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INTRODUCTION |
Excessive exposures to both nickel and sulfite are known to
produce serious toxic and carcinogenic effects in animals and humans
(1-4). The generation of potentially harmful radicals from sulfite
oxidation by transition metal catalysis has been well established (5).
Nickel salts are physiologically redox-inactive, and therefore nickel
toxicity in vivo is inferred to involve activation by
coordination with peptides and proteins. A variety of catalytic oxidative properties of Ni(II)/peptide complexes have been reported in
the literature. These include decarboxylation of the Ni(II)-Gly-Gly-His complex in the presence of molecular oxygen (6), the ability of
Ni(II)-histidyl peptide complexes to function as Fenton reaction catalysts (7, 8), and the ability of the
Ni(II)-Gly-Gly-L-His complex to catalyze the oxidation and
cleavage of DNA (9, 10) and to promote the formation of intermolecular
protein cross-links (11, 12). Muller et al. (13) and Liang
et al. (14) have recently reported that autooxidation of
sulfite catalyzed by the Ni(II)-complexed Lys-Gly-His-amide tripeptide
can oxidatively damage DNA. They postulate in situ formation
of monoperoxysulfate, a strong oxidant, as an active intermediate in
the damaging effect.
We have recently reported on the identification of a unique Ni(II)
binding site on hemoglobin that, in the presence of monoperoxysulfate, produces N-terminal oxidative deamination, as well as intramolecular cross-linking, both specific to the
-globins (15). In the present study, we have employed small model peptides in order to verify the
structural assignment of oxidative deamination, as well as to identify
the minimal sequence requirements for reaction susceptibility. Additionally, we have tested a system that substitutes sulfite (SO32
) and oxygen (O2) for
potassium peroxymonosulfate
(oxone).1 Our findings show
clearly that histidine at position 2 is a fundamental requirement for
Ni(II)-catalyzed oxidative N-terminal deamination and that sulfite and
ambient oxygen can readily substitute for the potent peroxyl oxidant
oxone. Furthermore, by contrasting some of the differences between
products produced by the sulfite/oxygen and the oxone reactions with
Ni(II) peptides, we can exclude the formation of diffusible
monoperoxysulfate (HSO5
) as a mediator
in SO32
/O2-promoted deamination.
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EXPERIMENTAL PROCEDURES |
Materials--
Sodium sulfite was purchased from Fluka
Biochemika. Ammonium sulfite, potassium peroxymonosulfate (oxone),
nickel chloride, butylated hydroxyanisole, tert-butyl
alcohol, EDTA, mannitol, ethanol, sodium cyanoborohydride, sodium
phosphate (as mono- and dibasic salts), and thiourea were from Sigma.
The peptides bursin (KHG-amide), carnosine (
-alanyl-histidine), and
homocarnosine (
-amino butyryl-histidine) were purchased from Sigma.
KGH-amide was synthesized by SynPep Corp. All other peptides were
purchased from Bachem Bioscience Inc. and used without repurification.
Sulfite Reaction Standard Conditions--
Unless otherwise
noted, reaction mixtures contained 30 mM peptide and 5 mM nickel chloride, to which sodium sulfite was added from
a 10× stock to a final concentration of 60 mM, all in 0.1 M sodium phosphate, pH 8.2. 300-µl reactions were
maintained at room temperature for 24 h under ambient air in
16 × 100-mm glass test tubes protected from light. Reactions were
quenched by addition of 1: 10 (v/v) of 0.5 M EDTA, pH 8, and then diluted 1:10 into 2% formic acid in water and placed at
4 °C until analysis by reversed phase chromatography.
Oxone Reaction Standard Conditions--
Initial reaction
mixtures contained 30 mM peptide and 30 mM
nickel chloride in 0.1 M sodium phosphate, pH 8.2, to which
oxone, also in 0.1 M sodium phosphate, pH 8.2, was added as
a 10× stock to a final concentration of 30 mM. After
approximately 5 min at room temperature, the oxone reactions were
quenched by the addition of (at 1:10 (v/v)) 0.5 M EDTA, pH
8, and then diluted 1:10 into 2% formic acid in water and placed at
4 °C until analysis by reversed phase chromatography.
With time, optimized conditions for the oxone reactions were
established. Where noted as optimized oxone conditions, the peptides, NiCl2, and oxone stocks were made up in 0.2 M
sodium bicarbonate, pH 8.2. Additionally, under optimized conditions,
the oxone was added in five equal increments 2 min apart, rather than
as a single bolus.
Amino Acid Analysis--
Peptides were subjected to gas phase
hydrolysis at 165 °C for 1 h in the presence of HCl containing
1% phenol, using a Savant AP100AminoPrep Hydrolyzer. Amino acids were
analyzed using precolumn derivatization with AQC as described
previously (16).
Derivatization with
2,4,-Dinitrophenylhydrazine--
Derivatizations were performed as
described previously (15).
LC-MS--
Electrospray mass spectrometry was performed using a
Finnigan Mat LCQTM ion trap mass spectrometer interfaced
with an HP1090M HPLC. The LCQTM spectrometer was calibrated as per the
manufacturer's recommendations, which provide for a specified accuracy
of 0.01% for m/z masses of 100-2000 Da. Peptides were
separated at room temperature using a Zorbax 300SB C18 column with a
gradient of 1-40% acetonitrile in 0.1% trifluoroacetic acid/water
(v/v) in 20 min at 1 ml/min, following an initial 4-min isocratic (1%) hold.
NMR Analysis--
1H and 13C NMR spectra
were collected on a Varian VXR-300S instrument. Peptides were dissolved
in either deuterium oxide or deutero-Me2SO. Chemical shifts
are reported relative to DSS for deuterium oxide samples or
tetramethylsilane for deutero-Me2SO samples. 1H
chemical shift versus pH studies were performed using a
D2O-phosphate system. No corrections of pH measurement for
D2O content were made.
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RESULTS AND DISCUSSION |
Reaction of the Ni(II)-complexed Tripeptide Ala-His-Ala with
Peroxymonosulfate Produces N-terminal Oxidative
Deamination--
Reaction of the tripeptide Ala-His-Ala with Ni(II)
and oxone under initial standard conditions resulted in >60%
modification of the peptide. The modified peptide eluted as a broad,
asymmetric peak several minutes later than the parent peptide peak when
analyzed by reversed phase C18 HPLC (Fig.
1, A and B). The
modified peptide displayed significantly increased absorption at 245 nm. Also, the modified peptide displayed a mass of 296.1 Da when
analyzed by in-line mass spectrometry. This represents a discrete loss of 1 Da when compared with the parent peptide (296.1 versus
297.1 Da for the parent peptide). MS2 fragmentation mapped
the 1-Da loss to the A2, B2, and B3
fragment ions (Fig. 2, A and
B). Y-type ions were not detected in either case. The ion of
m/z 227, which can be attributed to Y2
structure, was instead assigned to loss of the C-terminal Ala residue
from the AHA tripeptide (17). Ion fragment assignments were confirmed by MS3. A significant recovery of the histidine immonium
fragment ion was observed (of m/z 110), which further
indicated that the histidine residue was unaffected. These findings all
suggested that the 1-Da mass loss was located on the the N-terminal
alanine, and the histidine residue remained unchanged.

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Fig. 1.
Reversed phase C18 HPLC separation of
Ala-His-Ala reactions under standard conditions in 0.1 M
phosphate buffer, pH 8.2. A, parent peptide;
B, after reaction with Ni(II)/oxone; C,
Ni(II)/oxone oxidation followed by treatment with NaCNBH3;
D, after reaction with oxone, without Ni(II).
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Fig. 2.
MS2 fragmentation spectra.
A, parent AHA peptide; B, Ni(II)/oxone-reacted
peptide ( -ketoamide); C, oxidized and NaCNBH3
reduced peptide ( -hydroxyamide) eluting at 6.4 (solid
line) and 7.3 (dotted line) min as shown in Fig.
1C; D, modified peptide from oxone reaction
without Ni(II) (oxime). Annotation of fragment ions refers to parent
peptide.
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The modified peptide was blocked to Edman protein sequencing,
suggesting that a chemical modification had occurred at the N terminus.
This finding was corroborated by a lack of reactivity with AQC reagent,
which was used to probe for the presence of primary or secondary
amines. Amino acid analysis of the modified peptide showed
approximately one-half of the yield of alanine relative to histidine
that was observed for the parent peptide. Also in contrast to the
parent peptide peak, the modified peptide peak reacted quantitatively
with DNP, yielding a derivatized peptide with 400 nm absorbance and
mass of 476.1 Da (Fig. 3). This
represents a mass gain of 180 Da, which is characteristic of a
dinitrophenylhydrazone product and therefore indicative of the presence
of a free carbonyl on the modified peptide. The presence of a free
carbonyl, the apparent loss of free amino group reactivity, and the
1-Da mass decrease all indicated that the modified peptide was
oxidatively deaminated at the N terminus, thus resulting in an
-ketoamide peptide (Reaction RI).

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Fig. 3.
Reversed phase separation of DNP-reacted
peptides tested for the presence of a free carbonyl group.
A, blank; B, parent AHA peptide; C,
Ni(II)/oxone-reacted peptide ( -ketoamide). Results indicate that
oxidized Ala-His-Ala peptide contains a reactive carbonyl group. The
observed mass of 476.1 Da is consistent with a
dinitrophenylhydrazone derivative of the oxidized peptide
( -ketoamide).
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1H and 13C NMR analyses of the modified
peptide peak were used to further verify this structural assignment.
The 1H spectrum of the (putative) oxidatively deaminated
peptide displayed a shift of the N-terminal alanine methyl group
downfield from 1.23 to 2.32 ppm along with a resonance splitting change
from a doublet to a singlet, consistent with formation of a ketone at
the
-carbon of Ala-1 (Table I). The
C-1 vinyl proton of the histidine imidazole ring was observed to be
significantly deshielded relative to the parent peptide. However, an
additional study that examined the effect of pH versus
1H chemical shift of the histidine C-2 vinyl proton
demonstrated no significant change in pKa of the
imidazole group following Ni(II)/oxone oxidation, yielding further
evidence that the histidine side chain was not modified in the reaction
(Fig. 4). 13C NMR also
indicated the presence of an intact histidine imidazole ring, two amide
carbons, one carboxylic acid carbon, and a downfield ketone carbonyl
resonance at 196 ppm, all in agreement with oxidative deamination
(Table II).

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Fig. 4.
pH dependence of the 1H chemical
shift of the C-2 vinyl proton of His-2 imidazole side chain of parent
AHA and Ni(II)/oxone-reacted peptide
( -ketoamide).
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Sodium cyanoborohydride treatment of the modified peptide peak yielded
quantitative reduction of the Ni(II)/oxone-oxidized AHA peptide,
resulting in a species that eluted earlier in the reversed phase
gradient as a doublet of two apparent diastereomers, both exhibiting
mass gains of 2 Da (Fig. 1C). These peaks were structurally
assigned as diastereomeric alcohols derived from reduction of the
-keto carbonyl. MS2 fragmentation (Fig. 2C),
as well as 1H NMR analysis of these peaks, confirmed this assignment.
AQC derivatization of the AHA deamination reaction mixture analyzed by
LC-MS yielded evidence of free ammonia released in the reaction. The
amount detected was commensurate with the amount of peptide oxidatively
modified, showing that deamination was concomitant with the
proportional release of free ammonia.
The same oxone reaction on the AHA tripeptide in the absence of added
Ni(II) was tested. It was found that when Ni(II) was left out of the
reaction, under otherwise identical conditions, about 50% of the
peptide was modified. The modified species exhibited an increased
absorbance at 250 nm and eluted during the reversed phase HPLC gradient
as a sharp, symmetrical peak later than either the parent peptide or
the oxidatively deaminated species (Fig. 1D). LC-MS analysis
of this peak showed a mass gain of 14 Da over the parent peptide mass
(311.1 versus 297.1 Da for the parent). This modified peak
was found to be blocked to Edman chemistry sequencing, suggesting that
a chemical modification had occurred at the N terminus. In contrast to
the oxidatively deaminated AHA peptide peak, this modified peptide peak
was unreactive with DNP, suggesting the absence of any free carbonyl
group. MS2 ion trap fragmentation mapped the 14-Da gain to
the A2, B2, and B3 fragment ions
(Fig. 2D). From these findings, we postulated that this
modification was likely the result of chemical oxidation of the
terminal amine nitrogen to an oxime (18). 1H proton NMR
analysis confirmed this assignment. The N-terminal methyl resonance was
shifted downfield from 1.23 to 1.85 ppm and resonated as a singlet.
Also observed was the appearance of a single, sharp resonance far
downfield at 11.86 ppm, which is consistent with an oxime proton.
Reaction of the Ni(II)-complexed Tripeptide Ala-His-Ala with
Sulfite and Oxygen Produces Identical N-terminal Oxidative
Deamination--
Muller et al. (13) have postulated the
in situ formation of HSO5
from SO32
in the presence of ambient
oxygen, Ni(II), and the tripeptide Lys-Gly-His-amide. Although Ni(II)
complexes of this sequence motif have been reported to exhibit
catalytic properties different from that of histidine at position 2 (7), we decided to test for indications of peroxymonosulfate formation
from sulfite autooxidation on a peptide with histidine at position 2.
The tripeptide Ala-His-Ala was incubated with sodium sulfite in the
presence of nickel under the standard conditions described above. This
treatment was observed to produce modification of >60% of the
peptide. The modified species displayed the same elution time, 1-Da
mass loss, and MS2 fragmentation pattern by LC-MS analysis
as the oxidatively deaminated
-ketoamide species produced using
oxone. Additional chemical tests, including Edman sequencing, DNP, and
AQC reactivity, as described above for the modified product of AHA
using Ni(II) and oxone (excepting proton NMR analysis), confirmed that
an identical oxidatively deaminated AHA peptide was produced using
Ni(II), sulfite, and ambient oxygen.
The sulfite reaction with AHA in the absence of added nickel was
tested. It was found that in contrast to the oxone reaction, no oxime
formation was observed. In fact, when nickel was omitted from the
reaction of the AHA tripeptide with sulfite, no detectable modification
of any kind was observed.
Reaction of the Ni(II)-complexed Tripeptide Ala-Ala-Ala Shows No
Evidence of Oxidative Deamination with Either
SO32
/O2 or Oxone--
We have
previously postulated that histidine at position 2 is an essential part
of an oxidatively reactive Ni(II) binding site on the
-chain of
human hemoglobin (15). Therefore, as a comparison with the AHA peptide,
we examined the reactivity of the tripeptide AAA. Reaction of the AAA
tripeptide with Ni(II) and oxone under standard conditions also
resulted in >60% modification of the peptide. This modified peptide
eluted primarily as a sharp, symmetrical peak several minutes later
than the unmodified parent peptide peak when analyzed by reversed phase
C18 HPLC (Fig. 5, A and
B). The modified peptide displayed increased absorption at
250 nm and a discrete mass gain of 14 Da when analyzed by LC-MS compared with the parent peptide (245.1 versus 231.1 Da for
the parent peptide.) This species was found to be blocked to Edman sequencing. MS2 fragmentation also indicated that the
modification was located on the N-terminal amino acid. Based on these
findings, we concluded that this modification was an oxime on the
N-terminal nitrogen. When nickel was omitted from the oxone reaction
with the Ala-Ala-Ala tripeptide, an identical product was observed
(Fig. 5C). Therefore, oxime formation appears to be entirely
non-nickel-dependent.

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Fig. 5.
Reversed phase C18 HPLC separation of
Ala-Ala-Ala tripeptide reacted under standard conditions in 0.1 M phosphate buffer, pH 8.2. A, parent
peptide; B, after reaction with Ni(II)/oxone; C,
after reaction with oxone, without Ni(II).
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Reaction of the tripeptide Ala-Ala-Ala with Ni(II) and sodium sulfite
under standard (SO32
) conditions
produced no detectable modification of any kind. This finding suggests
that in the case of the Ala-Ala-Ala tripeptide, no
peroxymonosulfate-like species is generated by that peptide in the
presence of Ni(II), SO32
, and ambient
oxygen. Histidine at position 2 therefore appears to be required for
both susceptibility to oxidative deamination, as well as the apparent
ability to generate a peroxymonosulfate-like species from sulfite and oxygen.
Sequence Effects Studies Using Small Peptides Reacted with Ni(II)
and Either SO32
/O2 or
Oxone Show an Absolute Requirement for Histidine at Position 2 for
Oxidative Deamination--
To further delineate sequence effects and
requirements for susceptibility to oxidative deamination, we studied
reactions of Ni(II) and a series of small peptides with either
SO32
/O2 or oxone as the
oxidant. We examined the reactions for any evidence of modification of
the parent peptide. The results of these experiments are shown in Table
III.
Almost all of the peptides with histidine at position 2 demonstrated
significant susceptibility to oxidative deamination under both sets of
conditions, whereas none of the peptides lacking histidine at position
2 demonstrated any oxidative deamination under either condition. It
should be noted that in the cases of both AHA and GHG, a significant
amount of the deaminated product from reaction at 30 mM
peptide was found to be in the form of apparently diastereomeric pairs
of relatively stable dimers (exhibiting a mass of 592 Da in the case of
AHA peptide). These dimers were not observed when the
SO32
/O2 reaction was
performed at a peptide concentration of 3 mM, indicating
that their formation was (peptide) concentration-dependent. Further characterization of the dimer by 1H NMR indicated
it to be a product of aldol condensation of two molecules of
-ketoamide (data not shown). These dimers were not observed when
oxone was used as the oxidant.
Unlike Ala-His, the dipeptides carnosine (
-alanyl-histidine) and
homocarnosine (
-amino butyryl-histidine) were found to be
nonreactive to
Ni(II)/SO32
/O2-promoted
deamination. These results suggest that only
-amines are susceptible
to this pathway of deamination. Interestingly, these peptides showed
very little oxime formation in the reaction with oxone.
Peptides with proline as the first residue also produced noticeably
different results. N-terminal proline, being a secondary amine, is not
likely to be as susceptible to the oxidative release of ammonia as
other amino acids. No released ammonia in either reaction with PHA was
detected, and in both cases, a modified peptide that was formed
demonstrated a loss of 2 Da (321 versus 323 Da for the
parent peptide). The modified peptide was unreactive with AQC,
indicating that the secondary amine was no longer present. This result
would be consistent with formation of a cyclic imine species that was
stable against spontaneous hydrolysis.
Peptides GGH and KGH-amide with a histidine residue in position 3 were
also tested. Under similar conditions, these peptides have been shown
by Muller et al. (13) and others (12, 14, 19) to produce
radicals capable of causing DNA damage and protein cross-linking. No
deamination of either the GGH or the KGH-amide peptides were detected
in reactions with
Ni(II)/SO32
/O2. The
KGH-amide peptide was modified in the reaction to an earlier eluting
product exhibiting a mass gain of 80 Da, consistent with the
displacement of one proton by an SO3H group. This product was not further characterized. In the reactions with oxone,
decarboxylation of GGH and other peptide degradation of both peptides
were observed, none of which appeared to include deamination. By
contrast, the tripeptide KHG-amide (bursin) appeared to readily
N-terminally deaminate with both
Ni(II)/SO32
/O2 and
Ni(II)/oxone. However, the ketoamide product appeared to spontaneously
form an intramolecular Schiff's base with the
-amine of the lysyl
side chain (Reaction RII). This was confirmed by LC-MS, Edman
sequencing, and reaction with DNP. Under these conditions, no evidence
of
-amino side chain deamination was found.
Peptides with methionine and tyrosine as the N-terminal residues
also appeared to present exceptions to susceptibility to oxidative
deamination. The lack of reactivity of MH using sulfite is possibly due
to sulfoxide formation acting as a "sink" for oxidative
modification, although no substantial difference in the amount of
methionine sulfoxide formed was seen relative to ML with either oxidant
(20, 21). It is also conceivable that other chemical and electronic
properties of the sulfur atom of the methionine side chain could affect
the reaction pathway. The lack of deamination of the dipeptide YH is
less clear. Stable tyrosine radicals have been reported in proteins
(22, 23). However, no evidence of dityrosine, a known product of
oxidative degradation of tyrosine involved in protein cross-linking,
was detected (12, 22), and no evidence of tyrosine radical was found by
EPR or found to be trappable by the method of Fenwick and English (24).
However, with an increased excess of sulfite, we noticed significant
accumulation of a product of the YH reaction, which exhibited a mass of
354 Da (gain of 36 Da). Under standard conditions, except using 4:1
ratio of sulfite over peptide, the reaction resulted in a 30% yield of
this modification. Mass gain, MS2 fragmentation, and Edman
protein sequencing were all consistent with C-terminal decarboxylation
followed by the attachment of the SO3H group forming a
sulfo-peptide (Reaction RIII). We believe that such a product would most
likely be formed through a radical type of reaction, potentially
promoted by the tyrosine side chain (25). Analogous products were
detectable with other first residue side chains in dipeptides
containing His-2, but with yields at least 1 order of magnitude lower.
The YH sulfo-peptide product was not observed when Ni(II) was omitted
from the reaction. The Ni(II)/oxone reaction with YH produced a small
but detectable yield of oxidative deamination. Additionally, a
significant amount of other uncharacterized degradation occurred
including formation of a yellow precipitate.
In all cases, oxone produced a significant yield of oxime in
peptides lacking histidine at position 2. This finding provided us with
a means to look for evidence of the generation of a diffusible peroxymonosulfate species from peptide/Ni(II) reactions with sulfite. This could be tested by including the AAA tripeptide in reactions with
His-2-containing peptides and monitoring for oxime formation. These
conditions resulted in substantial deamination of the susceptible peptides, as was seen with oxone addition. No trace of oxime formation was detectable on the AAA tripeptide when it was combined with the LH
dipeptide during standard
Ni(II)/SO32
/O2 conditions.
Therefore, it can be inferred that the oxidative deamination reaction
did not generate any appreciable quantities of diffusible
peroxymonosulfate. However, in the case of all His-2 peptides, an
in situ formation of a peroxymonosulfate-like species, which
is either constrained from diffusing or consumed in a subsequent reaction too quickly to do so, is plausible.
Nickel Titration Studies of LH Dipeptide Reacted with Ni(II) and
Either SO32
/O2 or
Oxone--
A nickel titration study of the
SO32
/O2 reaction was
conducted using the dipeptide LH under standard conditions. The results showed that in the absence of nickel, no oxidative deamination occurred. At a Ni(II) to peptide ratio of 1:10, about 40% of the parent peptide was deaminated (i.e. 4 equivalents of
deaminated peptide per 1 equivalent of Ni(II)), indicating a catalytic
turnover of 4. Using a 1:10 ratio of nickel, and 4 equivalents of
sulfite, the catalytic turnover of Ni(II) increased to about 8.5. Increasing the Ni(II)/peptide ratio above 1:5 did not have a
significant effect on the yield of deamination (Fig.
6). Therefore, a 1:5 ratio of
Ni(II):peptide was selected as an optimal for the standard deamination
reaction with sulfite.

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Fig. 6.
Yield of oxidative deamination
versus Ni(II) to LH peptide ratio for oxone and
SO32 /O2 reactions under
optimized conditions as described under "Experimental Procedures"
(30 mM dipeptide and 30 mM oxone or
sulfite).
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A similar Ni(II) titration study using the LH dipeptide was conducted
with oxone. In contrast to the
SO32
/O2 system, under
initial conditions, maximal oxidative deamination required nearly 1:1
Ni(II):peptide when oxone was used. However, under optimized conditions
for the oxone reaction, the oxone was added to the reaction in five
equal increments, rather than as a single bolus. In this case, 30%
deamination was achieved using approximately 1:5 Ni:peptide (catalytic
turnover of 1.5), suggesting that redox cycling of Ni(II) can be
achieved in the oxone reaction as well (Fig. 6). Chromatograms of this
"optimized" oxone reaction, along with a standard condition sulfite
reaction, both with the LH dipeptide at 1:5 Ni(II):peptide, are shown
in Fig. 7. It can be seen from these that
the sulfite reaction results in significantly decreased degradative
side products compared with oxone. It appeared that when nickel was
limiting, oxime formation was a potentially competing side reaction. We
therefore purified the LH oxime species and re-reacted it with Ni(II)
and oxone under standard conditions. No evidence of oxidative
deamination was observed, although significant heterogeneous,
uncharacterized peptide degradation occurred. This finding suggested
that oxime formation may be a competitive and kinetically relevant
"dead-end" side reaction when Ni(II) is limited during reaction
with oxone.

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Fig. 7.
Reversed phase C18 HPLC separation of
oxidative deamination reactions of LH dipeptide under optimized
conditions. Top panel, 24-h reaction with
Ni(II)/sulfite/oxygen; bottom panel, 10-min reaction with
Ni(II)/oxone.
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Reaction Kinetics for Oxidative Deamination of LH Dipeptide Reacted
with Ni(II) and Either
SO32
/O2 or
Oxone--
Studies were conducted to examine the kinetics of oxidative
deamination of the LH dipeptide comparing the use of either
SO32
/O2 or oxone (with
sequential addition) as oxidants (Fig.
8). As can be seen, the oxidative
deamination using oxone as the oxidant was completed within (at most)
several minutes of initiation. By contrast, the reaction using
SO32
and ambient oxygen displayed a
distinctly lower initial rate of deamination, which appeared to be
relatively constant during the first 40 min of reaction. No further
increase in deamination was observed beyond 6 h (up to 1 week).
When this reaction was repeated under an oxygen-enriched head space
(>95% O2 at approximately 2 atm), no difference in the
rate of peptide modification was observed. This finding suggested that
oxygen diffusion is not rate-limiting under the standard conditions
with sulfite. However, when oxygen was strictly excluded from the
sulfite reaction (under argon), no peptide modification was observed.
By contrast, exclusion of oxygen from the oxone reaction did not
inhibit oxidative deamination.

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Fig. 8.
Time course of oxidative deamination of LH
peptide for oxone and
SO32 /O2 reactions under
optimized conditions as described under "Experimental Procedures"
(30 mM dipeptide and 30 mM oxone of
sulfite).
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Quenching and Inhibition Studies of Deamination of LH Dipeptide
Reacted with Ni(II) and Either Sulfite or Oxone--
Further studies
were conducted to examine the effect of the addition of various
compounds on the yields of oxidative modification in the LH/Ni(II)
reactions using either sulfite or oxone. These additives included known
chelating agents, radical scavengers, and different anions. The results
of these experiments tabulated as percentage of inhibition of LH
deamination under the respective standard conditions are listed in
Table IV.
The lack of inhibition observed in the presence of ethanol, methanol,
tert-butyl alcohol, or mannitol argues strongly against the
involvement of diffusible SO
4 or OH· radicals mediating
the oxidative deamination in either reaction (2, 26). The inability of
catalase to inhibit either reaction suggests that it is unlikely that
oxidative deamination in either case proceeds through formation of
diffusible H2O2 followed by a Fenton-type
reaction, as in a mechanistic scenario for oxidative protein carbonyl
formation proposed by Stadtman (27). However, no test was conducted to
determine whether this enzyme was inactivated by the reaction
conditions, as has been reported for sulfite inactivation of several
copper-dependent monooxygenases (28). The complete inhibition of oxidative deamination by the metal chelating agent EDTA
would suggest that sequestration of nickel away from the peptide is the
mode of action by which it precludes the reactions. The mechanism for
inhibition exhibited by thiourea is less clear, but the differential
effects of CN
and HCO3
on reactions using SO32
/O2
versus using HSO5
begins to
suggest that these may act by perturbing the coordination structure of
the reactive nickel complexes (29). Such differential effects also
begin to suggest that the reactive complex that produces an actively
oxidizing species from SO32
may differ
from the one involved in the reaction with preformed HSO5
(oxone).
Comparative Sulfite and Oxone Titration Studies with
Ni(II)-complexed LH Dipeptide--
Titration studies under standard
conditions using oxone as the oxidant showed that approximately 1:1
oxone:peptide was sufficient to approach maximal deamination of the LH
dipeptide (Fig. 9). It was also observed
that
2:1 oxone:peptide resulted in significant, heterogeneous,
uncharacterized degradation of the peptide (much worse than the 1:1
shown in Fig. 7). By contrast, titration with SO32
under standard conditions
reproducibly showed that significantly more than 1 equivalent of
SO32
to peptide was required to
approach maximal deamination. With 3 equivalents of sulfite, more than
60% of the peptide was oxidatively deaminated, with less than 20% of
degradative side-products observed.

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Fig. 9.
Yield of oxidative deamination
versus oxidant to LH peptide ratio for oxone and
SO32 /O2 reactions under
optimized conditions as described under "Experimental Procedures"
(30 mM peptide and 6 mM Ni).
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pH Effect Studies of LH Dipeptide Reacted with Ni(II) and Either
Sulfite/O2 or Oxone Show High Relative Yields of Oxidative
Deamination throughout a Physiological pH Range--
Reactions with
the LH dipeptide/Ni(II) system using oxone or sulfite/O2
were examined for effects of reaction pH, the results of which are
shown in Fig. 10. Although the oxone
reaction showed inhibition below pH 6.5, both reactions displayed a
high relative yield of oxidative deamination over the physiologically
relevant pH range of 6.5-8.5. This finding lends support to the
possibility that this model system for oxidative peptide modification
may be relevant to mechanisms of nickel and sulfite toxicities in vivo (1, 3).

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Fig. 10.
pH versus yield of
oxidative deamination of LH peptide for oxone and
SO32 /O2 reactions under
optimized conditions as described under "Experimental
Procedures."
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Conclusions--
We have shown that the reaction of peptides
containing histidine at position 2 with Ni(II) and oxone produces
oxidative deamination of the peptides with a minimum of other
significant modifications of the peptide, and we found no evidence of
modification of the histidine residue (up to 1 equivalent of oxone,
above which other degradative modifications occur). When Ni(II) was
omitted from the reaction, oxime formation on the N-terminal nitrogen
was produced instead. In peptides lacking histidine at position 2, only
oxime formation was observed, regardless of whether Ni(II) was present or not.
The identical oxidative deamination of peptides containing His-2 can be
produced by using Ni(II), sulfite, and ambient oxygen. A
substoichiometric amount of nickel can serve to deaminate the peptides
in high yields, indicative of redox cycling of Ni(II) in the reaction.
However, when either nickel, sulfite, or oxygen was excluded, no
oxidative deamination or any other modification was observed. No
oxidative deamination was observed in any peptides lacking histidine at
position 2. Significant modifications other than deamination were
observed in reactions with both GGH and KGH-amide peptides. These
peptides have been reported to promote protein cross-linking and DNA
damage under similar conditions. Additionally, the GGH peptide is known
to undergo oxidative degradation, including decarboxylation in the
presence of ambient oxygen or other oxidizers (6, 14, 30). These
results, along with our finding of oxidative deamination only on
-carbon amines in His-2 peptides, indicate a unique structural
specificity for a sequence motif that was first discovered in the
-chain of human hemoglobin (15).
The finding that both sulfite/O2 and oxone produce an
identical
-ketoamide suggests that both pathways may share a common reactive intermediate. The Ni(II)-catalyzed formation of
peroxymonosulfate from sulfite and oxygen would appear to be consistent
with these results (13). However, co-incubation of peptides lacking
histidine at position 2 exposed to the Ni(II), sulfite, and oxygen
system showed no evidence of oxime formation, suggesting that no
diffusible peroxymonosulfate is produced during the deamination
reaction. Additional quenching studies using well characterized radical scavengers appear to rule out diffusible OH· or SO
4 as
mediating any of the oxidative deamination observed (2, 26).
We postulate that the reactions of Ni(II) complexed His-2 peptides with
either oxone or an in situ formed, nondiffusing,
peroxymonosulfate-like species generated catalytically by sulfite
autooxidation produce a common imine intermediate. All of our results
would be consistent with a pathway involving imine formation at the N
terminus, followed by spontaneous hydrolysis (with the exception of
proline, which apparently forms a stable cyclic imine). The finding of
ammonia release in all cases of oxidative deamination is consistent
with such a pathway. This postulated pathway is shown in Fig.
11. Imine formation in oxidative
deamination has been postulated by Stadtman (27) to be preceded by
radical formation on the carbon adjacent to the departing amine group.
If such is the case for Ni(II)-catalyzed N-terminal oxidative
deamination, the radical appears inaccessible to quenching by common
radical scavengers, i.e. a type of "caged" radical.
Results with YH dipeptide suggest that the (tyrosyl) side chain can
redirect the outcome of the reaction of such a complexed radical.

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Fig. 11.
Proposed scheme of Ni(II)-catalyzed
oxidative modification of peptides containing a His residue in position
2. R1, side chain of amino acid
residues other than Pro and Met. R2, side
chain of amino acid residues other than His. Im, imidazole
side chain of His residue.
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These findings are of interest from several perspectives. They provide
a defined, potentially physiologically relevant model system with which
to study the mechanism of a metal-catalyzed protein carbonyl formation,
a modification known to be a frequent consequence of oxidative damage
to proteins in vivo (31, 32). Such a defined system may
provide an opportunity for further elucidating mechanisms underlying
the known toxicities of sulfite, a ubiquitous environmental contaminant
(2, 3). Additionally, our findings point toward a facile method for the
intentional introduction of a carbonyl "handle" into natural and
engineered proteins and peptides, under conditions that produce
relatively few undesirable side reactions, a technology of
significant potential utility (33, 34).