Amyloid
Peptides Do Not Form Peptide-derived Free Radicals
Spontaneously, but Can Enhance Metal-catalyzed Oxidation of
Hydroxylamines to Nitroxides*
Sergey I.
Dikalov
§,
Michael P.
Vitek¶,
Kirk R.
Maples
, and
Ronald P.
Mason
From the
Laboratory of Pharmacology and Chemistry,
NIEHS, National Institutes of Health, Research Triangle Park, North
Carolina 27709, the ¶ Division of Neurology, Department of
Medicine, Duke University Medical Center, Durham, North Carolina
27710, and
Centaur Pharmaceuticals, Inc.,
Sunnyvale, California 94086
 |
ABSTRACT |
Amyloid
(A
) peptides play an important
role in the pathogenesis of Alzheimer's disease. Free radical
generation by A
peptides was suggested to be a key mechanism of
their neurotoxicity. Reports that neurotoxic free radicals derived from
A
-(1-40) and A
-(25-35) peptides react with the spin trap
N-tert-butyl-
-phenylnitrone (PBN) to form a
PBN/·A
peptide radical adduct with a specific triplet ESR
signal assert that the peptide itself was the source of free radicals.
We now report that three A
peptides, A
-(1-40), A
-(25-35),
and A
-(40-1), do not yield radical adducts with PBN from the
Oklahoma Medical Research Foundation (OMRF). In contrast to OMRF PBN,
incubation of Sigma PBN in phosphate buffer without A
peptides
produced a three-line ESR spectrum. It was shown that this nitroxide is di-tert-butylnitroxide and is formed in the Sigma PBN
solution as a result of transition metal-catalyzed auto-oxidation of
the respective hydroxylamine present as an impurity in the Sigma PBN. Under some conditions, incubation of PBN from Sigma with A
-(1-40) or A
-(25-35) can stimulate the formation of
di-tert-butylnitroxide. It was shown that A
peptides
enhanced oxidation of cyclic hydroxylamine 1-hydroxy-4-oxo-2,2,6,6-tetramethylpiperidine (TEMPONE-H), which was strongly inhibited by the treatment of phosphate buffer with Chelex-100. It was shown that ferric and cupric ions are effective oxidants of TEMPONE-H. The data obtained allow us to conclude that
under some conditions toxic A
peptides A
-(1-40) and
A
-(25-35) enhance metal-catalyzed oxidation of hydroxylamine
derivatives, but do not spontaneously form peptide-derived free radicals.
 |
INTRODUCTION |
As the leading cause of dementia, Alzheimer's disease
(AD)1 is characterized by a
loss of memory and neurons. These characteristics have been associated
with brain lesions known as neurofibrillary tangles composed of Tau
protein and amyloid plaques, which consist of amyloid
(A
)
peptide. In a small number of families, mutations in the genes for the
amyloid peptide precursor (APP, chromosome 21) to A
peptide for
presenilin-1 (PSEN-1, chromosome 14), presenilin-2 (PSEN-2, chromosome
1) and for an anonymous gene on chromosome 12 have been associated with
AD. In contrast, Saunders and colleagues (1) discovered that a
significant percentage of patients inheriting one or more of the
epsilon-4 alleles of apolipoprotein-E (APOE4, chromosome 19) were at
risk of acquiring AD at an earlier age than their counterparts
expressing the more common epsilon-3 allele of APOE. The observation
that AD patients with mutant genes for APP, PSEN-1, or PSEN-2 have
higher levels of A
peptide than non-diseased controls (2, 3) argues
that A
peptide may cause AD. Further supporting this idea is the
observation that APOE4 patients with AD display more amyloid plaques
than those with APOE3 (4). Based on neuropathological, genetic, and
biochemical associations, the presence of amyloid
peptide is a key
component of AD.
A crucial link between A
peptide and AD was provided by Yankner and
colleagues (5), who showed that fibrillar aggregates of A
peptide
were toxic to neurons. This seminal observation inspired a global quest
to define the mechanism by which fibrillar A
peptide mediated
neurotoxicity. One of the mechanisms proposed suggests that neurons
exposed to A
peptide suffer severe oxidative stress that may lead to
their death. Clear evidence of oxidative stress in Alzheimer's disease
has been provided by Smith et al. (6), who found that
neurons of AD patients contained nitrotyrosine modifications of
proteins, which were not detected in age-matched control brains.
Increased levels of lipid peroxides (7), reactive aldehydes such as
hydroxynonenal (8), and oxidized DNA (9) have also been reported in AD
brains, providing additional evidence of an oxidative stress component
in the disease.
The exact nature of the radical species generated in Alzheimer's
diseased brains is unknown. In one line of investigation, Behl et
al. (10) demonstrated that cells exposed to fibrillar A
peptide
responded by releasing hydrogen peroxide and dying, a process that
could be inhibited by the application of catalase to degrade the
released peroxide. Subsequent reports have demonstrated cellular
release of superoxide (11) and nitric oxide (12) in response to A
peptide treatment. The effect of these radicals may be potentiated
since A
peptides also appear to inhibit the cellular redox
mechanisms that normally protect cells from oxidative stress (13).
In a provocative hypothesis, Hensley et al. (14) suggest
that the A
peptide itself spontaneously generates free radicals that
can damage cells. By mixing the spin trap
N-tert-butyl-
-phenylnitrone (PBN) with
neurotoxic forms of the A
peptide such as A
-(1-40) and
A
-(25-35), they report the generation of ESR-detectable radical adducts in cell-free solutions (14-17). They suggest that these radicals are generated by methionine oxidation (14, 15) or by the
fragmentation of A
peptide into smaller oligopeptide radicals (14,
15) that act as "shrapnel" to damage and eventually kill cells.
Recently, these results were described as possibly artifactual due to
contaminants in some of the preparations (18).
We have reinvestigated the spin-trapping studies of spontaneous free
radical formation by A
peptides. In contrast to previous literature
(14-17), we now report that neurotoxic A
-(1-40) and A
-(25-35)
in the presence of the spin trap PBN do not form ESR-detectable radical
adducts spontaneously. Amyloid/PBN radical adducts (14-17) reported
earlier were found to be di-tert-butylnitroxide and
tert-butylhydronitroxide, which are formed by oxidation of
the corresponding hydroxylamines. We have investigated the
possibility that toxic A
may potentiate metal-catalyzed oxidation of
hydroxylamine derivatives. Transition metals like iron and copper
frequently contribute to reactions where molecules are oxidized, but
the transition metal oxidation of hydroxylamine derivatives has not
been previously studied. A body of emerging work demonstrates that
copper and iron are significantly associated with amyloid plaques in
the brains of patients with AD, but not with the neuropil lacking
plaques nor with control neuropil from healthy brains (19). Since A
is retained on metal chelate columns charged with copper (20), and
copper dramatically increases the rate of A
aggregation into amyloid
fibrils (21), then complexes of A
and copper may contribute to the
pathological oxidative stress associated with AD. We now report the
effects of neurotoxic A
-(1-40) and A
-(25-35) and non-toxic
A
-(40-1) on the oxidation of hydroxylamine derivatives in the
presence and absence of metals. The data obtained allows us to conclude
that the previously reported formation of amyloid/PBN radical adducts
(14-17) in the Sigma PBN treated with A
peptides can be explained
as A
peptide-stimulated, metal-catalyzed oxidation of the
corresponding hydroxylamine impurities in Sigma PBN.
 |
EXPERIMENTAL PROCEDURES |
Reagents--
PBN was obtained from the Oklahoma Medical
Research Foundation (OMRF) Spin Trap Source (Oklahoma City, OK). Two
different lots (101H3696 and 87H36602) of PBN from Sigma were used.
Deferoxamine (Desferal) was purchased from Sigma.
Di-tert-butylnitroxide (Aldrich), N-tert-butylhydroxylamine (TCI America, Portland,
OR) and analytical grade Chelex-100 (Bio-Rad) were used. Synthetic
A
-(1-40), A
-(25-35) and A
-(40-1) peptides were purchased
from Bachem (Torrance, CA). These lots of A
-(1-40) and
A
-(25-35), but not the A
-(40-1) peptides, have been previously
shown to kill neurons in a cell culture system (data not shown).
TEMPONE-H was obtained from Alexis Corporation (Läufelfingen,
Switzerland) (22). All other reagents were analytical grade.
Preparation of PBN Stock Solution--
Stock solutions of PBN
(150 mM) in HPLC grade water were used to prepare final 50 mM PBN solutions.
Preparation of TEMPONE-H Stock Solutions--
TEMPONE-H was
dissolved in oxygen-free HPLC grade water (30-min argon bubbled water)
with 1 mM Desferal. Desferal was used to decrease the
spontaneous oxidation of hydroxylamines catalyzed by traces of
transition metal ions. The concentration of TEMPONE-H in the stock
solutions was 1 mM. Prior to the experiments, the stock
solution was kept under a flow of argon in a cool, air-tight place.
Preparation of tert-Butylhydronitroxide
Solution--
tert-Butylhydronitroxide was obtained by
auto-oxidation of 1 mM
N-tert-butylhydroxylamine in water or by
oxidation of 10 µM solution of
N-tert-butylhydroxylamine with 10 µM K3[Fe(CN)6] in 0.15 M phosphate buffer at pH 7.4.
ESR Spin-trapping Experiments--
The ESR spectra were recorded
using a Bruker ECS-106 spectrometer operating at 9.76 GHz with a
modulation frequency of 50 kHz and a TM110 cavity.
Spin-trapping experiments were started by solubilizing A
peptides
into a solution of PBN or TEMPONE-H. All ESR samples were placed in the
17-mm flat cell, which contained A
peptides (1 mg/ml), PBN (50 mM), or TEMPONE-H (0.3 mM) in sodium phosphate
buffer (0.15 M) at pH 7.4. In order to perform experiments with a lower concentration of transition metals, phosphate buffer was
treated with 5 g of Chelex-100/100 ml of solution for 2 h, followed by filtration using Millex-HA 0.45-µm filters (Millipore Corp., Bedford, MA). The ESR instrumental settings for experiments with
PBN were as follows: field sweep, 70 G; microwave frequency, 9.76 GHz;
microwave power, 40 milliwatts; modulation amplitude, 0.32 G;
conversion time, 1310 ms; time constant, 1310 ms; receiver gain, 1 × 105. ESR signals were monitored for 6 h. The ESR
instrumental settings for TEMPONE-H experiments were the following:
field sweep, 50 G; microwave frequency, 9.76 GHz; microwave power, 20 milliwatts; modulation amplitude, 1 G; conversion time, 656 ms; time
constant, 2620 ms; receiver gain, 1 × 105.
Study of Iron-catalyzed Oxidation of Hydroxylamines--
The
presence of hydroxylamine impurities in Sigma PBN was determined by
studying iron's effect on nitroxide formation in solutions of PBN with
K3[Fe(CN)6] and the iron-chelating agent
Desferal. Oxidation of hydroxylamine impurities in Sigma PBN was
performed using 5 µM
K3[Fe(CN)6]. Inhibition of iron-catalyzed
oxidation of hydroxylamine impurities in Sigma PBN was carried out in
the presence of 2 mM Desferal.
Study of Ferric-catalyzed Oxidation of Hydroxylamines--
The
oxidation of TEMPONE-H by ferric ions was confirmed by the formation of
TEMPONE nitroxide during the incubation of TEMPONE-H with ferric ions.
The effect of 1 µM
Fe(NH4)(SO4)2 and chelating agent
Desferal on the TEMPONE nitroxide formation from 0.1 mM TEMPONE-H was studied. Inhibition of iron-catalyzed oxidation of
TEMPONE-H was carried out in the presence of 1 mM Desferal, which has its highest affinity for ferric ion.
Study of Cupric-catalyzed Oxidation of Hydroxylamines--
The
oxidation of TEMPONE-H by cupric ions was confirmed by the formation of
TEMPONE nitroxide during the incubation of TEMPONE-H with cupric ions.
In this experiment the effect of 1 µM CuSO4 and chelating agent Desferal on the TEMPONE nitroxide formation from
0.1 mM TEMPONE-H was studied. Inhibition of
cupric-catalyzed oxidation of TEMPONE-H was carried out in the presence
of 1 mM Desferal.
Computer Simulation--
Computer simulations and spin-trap data
base searches were performed using a computer simulation
program,2 the details of
which have been described elsewhere (23).
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RESULTS |
Reinvestigation of Spontaneous Free Radical Formation by A
Peptides--
The ability of A
peptides to spontaneously form free
radicals was studied by ESR using the spin trap PBN from the OMRF Spin Trap Source. A
-(1-40), A
-(40-1), and A
-(25-35) peptides
were tested in parallel with a control that did not contain A
peptides (Fig. 1). ESR spectra were
collected every 40 min over 6 h, and no ESR signals were observed
in any mixtures (Fig. 1, A-D). Therefore, A
peptides do
not form ESR-detectable radical adducts spontaneously.

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Fig. 1.
ESR spectra of A
with high quality OMRF PBN (A-D) or with Sigma
PBN (E-H). A, ESR spectrum obtained
after 6 h of incubation of 50 mM Sigma PBN in
Chelex-100-treated 0.15 M sodium-phosphate buffer (pH 7.4)
without A peptides. B, ESR spectrum obtained after 6 h of incubation of 50 mM PBN with 1 mg/ml A -(1-40).
C, ESR spectrum obtained after 6 h of incubation of 50 mM PBN with 1 mg/ml A -(40-1). D, ESR
spectrum obtained after 6 h of incubation of 50 mM PBN
with 1 mg/ml A -(25-35). E, ESR spectrum obtained after
6 h of incubation of 50 mM Sigma PBN in
Chelex-100-treated 0.15 M sodium-phosphate buffer (pH 7.4)
without A peptides. F, ESR spectrum obtained after 6 h of incubation of 50 mM PBN with 1 mg/ml A -(1-40).
G, ESR spectrum obtained after 6 h of incubation of 50 mM PBN with 1 mg/ml A -(40-1). H, ESR
spectrum obtained after 6 h of incubation of 50 mM PBN
with 1 mg/ml A -(25-35).
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In contrast to the OMRF PBN results, ESR spectra from mixtures of Sigma
PBN alone, plus A
-(1-40), plus A
-(40-1), or plus A
-(25-35)
peptides all yielded a triplet ESR spectrum with the A
peptide
inhibiting radical formation (Fig. 1, E-H). This triplet spectrum has a nitrogen hyperfine-coupling constant of 17.14 G. The
formation of this triplet spectrum with Sigma PBN, but not OMRF PBN, is
consistent with the spectrum arising from an impurity in Sigma PBN.
Formation of Di-tert-butylnitroxide and tert-Butylhydronitroxide
from Impurities in Sigma PBN--
PBN from Sigma was checked for the
presence of impurities that might lead to the formation of ESR signals.
After a 6-h incubation of 50 mM aqueous Sigma PBN, a strong
ESR spectrum was observed (Fig.
2A) that consisted of ESR
spectra from two nitroxides (Fig. 2, B-D). One nitroxide
has a triplet ESR spectrum (Fig. 2C) with a nitrogen
hyperfine coupling constant of 17.16 G, which is very close to the
reported nitrogen hyperfine coupling constant for di-tert-butylnitroxide. The second nitroxide has a four-line
ESR spectrum (Fig. 2D) that is consistent with a nitrogen
hyperfine coupling constant of 14.61 G and a hydrogen hyperfine
coupling constant of 13.93 G, which are very close to the reported
hyperfine coupling constants for tert-butylhydronitroxide
(24, 25).

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Fig. 2.
Formation of
di-tert-butylnitroxide and
tert-butylhydronitroxide in an aqueous solution
prepared using Sigma PBN. A, ESR spectrum obtained
after a 6-h incubation of 50 mM Sigma PBN. B,
computer simulation of A obtained by adding spectra of two
nitroxides. C, computer simulation of ESR spectrum of
nitroxide with a nitrogen hyperfine coupling constant of 17.16 G. D, simulation of ESR spectrum of nitroxide using hyperfine
coupling constants of 14.61 G for nitrogen and 13.93 G for hydrogen.
E, ESR spectrum of 200 nM
di-tert-butylnitroxide. F, ESR spectrum of
tert-butylhydronitroxide from 10 µM
N-(tert-butyl)hydroxylamine.
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To confirm the chemical structure of nitroxide radicals observed in the
solution of Sigma PBN, we obtained experimental ESR spectra of
di-tert-butylnitroxide (Fig. 2E) and
tert-butylhydronitroxide (Fig. 2F). Experimental
ESR spectra of di-tert-butylnitroxide and
tert-butylhydronitroxide were identical to the ESR spectrum of the PBN solution shown above (Fig. 2). The ESR spectra from the
Sigma PBN solution (Fig. 2A) can be described as a
combination of two specific ESR spectra: those of
di-tert-butylnitroxide (Fig. 2E) and
tert-butylhydronitroxide (Fig. 2F). Thus, during
the incubation of Sigma PBN in water, both
di-tert-butylnitroxide and
tert-butylhydronitroxide were formed.
In order to analyze the mechanism of nitroxide formation in the PBN
solution, the effects of the chelating agent Desferal and of ferric
iron addition were studied. Fresh solutions of Sigma PBN in
Chelex-100-treated phosphate buffer contained trace amounts of
di-tert-butylnitroxide as identified by its ESR spectrum
(Fig. 3A). After just 2 h
of incubation, the ESR signal was increased dramatically (Fig.
3B). A parallel 2-h incubation in the presence of the
chelating agent Desferal significantly inhibited formation of
di-tert-butylnitroxide (Fig. 3C). Addition of 5 mM K3[Fe(CN)6] to the fresh PBN
solution caused greater nitroxide formation (Fig. 3D) than
that of the 2-h incubation (Fig. 3B).

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Fig. 3.
Formation of
di-tert-butylnitroxide during transition
metal-catalyzed auto-oxidation of
di-tert-butylhydroxylamine in a solution of Sigma
PBN. A, ESR spectrum obtained after 10 min of
incubation of 50 mM Sigma PBN in Chelex-100-treated
phosphate buffer. B, ESR spectrum obtained after 2 h of
incubation. C, ESR spectrum obtained after 2 h of
incubation of 50 mM Sigma PBN with 2 mM
Desferal; D, ESR spectrum obtained after addition of 5 µM K3[Fe(CN)6] to
A.
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Linewidth Dependence on the ESR Instrumental
Settings--
Previously, it was reported that A
-(1-40) and
A
-(25-35) peptides formed radical adducts with identical triplet
ESR spectra and nitrogen hyperfine coupling constants of 17.1 G (16),
which is actually the same as that reported for
di-tert-butylnitroxide (Fig. 2E). Based on the
much greater linewidth of the A
peptide/PBN reaction product (1.6 G), these authors concluded that this product was not
di-tert-butylnitroxide (17). However, the linewidth is not a
specific parameter of a nitroxide ESR spectrum (26). Linewidth is very
dependent on the ESR instrumental settings, mainly modulation amplitude
and microwave power. In order to show that authentic
di-tert-butylnitroxide and the nitroxide derived from Sigma
PBN can have the same ESR linewidth as has been described for a
PBN/·A
peptide radical adduct, the ESR spectra were obtained
using different modulation amplitude settings (Fig.
4). The linewidth of the ESR spectra of
di-tert-butylnitroxide was equal to that of the nitroxide
from Sigma PBN (Fig. 4, A and D) when ESR spectra were collected under identical instrument settings. Moreover, both
spectra displayed the same linewidth dependence on the modulation amplitude setting. We observed peak-to-peak linewidths of 0.51, 0.96, and 1.56 G when using modulation amplitudes of 0.32, 1.00, and 1.59 G,
respectively (Fig. 4).

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Fig. 4.
Dependence of ESR spectra of
di-tert-butylnitroxide on modulation amplitude.
ESR spectra of 50 mM Sigma PBN after 6 h of incubation
in Chelex-100-treated phosphate buffer using the following modulation
amplitudes: A, 0.32 G; B, 1.00 G; C,
1.59 G. ESR spectra of 200 nM
di-tert-butylnitroxide using the following modulation
amplitudes: D, 0.32 G; E, 1.00 G; F,
1.59 G.
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Previously, it was also reported that non-toxic A
-(40-1) peptide
formed a radical adduct with a quartet ESR spectrum and equivalent
nitrogen and hydrogen hyperfine coupling constants of 14.5 G (16, 17),
which are very close to those reported for
tert-butylhydronitroxide (Fig. 2F). Based on the
much greater linewidth of the PBN/·A
peptide reaction
product, it was concluded that this product was not
tert-butylhydronitroxide (17). In order to show that tert-butylhydronitroxide and the nitroxide from Sigma PBN
solution could have the same ESR spectrum linewidth as the radical
described as the PBN/·A
peptide-(1-40) reaction product, the
ESR spectra were obtained using different modulation amplitude settings
for the ESR spectrometer (Fig. 5). The
linewidth of the ESR spectrum of tert-butylhydronitroxide was the same as for the nitroxide species from the Sigma PBN solution (Fig. 5, A and D). Moreover, the dependence of
linewidth on the modulation amplitude was the same. Using modulation
amplitudes of 0.32, 1.00, and 1.59 G, the widths of the low-field line
were observed to be 0.76, 0.98, and 1.53 G, respectively (Fig. 5).

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Fig. 5.
Dependence of ESR spectra of
tert-butylhydronitroxide on modulation amplitude.
ESR spectra of 50 mM Sigma PBN after 6 h of incubation
in phosphate buffer using the following modulation amplitudes:
A, 0.32 G; B, 1.00 G; C, 1.59 G. ESR
spectra of tert-butylhydronitroxide using the following
modulation amplitudes: D, 0.32 G; E, 1.00 G;
F, 1.59 G.
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A
Peptide Stimulation of Oxidation of
Di-tert-butylhydroxylamine Impurity in Sigma PBN--
The ability
of A
peptides to stimulate oxidation of
di-tert-butylhydroxylamine was studied by ESR using PBN from
Sigma. For these experiments, solutions of A
-(1-40), A
-(25-35),
and A
-(40-1) with 50 mM PBN and 1 mM
Desferal were tested in parallel with a control that did not contain
A
peptides (Fig. 6). Incubation of PBN
(Sigma) with A
peptides for 2 h in phosphate buffer with Desferal gave rise to the ESR spectra shown in Fig. 1. Neurotoxic A
-(1-40) and A
-(25-35) produced triplet ESR spectra (Fig. 6, A and B) in agreement with Butterfield and
co-workers (14-17). Computer simulation of these ESR spectra (Fig. 6)
revealed an ESR spectrum of one nitroxide compound which displays a
triplet ESR spectrum with a nitrogen hyperfine coupling constant of
17.16 G, which is identical to the nitrogen hyperfine coupling constant for di-tert-butylnitroxide. Non-toxic A
-(40-1) produced
a much weaker nitroxide triplet and the four-line
tert-butylhydronitroxide (Fig. 6C). ESR spectra
of a control sample without A
peptides did not reveal any nitroxide
formation (Fig. 6D). Under these conditions the formation of
this triplet spectrum with Sigma PBN was dependent on the presence of
toxic A
-(1-40) and A
-(25-35).

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Fig. 6.
Formation of
di-tert-butylnitroxide during the incubation of
A peptides with Sigma PBN. ESR spectra
obtained after a 2-hr incubation of 50 mM PBN in 0.15 M sodium phosphate buffer (pH 7.4) plus 1 mM
Desferal: A, with 1 mg/ml A -(1-40); B, with 1 mg/ml A -(25-35); C, with 1 mg/ml A -(40-1);
D, without A . ESR receiver gain was 5 × 105.
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It was previously shown that di-tert-butylnitroxide can be
formed in a solution of PBN by the metal-catalyzed auto-oxidation of
di-tert-butylhydroxylamine. Therefore, in the presence of 1 mM Desferal in phosphate buffer, A
-(1-40) and
A
-(25-35) stimulate the oxidation of the
di-tert-butylhydroxylamine impurity to form di-tert-butylnitroxide.
A
-stimulated Oxidation of TEMPONE-H--
In order to study the
mechanism of A
-stimulated hydroxylamine oxidation, we used TEMPONE-H
(22). The time course of ESR spectra and the effect of removing
transition metals from the phosphate buffer by treatment with chelating
resin Chelex-100 were studied.
TEMPONE-H was incubated with A
peptides in phosphate buffer with 1 mM Desferal (Fig. 7). It was
found that after a 1-h incubation of TEMPONE-H (0.3 mM)
with A
-(25-35) and A
-(1-40), the ESR amplitude of TEMPONE
nitroxide increased up to 4 times in comparison with the control (Fig.
7, A, B, and D). Incubation of
TEMPONE-H (0.3 mM) with A
-(40-1) increased the ESR
amplitude in the range of 50-100% compared with the control (Fig. 7
C, D). Therefore, neurotoxic A
-(1-40) and A
-(25-35)
significantly stimulated oxidation of TEMPONE-H to TEMPONE nitroxide
(Fig. 7, A and B). Non-toxic A
-(40-1) caused
relatively minor changes in the formation of TEMPONE nitroxide (Fig.
7C) compared with a control sample without A
peptides
(Fig. 7D).

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Fig. 7.
Effect of A peptides
on the TEMPONE formation during transition metal-catalyzed oxidation of
TEMPONE-H. A-D, 0.15 M sodium phosphate
buffer plus 1 mM Desferal (DF). E-H,
Chelex-100-treated 0.15 M sodium phosphate buffer plus 1 mM DF. ESR spectra obtained after a 1-h incubation of 0.3 mM TEMPONE-H with A (1 mg/ml). ESR receiver gain was
1 × 105.
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In order to clarify the role of transition metals in A
-mediated
stimulation of hydroxylamine oxidation, phosphate buffer was pretreated
for 1 h with chelating resin Chelex-100 to remove trace-metal
contaminants. ESR spectra obtained after the incubation of TEMPONE-H
(0.3 mM) with A
peptides in Chelex-100-treated phosphate buffer are shown in Fig. 7, E-H. In Chelex-100-treated
phosphate buffer, A
peptides did not stimulate oxidation of
TEMPONE-H, implying that A
peptides were not the primary source of
trace transition metals.
Fig. 8 shows the time dependence of
TEMPONE formation during the incubation of TEMPONE-H with A
peptides. Formation of TEMPONE nitroxide in the samples with
A
-(1-40) and A
-(25-35) was significantly faster than with
A
-(40-1) and in the control (Fig. 8A). Removing transition metals from the solution by pretreatment of phosphate buffer
with Chelex-100 strongly inhibited A
stimulation of TEMPONE formation (Fig. 8B). Therefore, in phosphate buffer with 1 mM Desferal, A
-(1-40) and A
-(25-35) enhanced
TEMPONE-H oxidation, which was inhibited by Chelex-100 treatment.

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Fig. 8.
Time course of TEMPONE formation during
incubation of TEMPONE-H with A peptides (1 mg/ml). A, formation of TEMPONE in 0.15 M
sodium phosphate buffer plus 1 mM Desferal. B,
formation of TEMPONE in Chelex-100-treated 0.15 M sodium
phosphate buffer plus 1 mM Desferal.
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Effect of Ferric Ions on TEMPONE-H Oxidation--
The potential
role of ferric ion in A
-stimulated hydroxylamine oxidation was
studied (Fig. 9). ESR spectra of the
fresh solution of TEMPONE-H revealed only a relatively small
amount of TEMPONE (Fig. 9A). Addition of 1 µM
Fe3+NH4(SO4)2 to
TEMPONE-H led to a 10-fold increase in the ESR amplitude of the TEMPONE
spectra (Fig. 9B). The chelating agent Desferal (1 mM) completely inhibited oxidation of TEMPONE-H in
solutions containing 1 µM
Fe3+NH4(SO4)2 (Fig.
9C). Therefore, ferric ion can oxidize hydroxylamine TEMPONE-H to form nitroxide TEMPONE and, presumably, ferrous ion (Reaction 1), which is air-oxidized (Reaction 2) (27). Hydrogen peroxide did not directly oxidize the hydroxylamine TEMPONE-H in
solution containing Desferal (data not shown).
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Fig. 9.
Effect of ferric ion on TEMPONE-H
oxidation. A, control. B, in the presence of
1 µM
Fe3+NH4(SO4)2;
C, in the presence of 1 µM Desferal plus 1 µM
Fe3+NH4(SO4)2. ESR
spectra were recorded after a 20-min incubation. ESR receiver gain was
1 × 105.
|
|
Effect of Cupric Ions on TEMPONE-H Oxidation--
The possible
role of cupric ions in A
-stimulated hydroxylamine oxidation was
studied (Fig. 10). Addition of 1 µM Cu2+SO4 to TEMPONE-H led to a
drastic increase in the ESR amplitude of the TEMPONE spectrum (Fig. 10,
A, and D), which was much more pronounced than
the effect of 1 µM
Fe3+NH4(SO4)2 (Fig.
10B). The chelating agent Desferal (1 mM)
completely inhibited oxidation of TEMPONE-H by 1 µM
Cu2+SO4 (Fig. 10C). Therefore,
cupric ion can oxidize hydroxylamine TEMPONE-H to form nitroxide
TEMPONE and cuprous ion (Reaction 3), which is oxidized by oxygen
(Reaction 4) (28).

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|
Fig. 10.
Effect of cupric ion on TEMPONE-H
oxidation. A, in the presence of 1 µM
Cu2+SO4. B, in the presence of 1 µM
Fe3+NH4(SO4)2.
C, in the presence of 1 mM Desferal plus 1 µM Cu2+SO4. D,
control. ESR spectra were recorded after a 20-min incubation. ESR
receiver gain was 1 × 104.
|
|
Auto-oxidation of Hydroxylamines--
In order to clarify the
mechanism of auto-oxidation of hydroxylamine derivatives, formation of
TEMPONE was determined in the presence of superoxide dismutase,
catalase, or Desferal (Fig. 11).
TEMPONE-H was incubated in phosphate buffer for 30 min. ESR spectra of
the control sample of TEMPONE-H revealed a small amount of TEMPONE
(Fig. 11A). The addition of superoxide dismutase (100 units/ml) to TEMPONE-H led to a 2-fold decrease in the ESR amplitude of
the TEMPONE spectra (Fig. 11B). The effect of superoxide
dismutase supports the role of Reaction 5 in the auto-oxidation of
hydroxylamines (22).
Addition of catalase (10 µg) to TEMPONE-H did not significantly
change the content of TEMPONE in the sample (Fig. 11C).
Addition of the chelating agent Desferal (100 µM) to
TEMPONE-H greatly inhibited the TEMPONE formation (Fig.
11D). Therefore, transition metals
(Mn+1) can oxidize hydroxylamine TEMPONE-H to
form TEMPONE (Reaction 6). Reduced transition metals
(Mn+) are oxidized by oxygen (Reaction 7).
Decomposition of hydrogen peroxide by catalase did not affect the
oxidation of hydroxylamine TEMPONE-H, excluding a role for a direct
oxidation of TEMPONE-H by hydrogen peroxide.

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|
Fig. 11.
Formation of TEMPONE nitroxide during the
auto-oxidation of TEMPONE-H. ESR spectra obtained after a 30-min
incubation of 0.1 mM TEMPONE-H in 0.15 M sodium
phosphate buffer (pH 7.4). A, control. B,
TEMPONE-H (0.1 mM) + SOD (100 units/ml). C,
TEMPONE-H (0.1 mM) + catalase (10 µg/ml). D,
TEMPONE-H (0.1 mM) + Desferal (0.1 mM). ESR
receiver gain was 2 × 105.
|
|
 |
DISCUSSION |
Despite many studies on A
peptide-mediated neurotoxicity, its
exact mechanism of action is still undefined. One intriguing hypothesis
suggests that A
peptides in solution spontaneously form free
radicals which damage and kill cells (14, 15). We did not observe any
radical adducts when the A
peptides A
(1-40), A
-(40-1), and
A
-(25-35) were mixed with high quality PBN (OMRF Spin Trap Source).
Previously, it was reported that ESR signals formed during the
incubation of A
peptides with PBN were due to PBN/·A
peptide radical adducts (14-17). However, according to the current literature, there are no reported radical adducts of PBN that have
similar ESR parameters (aN = 17.1 G). Using
Sigma PBN, both the triplet and quartet spectra of
di-tert-butyl- and tert-butylhydronitroxides, respectively, were detected. These nitroxides had previously been misinterpreted as novel radical adducts formed from A
radicals (14-17).
Recently, it was suggested that PBN/·A
peptide radical
adducts decomposed to an alkoxyl nitroxide (15, 17). This alkoxyl nitroxide would have the same structure as the alkoxyl adduct of
2-methyl-2-nitrosopropane. This assignment is also an error, because
the nitrogen hyperfine coupling constant of alkoxyl adducts of
2-methyl-2-nitrosopropane is about 27 G (29, 30), which is quite
different from the 17.1 G for the radical observed. Moreover, according
to the data base (http://epr.niehs.nih.gov/), the hyperfine coupling
constant of 17.1 G is very specific to
di-tert-butylnitroxide, which is directly supported by the
ESR experiments with synthetic di-tert-butylnitroxide
presented here (Fig. 2E).
We show in this work that these ESR signals do not appear during the
incubation of A
peptides with high quality OMRF PBN. Moreover, these
ESR signals did appear during the auto-oxidation of impurities only in
the Sigma PBN. These data strongly suggest that the ESR spectra
reported as PBN/·A
peptide radical adducts actually result
from the formation of di-tert-butylnitroxide and
tert-butylhydronitroxide radicals. These nitroxide radicals
are formed during metal-catalyzed auto-oxidation of
di-tert-butylhydroxylamine and
N-tert-butylhydroxylamine (Scheme 1), which are impurities in PBN from
Sigma.
The spin trap PBN is usually synthesized by condensation of
benzaldehyde with N-tert-butylhydroxylamine (31).
Therefore, the trace amount of
N-tert-butylhydroxylamine in preparations of PBN
is probably due to incomplete purification.
The appearance of di-tert-butylhydroxylamine in PBN can be
explained by the presence of di-tert-butylhydroxylamine in
the N-tert-butylhydroxylamine commonly used
for the synthesis of PBN. Scheme 2
illustrates the mechanism of di-tert-butylhydroxylamine formation from N-tert-butylhydroxylamine.
N-tert-butylhydroxylamine is a good
reducing agent, which, after two one-electron oxidations, produces
2-methyl-2-nitrosopropane. 2-Methyl-2-nitrosopropane is an unstable
compound, which is readily decomposed by heat or light to the
tert-butyl radical and nitric oxide (32, 33). The
tert-butyl radical will react with 2-methyl-2-nitrosopropane (which is actually a well known spin trap) to form
di-tert-butylnitroxide (32, 33).
Di-tert-butylnitroxide can easily be reduced to di-tert-butylhydroxylamine even by excess
N-tert-butylhydroxylamine. The presence of
di-tert-butylhydroxylamine in the sample of
N-tert-butylhydroxylamine was supported by the
fact that the ESR spectra of
N-tert-butylhydroxylamine oxidized by
K3[Fe(CN)6] contained small traces of the
triplet signal of di-tert-butylnitroxide (Fig.
3F). Therefore, trace amounts of
di-tert-butylhydroxylamine are present in many preparations of PBN. Due to the intrinsic sensitivity of ESR, oxidation of trace
levels of these impurities can lead to readily detected ESR signals.
The assignment of the structure of these nitroxides to specific
compounds excludes the possibility that these ESR spectra are derived
from amyloid
peptides.
In this work we investigated the effect of neurotoxic A
-(1-40) and
A
-(25-35) and non-toxic A
-(40-1) on the oxidation of hydroxylamine derivatives. We previously showed that, in the presence of Desferal, incubation of A
-(1-40) and A
-(25-35) with PBN
(Sigma) led to the formation of di-tert-butylnitroxide,
which was produced after oxidation of
di-tert-butylhydroxylamine. Stimulation of oxidation of
hydroxylamine derivatives by A
-(1-40) or A
-(25-35) was also
demonstrated using TEMPONE-H. Oxidation of TEMPONE-H to TEMPONE
nitroxide in the presence of A
-(1-40) or A
-(25-35) was
significantly more intensive than in the control without A
. The treatment of phosphate buffer with chelating resin Chelex-100 inhibits the A
effect on TEMPONE-H oxidation. These data support a
catalytic role of phosphate buffer-derived transition metal contaminants in the A
-stimulated oxidation of hydroxylamine derivatives.
It is known that ferricyanide is able to oxidize hydroxylamine
derivatives (34). Cupric ion is another candidate for the oxidation of
hydroxylamines. In this work we checked the possible role of both
ferric and cupric ions in oxidation of hydroxylamine derivatives. It
was found that both ferric and cupric ions were effective in oxidation
of TEMPONE-H. Moreover, oxidation of TEMPONE-H by cupric ions was
40-fold more effective than ferric ions. Therefore, both ferric and
cupric ions could be involved in A
-stimulated metal-catalyzed
oxidation of hydroxylamine derivatives.
The data obtained allow us to conclude that, under some conditions, the
toxic A
peptides A
-(1-40) and A
-(25-35) enhance transition
metal-catalyzed oxidation of hydroxylamine derivatives. Therefore, the
previously reported formation of radical adducts with A
peptides can
be explained as an A
-enhanced, transition metal-catalyzed oxidation
of hydroxylamine derivatives found as impurities in commercial PBN. In
addition, our data (Fig. 7) demonstrate that A
peptides can compete
with Desferal for transition metals such as copper, which implies that
A
peptides may bind redox-active transition metals in
vivo where trace metal concentrations are extremely low. Any
biological significance of the catalytic role of the toxic A
peptides in the transition metal-mediated oxidation of hydroxylamine is
yet to be determined. Although these hydroxylamines do not occur
naturally, other easily oxidized substances exist in vivo
such as ascorbate and GSH.
Moreover, it is known that copper and iron ions catalyze protein damage
and may be partly responsible for the alterations of protein damage
in vivo (35). Metal-catalyzed protein damage is associated
with oxidative modification of amino acids, for example, formation of
protein carbonyl groups (35). Therefore, the previously reported
inactivation of glutamine synthetase and creatine kinase incubated with
A
-(25-35) (14) could be explained on the basis of metal-catalyzed
protein damage.
Previously, it was shown that iron facilitates A
toxicity to
cultured cells (36). Moreover, it was found that iron metabolism is
altered in Alzheimer's disease (37), which, in combination with
accumulation of A
peptides, could result in peroxidative stress in
the brains of Alzheimer's disease patients. It was suggested that
lowering the level of available iron could provide a therapeutic approach to Alzheimer's disease (38). Furthermore, it was shown that
A
-(25-35) enhances iron- and copper-catalyzed generation of
reactive oxygen species (39). Therefore, our data concerning toxic
A
-stimulated metal-catalyzed oxidation of hydroxylamine derivatives
could give insight into the synergetic toxicity of transition metals
and A
peptides.
The data obtained lead to the conclusion that A
peptides do not form
ESR-detectable radical adducts spontaneously. Our spin-trapping results
and interpretation differ from the previous investigations (14-17).
Our data do not support the conclusion that A
peptides spontaneously
form free radicals, but demonstrate that hydroxylamine impurities in
the spin trap preparations are responsible for the observed ESR
spectra. Therefore, the reported spontaneous generation of PBN radical
adducts by A
peptides has been reinterpreted. Although the
spontaneous free radical model of A
neurotoxicity has been
criticized by Sayre et al. (40), the previous spin-trapping results and interpretations have not been challenged until now.
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed: Laboratory of
Pharmacology and Chemistry, NIEHS/National Institutes of Health, 111 Alexander Dr., P. O. Box 12233, Research Triangle Park, NC 27709. Tel.: 919-541-2687; Fax: 919-541-1043; E-mail: dikalov{at}niehs.nih.gov. Permanent address: Institute of Chemical Kinetics and Combustion, Novosibirsk 630090, Russia.
2
This program is available to the public via the
World Wide Web (http://epr.niehs.nih.gov/).
 |
ABBREVIATIONS |
The abbreviations used are:
AD, Alzheimer's
disease;
A
, amyloid
;
ESR, electron spin resonance;
OMRF, Oklahoma Medical Research Foundation;
PBN, N-tert-butyl-
-phenylnitrone;
APP, amyloid
peptide precursor;
PSEN, presenilin;
APOE, apolipoprotein E;
HPLC, high
pressure liquid chromatography;
TEMPONE-H, cyclic hydroxylamine
1-hydroxy-4-oxo-2,2,6,6-tetramethylpiperidine.
 |
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