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-

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

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

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

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

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

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

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

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

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

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

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

). 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.

)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

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

)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+
–

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

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

–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

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
+ 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

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

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

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.

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,

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

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