet-A and protection by antioxidants.
Photochem. Photobiol. 56, 357–363.
47. Knight, K.L. & Mudd, J.B. (1984) The reaction of ozone with
glyceraldehyde-3-phosphate dehydrogenase. Arch. Biochem. Bio-
phys. 229, 259–269.
48. Van der Zee, J., Van Steveninck, J., K oster, J.F. & Dubbelman,
T.M. (1989) Inhibition of e nzymes and o xidative damage of re d
blood cells induced by t-butylhydroperoxide-derived radicals.
Biochim. Biophys. Acta. 980, 175–180.
49. Buchanan, J.D. & Armstrong, D.A. ( 1978) The radiolysis o f gly-
ceraldehyde-3-phosphate dehydrogenase. Int. J. Radiat. Biol. 33,
409–418.
50. Gutierrez-Co rrea, J. & Stoppani , A.O. (1997) Inactiva tion of yeast
glutathione reductase by Fenton systems: effect of metal chelators,
catecholamines and thiol compounds. Free Ra dic. Res. 27, 543–
555.
51. Tabatabaie, T. & Floyd, R.A. (1994) Susceptibility of glutathione
peroxidase and glutathione reductase to oxidative damage and the
protective effect o f spin t rapping agents. Arch. Biochem. Biophys.
314, 112–119.
52. Ogino, T. & Okada, S. (1995) Oxidative damage of bovine serum
albumin and other enzyme proteins by iron-chelate complexe s.
Biochim. Biophys. Acta. 1245, 359–365.
53. Dicker, E. & Cederbaum, A.I. (1988) Increased oxygen radical-
dependent inactivation of metabolic enzymes by liver microsomes
after chronic ethanol consumption. FASEB. J. 2, 2901–2906.
54. Ehring, R. & Colowick, S.P. (1969) The two-step formation

and inactivation of acylphosphatase by agents acting on
glyceraldehyde ph osphat e dehydrogenase. J. Biol. Chem. 244,
4589–4599.
55. Wassarman, P.M. & Major, J.P. (1969) The reactivity of the
sulfhydryl groups of lobster muscle glyceraldehyde 3-phosphate
dehydrogenase. Biochemistry 8, 1076–1082.
56. Garner, B., Witting, P.K., Waldeck, A.R., Christison, J.K., Raf-
tery, M. & Stocker, R. (1998) Oxidation of high density lipopro-
teins. I. Formation of methionine sulfoxide in apolipoproteins AI
and AII is an early event that accompanies lipid peroxidation and
can be enhanced by alpha-tocopherol. J. Biol. Chem. 27 3, 6080–
6087.
57. Garner, B., Waldeck, A .R., Witting, P.K., Rye,K A. &Stocker, R.
(1998) Oxidation of high density lipoproteins. II. Evidence for
direct reduction of lipid hydroperoxides by methionine residues of
apolip op rot ein s AI and AII. J. Biol. Chem. 273, 6088–6095.
58. Truscott, R.J. & Augusteyn, R.C. (1977) The state of sulfydryl
groups in normal and cataractous human lenses. Exp. Eye Res. 25,
139–148.
59. Garner, M.H. & Spector, A. (1980) Selective oxidation of cysteine
and methionine in normal and senile cataractous lenses. Proc. Nat l
Acad.Sci.USA77, 1274–1277.
60. Dillon, J., Roy, D. & Spector, A. (1985) The photolysis of lens
fiber membranes. Exp. Eye R es. 41, 53–60.
61. Sweeney, M.H. & Truscott, R.J. (1998) An impediment
to glutathione diffusion in older normal human lenses: a
possible precondition for nuclear cataract. Ex p. Eye Res. 67,
587–595.
62. Didier, C., Pouget, J.P., Cadet, J., Favier, A., Beani, J.C. &
Richard, M.J. (2001) Modulation o f e xogenous and endogenous

levels of thioredoxin in human skin fibroblasts prevents DNA
damaging effect of ultraviolet A radiation. Free Radic. Biol. Med.
30, 537–546.
63. Didier, C., Kerblat, I., Drouet, C., Favier, A., Beani, J. & Richard,
M. (2001) Induction of thioredoxin by ultraviolet-A radiation
prevents oxidative- mediated cell death in human skin fibroblasts.
Free Radic. Biol. Med. 31, 585–598.
Ó FEBS 2002 Enzyme inhibition by
1
O
2
-mediated protein peroxides (Eur. J. Biochem. 269) 1925