Davies1 1 EPR and2Cell Biology Groups, The Heart Research Institute, Sydney, New South Wales, Australia Reaction of certain peptides and proteins with singlet oxygen generated by visible
Trang 1Inhibition of glyceraldehyde-3-phosphate dehydrogenase by peptide and protein peroxides generated by singlet oxygen attack
Philip E Morgan1, Roger T Dean2and Michael J Davies1
1
EPR and2Cell 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 that this inhibition is concentration,
peroxide, and time dependent, with H2O2less efficient than
some peptide peroxides Enzyme inhibition correlates with
loss of both the peroxide and enzyme thiol residues, with a
stoichiometry of two thiols lost per peroxide consumed
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 the enzyme and
the peroxide A number of low-molecular-mass compounds
including thiols and ascorbate, but not Trolox C, can pre-vent inhibition by removing the initial peroxide, or species derived from it In contrast, glutathione reductase and lac-tate dehydrogenase are poorly inhibited by these peroxides
in the absence of added Fe2+–EDTA The presence of this metal-ion complex enhanced the inhibition observed with these enzymes consistent with the occurrence of radical-mediated reactions Overall, these studies demonstrate that singlet oxygen-mediated damage to an initial target protein can result 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 of cellular dys-function as a result of photo-oxidation
Keywords: protein oxidation; protein peroxides; protein radicals; singlet oxygen; photo-oxidation
Singlet oxygen (molecular oxygen in its1Dg state; 1O2) 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.1O2
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,1O2has
been suggested to play a key role in the development of a
number of human pathologies including cataract, sunburn,
some skin cancers and aging [7–12]
1O2reacts 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, His and Tyr residues has been shown to yield peroxides, although the structure of some of these materials remains 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] 1O2-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 Wright, 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 results)
Previous studies with protein peroxides generated by high-energy radiation (e.g c-sources, X-rays), metal 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: m.davies@hri.org.au
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;1O 2 , molecular oxygen in its first excited singlet
(1D 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 November 2001, revised 12 February 2002, accepted 20
February 2002)
Trang 2antioxidants 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 and
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 to1O2-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),
by1O2-generated peroxides formed on peptides and proteins
These data have been compared with those obtained using
H2O2 The role of radical vs nonradical processes has been
investigated, as has the prevention of such damage
M A T E R I A L S A N D M E T H O D S
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 3.6M
(NH4)2SO4, pH 7.0], GAPDH (rabbit muscle, lyophilized
powder) and LDH [rabbit muscle, in 3.2 M (NH4)2SO4,
pH 6.0, or lyophilized powder] were from Sigma GAPDH
[rabbit muscle, in 3.2M (NH4)2SO4+ 0.1 mM EDTA,
pH 7.5], NADPH (tetrasodium salt), NAD+ (free acid),
NADH (disodium salt) were from Boehringer Mannheim
These preparations did not contain materials that interfered
with peroxide formation or enzyme 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 Fe2+–EDTA (1 : 1 complex) were prepared using
de-oxygenated water and maintained under oxygen-free N2
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 mM), 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 lM 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, catalase (Sigma, bovine liver, 5 lgÆmL)1for
BSA, 50 lgÆmL)1for lysozyme and RNase A, 250 lgÆmL)1
for peptides) was added, unless stated otherwise, to remove
H2O2 and the samples incubated for 30 min at room
temperature before freezing ()80 °C) in aliquots
Peroxide concentrations were determined by a modified
FOX (FeSO4/xylenol orange) assay, using H2O2standards
[36] This assay gives similar 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 over protein concentration
for 30 min at room temperature Control samples were incubated under identical conditions Immediately 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 A280values)
Thiol groups on GAPDH (2 mgÆmL)1) were blocked 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 mgÆmL)1GAPDH with 500 lM 5,5¢-dithiobis(2-nitro-benzoic acid) (in 100 mM 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 600M )1Æcm)1 [37]
Electron paramagnetic resonance (EPR) samples were prepared by addition of peroxide (200 lM) to the enzyme in the presence of the spin trap N-t-butyl-a-phenylnitrone (PBN) (9.4 mMin 50 mMphosphate buffer, pH 7.4) Fe2+– EDTA (100 lM, 1 : 1 complex) was added where stated Samples were incubated for 5 min at 20°C before exami-nation in a standard, flattened, aqueous solution cell (WG-813-SQ; Wilmad, Buena, 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· 106, 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 power 25.0 mW, frequency 9.7 GHz, with four acquisitions aver-aged
GR activity was examined at 37°C in 50 mMphosphate buffer, pH 7.4, containing 0.025 UÆmL)1GR, 20 lMFe2+– EDTA (where indicated), peroxide (20–200 lM), and antioxidant (10-fold excess over peroxide concentration) Aliquots were removed as indicated, and residual activity assayed by the sequential addition of oxidized glutathione (1 mM) and NADPH (0.1 mM), with consumption of the latter measured at 340 nm and 37°C over the 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 mM) and NADH (0.1 mM) added to the aliquots, and the loss of the latter monitored at
340 nm GAPDH activity was assessed in 50 mM pyro-phosphate buffer, pH 7.4, with 0.15 U mL)1enzyme and 15–100 lMperoxides Aliquots were removed as indicated, glyceraldehyde-3-phosphate (1 mM), NAD+(0.5 mM), and sodium arsenate (25 mM, 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 corres-ponding nonoxidized substrate Statistical analyses com-paring multiple enzyme activities were performed using a one-wayANOVAand Dunnett’s posthoc test Other analyses comparing multiple conditions were performed using a one-wayANOVAand Newman–Keuls posthoc test Where
Trang 3only 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
R E S U L T S
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 O2
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 H2O2
generated during the photolysis (e.g [38]) The subsequent
slow decay is assigned 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 H2O2appears
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 H2O2 before the
peroxides were used in other experiments Omission of the
rose bengal, photolysis in the absence of O2, or incubation
of complete samples in the absence of light, resulted in
peroxide concentrations of < 5 lM(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 on
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 in the place of 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 NaBH4
(approx 20-fold molar excess relative to protein
concentra-tion) for 30 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 Fe2+–EDTA, reduced glutathione (GSH), ebselen and other thiols also 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, which 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 in 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) were photolysed with visible light for 60 min in the presence of rose bengal (10 l M ) with continuous gassing with air at 4 °C Immediately after the cessation of photolysis catalase was added to part of the sample (50 lgÆmL)1for 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
± SD; 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.
Trang 4could not be readily removed from the reaction mixture,
and lysozyme was unable to be analysed as the protein
precipitated on addition of NaBH4
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 lMat 30 min), is approxi-mately double that of the peroxides lost under identical conditions (11.2 ± 1.1 lMat 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 °C in the
absence or presence of added GAPDH Peptide peroxide samples were
generated as described in Fig 1 and the text Immediately after
ces-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 measured at the indicated times for either
untreated controls (d), or samples with added GAPDH (n;
1 mgÆmL)1), using a modified FOX assay (A) N-Ac-Trp-OMe
per-oxides; (B) Gly-His-Gly perper-oxides; (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 on 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 mgÆmL)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-nitrobenzoic acid) 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 per GAPDH tetramer were detected 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 (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 (control, 42 ± 6% of thiols free; NEM-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) Peroxide loss observed in presence of control (non-NEM treated) GAPDH; (·) peroxide loss observed in presence of NEM-treated GAPDH; (d) peroxide loss in absence of added
GAP-DH (cf Fig 2A) Data are means ± SD; 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 < 0.05 level.
Trang 5the blocking of 50% of the free thiols on the enzyme when
compared to controls Complete blocking of all thiol groups
was not attempted as the requirement for high
concentra-tions of NEM can result in other modificaconcentra-tions [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 generated 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 Fe2+–EDTA and the residual enzymatic activity
determined (Fig 4, Table 1) Lower concentrations of
these enzymes were employed in these studies, compared to
those reported above, to prevent substrate depletion
Comparative studies were also carried out with H2O2
The EDTA complex of Fe2+ was employed to prevent potential binding of Fe2+to the target enzymes; omission
of the EDTA resulted in less efficient enzyme inhibition (data not shown)
GAPDH was rapidly inactivated, in a time-dependent manner, by all the peroxides tested, in both the absence and presence of added Fe2+–EDTA The rate of inhibition of GAPDH by N-Ac-Trp-OMe peroxides was concentration dependent over the range tested (20–200 lM peroxide) Incubation of GAPDH with nonphotolysed samples of the peptide or proteins (with, or without, 20 lMFe2+–EDTA),
or with Fe2+–EDTA alone, resulted in slow loss of enzyme activity, presumably owing to slow denaturation (Fig 4A)
GAPDH was readily inhibited by H2O2, which was employed as a positive control, in either the presence, or absence, of Fe2+–EDTA Comparison of the data obtained with H2O2 and N-Ac-Trp-OMe peroxides showed that fivefold higher concentrations of H2O2 needed to be employed to generate a similar rate and extent of inhibition (Fig 4B) 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-Trp-OMe peroxides (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 min with (f) lysozyme peroxides (20 l M ).
Fe 2+ –EDTA (20 l M ) was added where indicated Control samples contained equal concentrations of nonphotolysed materials, or water (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) nonphotolysed/non H 2 O 2 containing samples in presence of added Fe 2+ –EDTA; (r) nonpho-tolysed/non H 2 O 2 containing samples in absence of added Fe 2+ –EDTA Statistical analyses (one-way ANOVA with Dunnett’s posthoc test) compared all conditions to the nonphotolysed/non-H 2 O 2 control without added Fe2+–EDTA, ** P < 0.01 Where no error bar is visible it is obscured by the symbol.
Trang 6identical peroxide concentrations demonstrating that
per-oxide size and/or electronic charge play a role in
determin-ing the rate of inhibition
Identical studies using GAPDH with samples of RNase
A peroxides which had been pretreated with NaBH4 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-OMe
peroxides, but only at the highest concentrations tested
(200 lM) (Table 1) This inhibition was not stimulated by
added Fe2+–EDTA In contrast, rapid inhibition of GR by
H2O2was only observed in the presence of Fe2+–EDTA
(Table 1) LDH was not inhibited by the highest
concen-trations of peptide and protein peroxides (200 lM) tested, in
either the presence or absence of Fe2+–EDTA (data not
shown), whereas this enzyme was readily inhibited by H2O2
in the presence, but not absence, of Fe2+–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 inactivation
Examination of the role of peroxide-derived radicals
in enzyme inhibition
To examine whether radical species were generated during
the inactivation of GAPDH and LDH by peroxides in the
absence of added metal ions, GAPDH (24 mgÆmL)1) and
LDH (6 mgÆmL)1) were incubated with the spin trap PBN
(9.4 mM) and N-Ac-Trp-OMe peroxides or H2O2 (both
200 lM) 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 Fe2+–
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 peroxides or H2O2(Table 2) GSH, N-(2-mercaptopropionyl)glycine, ascorbic acid and dithiothreitol (all 200 lM) all offered highly significant protection against the inhibition of GAPDH induced by
20 lMN-Ac-Trp-OMe peroxides in the presence of 20 lM
Fe2+–EDTA (Table 2, cf Figure 4A) Methionine, at an identical concentration, had a much less marked, although still statistically significant, effect Trolox C was ineffective All the compounds tested showed a significant protective effect at 2 mM in the LDH/Fe2+–EDTA/H2O2 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
D I S C U S S I O N
Exposure of amino acids, peptides and proteins to radiation (ionizing, UV, or visible light in the presence of a photosensitiser) in the presence of O2, gives rise to peroxides [25–27,38] With 1O2, 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 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 studies have shown that protein peroxides are 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 Fe2+–EDTA Samples containing glutathione reductase (0.025 UÆmL)1) were incubated at 37 °C for 120 min with N-Ac-Trp-OMe peroxides (200 l M ) and H 2 O 2 (200 l M ) Samples containing lactate dehydrogenase (0.05 UÆmL)1) were incubated at 37 °C for 30 min with H 2 O 2
(200 l M ) 20 l M Fe 2+ –EDTA was present where indicated Control solutions contained equal concentrations 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 Fe2+– EDTA Statistical analyses (one-way ANOVA with Dunnett’s posthoc test) compared all conditions to the nonphotolysed N-Ac-Trp-OMe/H 2 O control without added Fe 2+ –EDTA; * P < 0.01.
Enzyme Added agents Initial control activity (%)
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*
H 2 O + Fe2+–EDTA 97 ± 4
H 2 O 2 + Fe 2+ –EDTA 23 ± 5*
H 2 O + Fe2+–EDTA 70 ± 6
H 2 O 2 + Fe 2+ –EDTA 10 ± 1*
Trang 7generated in cells on exposure to 1O2 (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 of1O2-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 one protein
can result in 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 recycles
oxidized glutathione 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 lM) on proteins in viable rose-bengal loaded THP-1 cells exposed to visible light (A Wright, C L Hawkins & M J Davies, unpublished results)
Previous studies (reviewed in [42]) have shown that GAPDH is rapidly, and specifically, inhibited in myocytes, aortic endothelial and U937 (pro-monocyte) cells on exposure to H2O2in the absence of added metal ions [43– 45] This inactivation arises via direct reaction of H2O2with
a particularly reactive Cys residue (Cys149), which has a
pKaof 5.4 owing to interaction with His176, in the active site of the enzyme 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], nitric 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 °C in the absence, or presence, of a 10-fold excess (over peroxide concentration) of putative antioxidant Samples containing GAPDH (0.15 UÆmL)1) or LDH (0.05 UÆmL)1) were incubated for 30 min; those containing GR (0.025 UÆmL)1) for 120 min All incubations contained 20 l M Fe 2+ –EDTA Control samples contained nonphotolysed 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 + Fe2+–EDTA H 2 O (control) 67 ± 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 + Fe2+–EDTA H 2 O (control) 97 ± 4
H 2 O 2 + Methionine 109 ± 6
H 2 O 2 + Trolox C 80 ± 1
H 2 O 2 + Dithiothreitol 55 ± 1*
LDH + Fe2+–EDTA H 2 O (control) 70 ± 6
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
Trang 8reactions (e.g involving HOÆ [46] or O2– Æ [42,49]) that
involve oxidation of Cys-149 to cysteic acid [47]
GR also contains an active site Cys residue [50] GR is
less-readily inhibited than GAPDH by H2O2, 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 presence of metal ions [53], but is less sensitive to
inhibition than GAPDH [42] A similar pattern appears to
hold with the peptide and protein peroxides investigated in
the current study, with GAPDH being more sensitive than
GR and LDH, and inactivation of GAPDH being
metal-ion independent, whereas inhibitmetal-ion of GR and LDH by
H2O2occurs most rapidly in the presence of metal ions
It is proposed that the inhibition of GAPDH and GR by
these1O2-generated peptide and protein peroxides occurs
via direct (nonradical) oxidation of the active site Cys
residues (i.e reaction 1, see below) This is supported 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 prevents enzyme inhibition 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 inhibition in the absence of metal ions is
confirmed by the EPR studies where no radicals were
detected; previous studies have shown that radicals formed
from these peroxide 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 sulfenic 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 during 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 H2O2 is believed to occur via radical-mediated
reactions, catalysed by the added Fe2+–EDTA
Enzyme-SHþ Peptide-/Protein-OOH ! Enzyme-S-OH
Enzyme-S-OHþ 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 1O2-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 free Met in the inhibition experiments carried out with N-Ac-Trp-OMe peroxides and GAPDH (cf Table 2, P < 0.01 by one-wayANOVAwith Dunnett’s posthoc test, when the Met treated sample was compared to photolysed N-Ac-Trp-OMe with added Fe2+–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
1O2on peptides and proteins are not removed by catalase [25], and this has been 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 and antioxidants
is therefore likely to be the major route 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 1O2-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 protein peroxides [26,27,29] It has also been shown 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 protection offered by thiols is owing to direct scavenging of 1O2, removal of peroxides (H2O2and/or protein), or repair of reversibly damaged targets, such as the enzymes investi-gated here, remains to be established
A C K N O W L E D G E M E N T S
The authors are grateful to the Australian Research Council and the Juvenile Diabetes Foundation International for financial support, and
to Dr Clare Hawkins for helpful discussions.
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