Complexation of Ni(II) with native state
recombinant hemoglobin is shown to produce
NH2-terminal deamination and globin cross-linking in
the presence of the oxidant potassium peroxymonosulfate
(OxoneTM). Both the oxidative deamination and cross-linking
are exclusive to the
chains. Recombinant hemoglobin mutants have
been created to identify protein sequence requirements for these
reactions. It was found that His-2 of the
globin is required for
redox active Ni(II) complexation, oxidative deamination, and
cross-linking. The oxidative deamination results in the formation of a
free carbonyl in place of the NH2-terminal amine of the
chain. Most cross-linking of the
globin occurs intramolecularly,
forming
globin dimers. Structural characterization of the
globin dimers indicates the presence of heterogeneous cross-links
within the central hemoglobin cavity between the NH2
terminus of one
chain and the COOH-terminal region of the
other.
 |
INTRODUCTION |
Transition metal-catalyzed oxidative damage to proteins has been
implicated in a variety of adverse physiological processes including
aging (1), artherosclerosis (2), and ischemic reperfusion injury (3).
The metal binding site-specific nature of metal-catalyzed oxidation of
proteins has been well established (4, 5). Oxidative modification of
proteins in vivo has been postulated to produce carbonyl
groups on amino acid side chains, including the formation of
-glutamyl semialdehyde from arginine (6), and 2-amino adipic
semialdehyde from lysine (7). Investigators have used model systems
employing Fe(II) and H2O2 to study oxidative modification of proteins and peptides in vitro, and the
physiological relevance of such Fenton chemistry models in elucidating
mechanisms of in vivo oxidation of proteins is widely
accepted (7, 8).
An application of this type of chemistry to intentionally effect
oxidative intermolecular cross-linking specific to complexed proteins
in vitro has been recently reported (9-12). That technique typically employs the addition of an exogenous tripeptide GGH, nickel(II), and strong oxidant (peracid), which together form a high
valent metal complex that propagates covalent bond formation between
associated proteins. Nickel(II) complexes of histidyl peptides have
also been reported to exhibit Fenton reaction activity with hydrogen
peroxide (13).
Previously, the investigation of metal-catalyzed protein oxidation of
hemoglobin and other heme proteins has been complicated by the
interaction of the heme iron with peroxides. This reaction can produce
significant heme oxidation and protein degradation through the
formation of the highly unstable ferryl-heme species (14). We report
here a reaction of hemoglobin and oxidants under conditions in which
oxidative cross-links between protein subunits can be generated without
causing appreciable heme iron oxidation, protein denaturation, or
aggregation. A metal binding site on hemoglobin appears to promote
covalent bond formation between the
globin subunits upon addition
of oxone1 in the presence of
nickel. This reaction also results in a substantial yield of a
modification that we have identified as oxidative deamination of the
globin amino terminus, generating in its place an
-ketoamide. Results from our studies indicate that the
globin His-2 is required for redox active nickel complexation, oxidative deamination, and intramolecular cross-linking.
 |
MATERIALS AND METHODS |
Recombinant Hemoglobins--
Expression of recombinant
hemoglobin in Escherichia coli has been previously reported
(15, 16). Recombinant human hemoglobin rHb69 was genetically engineered
with the replacement of the wild type valine residues at all
NH2 termini with methionine, and a fusion of two
globins with a single glycine amino acid linker. All other recombinant
hemoglobins were
globin variants of rHb69 and were expressed in the
same Escherichia coli background. In the expression system
used, the initiating methionine residue is quantitatively removed by
endogenous E. coli methionyl-aminopeptidase when the second
amino acid expressed was alanine but was fully retained when the second
residue was histidine or leucine (17). All mutants used in this study
had an identical di-
globin and the differences between them
occurred only in the amino- and carboxyl-terminal segments of the
globin, which are listed in Table I.
Directed mutagenesis by polymerase chain reaction was used to create
the
globin mutants with amino acid substitutions after the
initiating methionine codon. Separate oligonucleotides were used to
introduce these changes to the
subunit by amplification from a
rHb69
globin gene template, and subsequent cloning of the
polymerase chain reaction fragments into a high copy number vector for
expression (18). Candidates were screened by restriction digestion and confirmed by DNA sequencing. All recombinant hemoglobins were purified
from E. coli expression strains by a procedure previously reported (19). The hemoglobins were sparged with carbon monoxide and
buffer exchanged into 0.1 M sodium phosphate, pH 8.3, by
Sephadex G25 chromatography. Final primary sequence of each mutant was confirmed by both automated Edman sequencing and LC-MS analysis.
Cross-linking Reaction Conditions--
Cross-linking reactions
were carried out in 1.6-ml Eppendorf tubes in a total volume of 100 µl in 0.1 M sodium phosphate, pH 8.3, with final
hemoglobin concentration of 0.4 mM, and a final nickel
chloride (Aldrich) concentration of 0.8 mM. Hemoglobin solutions were retreated with carbon monoxide by a brief head space
sparge, after which a 16 mM solution of oxone (from Sigma) in 0.1 M sodium phosphate, pH 8.2, was added to a final
concentration of 1.6 mM, and the tubes were placed on ice
for 1 h under an argon head space. Reactions were quenched by
addition of EDTA to a final concentration of 10 mM. Where
noted, quenched reactions were treated with reductant by addition of 10 µl of 1 M DTT (Bio-Rad) or 10 µl of 1 M
sodium cyanoborohydride (Sigma) both in 0.1 M sodium phosphate, pH 8.2. The reactions with DTT and sodium cyanoborohydride were placed under argon at room temperature for 10 min or 2 h, respectively, prior to precipitation of the globins with acidified acetone (20).
Analytical C3 HPLC Separation--
Recombinant hemoglobin was
diluted to 2 mg/ml in HPLC grade water. Where specified, heme was
removed from globins by precipitation with 20 volumes of cold 0.6% HCl
in acetone and resolubilized in 2% (v/v) formic acid to a final
concentration of 2 mg/ml. Approximately 100 µg of protein was
injected onto an HP 1090IIM using a Zorbax 300SB-C3 HPLC analytical
column (0.46 × 25-cm) at 40 °C. Solvent A was 0.1% (v/v)
trifluoroacetic acid in water and solvent B was 0.1% (v/v)
trifluoroacetic acid in acetonitrile. The flow rate was maintained at 1 ml/min. Gradient conditions were as follows: isocratic 35% solvent B
for 5 min, then the percentage of solvent B was increased to 49% over
45 min. Eluant was monitored at 215, 280, and 400 nm. In some cases the
eluant was also monitored for detection of di-tyrosine by the method of
Heinecke et al. (21) using a Hewlett-Packard 1046A
programmable fluorescence detector employing excitation and emission
wavelengths of 325 and 410 nm, respectively.
SDS-PAGE Analysis--
Hemoglobin from native state reactions
(approximately 15 mg/ml) were diluted 1:5 with water and then 1:1 with
NOVEXTM SDS sample buffer. Where noted, the reducing (2×)
sample buffer contained 100 mM DTT. Sample lanes were
loaded with 30 µl/well. Lyophilized reversed phase HPLC globin
fractions were resolubilized in 1× reducing sample buffer containing
50 mM DTT and placed for 10 min at 70 °C. SDS-PAGE
analysis was performed using NOVEX 228 4-20% Tris/glycine gels per
manufacturer's instructions. Protein loads were about 20 µg/well.
Nondenaturing Size Exclusion Chromatography of Native State
Hemoglobin--
Hemoglobins, including those taken directly from
reactions, were diluted to 1 mg/ml in phosphate-buffered saline, pH
7.8. Injection volumes of 50 µl were analyzed using a Pharmacia
Superose 12 HR column (1 × 30-cm) with a flow rate of 0.5 ml/min
in phosphate-buffered saline, pH 7.8. Chromatography was performed
using an HP1090M (Hewlett-Packard) HPLC and monitored at 280 and 400 nm.
Electrospray Mass Spectrometry--
Electrospray mass
spectrometry (LC-MS) was performed using a Finnigan MAT LCQ 228 interfaced with an HP1090M as described previously (22).
Trypsin Mapping--
Tryptic mapping was performed using
Poroszyme trypsin cartridges (22). Separation was performed using a
Zorbax 300SB-C18 column (0.46 × 25-cm). The eluant was monitored
at 215, 280, and 400 nm.
Pepsin Mapping--
Pepsin mapping was performed using
immobilized enzyme from Pierce. Dried globins were resolubilized to
approximately 2 mg/ml in 2% formic acid, and enzyme suspension was
added to approximately 1:4 v/v. Digests were incubated at 30 °C with
gentle shaking for 2 h after which the immobilized enzyme was
removed by microcentrifugation. 50 µl of the reaction supernatant was
injected for reversed phase HPLC chromatography using a Vydac C18
column. Separation conditions were as follows: isocratic 1% B for 5 min, then buffer B was linearly increased to 70% B over 69 min at a
flow rate of 1 ml/min. Buffer A was 0.1% trifluoroacetic acid (v/v) in
water and buffer B was 0.1% trifluoroacetic acid (v/v) in
acetonitrile. The eluant was monitored at 215, 280, and 400 nm.
Detection of Ni(II) Complexation--
The stoichiometry of
Ni(II)-rHb binding in phosphate-buffered saline at pH 7.4, 25 °C,
rHb (4 µM) was determined by titrating with
Ni(II)SO4 (Sigma). The absorbance change at 240 nm was
monitored using a Shimadzu-2101 UV-visible spectrophotometer and
plotted as normalized change versus the ratio of
Ni(II):Hb.
Derivatization with 2,4-Dinitrophenylhydrazine--
Using a
modified procedure of Levine et al. (23), the reaction
mixture for derivatization of lyophilized reversed phase HPLC separated
peptides was prepared as follows: a room temperature saturated solution
of DNP (Aldrich) was prepared fresh in neat methanol and centrifuged
briefly at 10,000 × g. The supernatant was diluted 1:1
with deionized water and centrifuged again. The DNP supernatant was
added to substrate using 50 µl/100 µg of peptide. Concentrated HCl
was then added at 1 µl per 50 µl, and the reaction mixture placed
at room temperature for 1 h. Reactions were diluted 1:1 with 2%
formic acid in water prior to reversed phase HPLC-MS analysis.
In the case of reactions with native state hemoglobin, the DNP
supernatant was mixed 1:1 with the EDTA-quenched
hemoglobin/nickel/oxone reaction mixtures and the combined mixture was
placed at room temperature for 1 h. Hemoglobin was then
precipitated with acidified acetone prior to peptide mapping (22).
Protein Sequencing--
The sequence of proteins and peptides
was determined by automated Edman degradation chemistry using a Porton
2090 gas phase sequencer. Beckman peptide supports were used as
recommended by the manufacturer. PTH-amino acids were identified by
reversed phase chromatography on a modified Hewlett-Packard 1090L HPLC using a Hewlett-Packard AminoQuant column.
Amino Acid Analysis--
Protein and peptides were subjected to
gas phase hydrolysis at 165 °C for 1 h in the presence of HCl
containing 1% phenol, using a Savant AP100 AminoPrep hydrolyzer. Amino
acids were analyzed using precolumn derivatization with
6-aminoquinolyl-N-hydroxysuccinimidyl carbamate as described
previously (24).
 |
RESULTS |
Reaction of Human Hemoglobin A0 and Oxone in the
Presence of Nickel(II) Produces Nonreducible, Intramolecularly
Cross-linked Globin Dimers--
The reaction of carbon monoxide
liganded human hemoglobin A0, Ni(II), and oxone at pH 8.2, produced a substantial amount of dimerized globins as measured by
denaturing SDS-PAGE analysis (Fig. 1,
lanes 2 and 3). Addition of Ni(II) only showed no
change from the control. Following treatment with DTT, the reacted
hemoglobin still contained a similar amount of dimeric globin species,
which were not observed in the DTT-treated control reaction (Fig. 1, lanes 4 and 5). In contrast, nondenaturing size
exclusion chromatography of the Ni(II)/oxone-treated hemoglobin showed
less than 2% in total content of higher molecular weight species
(i.e. greater than 64 kDa), indicating that relatively
little intermolecular protein cross-linking was caused by the reaction
(data not shown). Spectrophotometric analysis of hemoglobin following
the reaction with Ni(II)/oxone did not indicate an increase in
methemoglobin content. CD spectroscopy revealed small but significant
changes in molar ellipticity in the Soret region with an isoelliptic
point at 430 nm (data not shown). These results suggest that the
reaction of human hemoglobin and oxone in the presence of Ni(II)
produces mainly intramolecular cross-linking of the protein, which is
not disulfide mediated. Interestingly, the hemes remain ferrous
throughout the cross-linking reaction. The changes detected in the
Soret region of the CD spectrum suggest some change in protein
structure, possibly indicative of changes in chain-chain interactions
or other structural perturbations influencing aromatic residues
surrounding the heme pockets (25, 26). These minor spectral changes did not reflect the dramatic change in oxygen affinity observed for hemoglobin A0 following the reaction with Ni(II)/oxone.
Oxygen affinity increased with p50 dropping from 10.0 to 4.1 torr, and cooperativity (Hill nmax) decreased from 2.8 to
1.0.

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Fig. 1.
SDS-PAGE of human hemoglobin A0
before and after reaction with Ni(II)/oxone. Lane 1, protein
size markers; lane 2, Hb Ao before reaction; lane
3, Hb Ao after reaction; lane 4, Hb Ao before reaction
under reducing condition with DTT; lane 5, Hb Ao after
reaction under reducing condition with DTT. Addition of nickel to
hemoglobin (2:1, Ni:Hb) showed no change from the control (not
shown).
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Reaction of Recombinant Hemoglobin and Oxone in the Presence of
Nickel(II) Produces Intramolecular Dimerization of the
Globins--
The recombinant hemoglobin rHb67 is structurally distinct
from human hemoglobin A0 in that the wild type
NH2-terminal valine of
globin is substituted by
alanine. Additionally, the
globins of rHb67 are genetically fused
by a single glycine amino acid linker and the wild type
NH2-terminal valine of the first
globin domain is
substituted by methionine. The reversed phase HPLC chromatograms of
both human hemoglobin A0, and the recombinant hemoglobin
rHb67, before and following the reaction with Ni(II) and oxone, are
shown in Fig. 2. It can be seen that the
genetic fusion of the
globins in rHb67 affords considerably
increased separation between elution of the two globin types. This
allows for discrete separation of a new peak formed in the reaction,
which elutes between them. Area integration of the chromatogram
indicates approximately 50% of the
globin in rHb67 was converted
to this form, as shown in Fig. 2. This peak was not resolved from
oxidized
globin peaks in the case of (reacted) human hemoglobin
A0. Fluorescence monitoring of this new peak by the method
of Heinecke et al. (21) yielded no evidence of di-tyrosine
formation. When 50 mM EDTA was included in the reaction
with Ni(II)/oxone, this modified globin peak was not observed (data not
shown).

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Fig. 2.
C3 reversed phase HPLC separation of globins.
Top panel, human hemoglobin Ao before (dashed
line) and after (solid line) reaction with
Ni(II)/oxone; bottom panel, recombinant hemoglobin rHb67
before (dashed line) and after (solid line)
reaction with Ni(II)/oxone. Addition of nickel to hemoglobin (2:1,
Ni:Hb) showed no change from the control (not shown).
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The reversed phase HPLC fraction containing the modified globin
peak formed in the reaction of rHb67 was collected and lyophilized. No
evidence of 2-oxohistidine (27) was found. Peptide mapping of this
fraction with trypsin yielded predominantly
globin tryptic peptides, along with a small level of di-
globin peptides (Fig. 3). The same lyophilized fraction was
analyzed by reducing SDS-PAGE and showed greater than 80% of the
protein migrated as a 32-kDa protein, apparently as a dimeric
globin (Fig. 4).

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Fig. 3.
HPLC separation of peptides produced by
tryptic mapping of isolated globin of rHb67 (top
panel), isolated oxidized beta globin of rHb67 as shown in Fig. 2
(middle panel), isolated dimer of rHb67 as shown in
Fig. 2 (bottom panel). Peaks annotated with "*"
correspond to oxidized peptides with sulfur-containing residues. Peaks
annotated with "#" correspond to low level di- globin-derived
peptides.
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Fig. 4.
Reducing SDS-PAGE of HPLC reversed phase
separated globin fractions of rHb67 before and after reaction with
Ni(II)/oxone as seen in Fig. 2. Lane 1, protein size
markers; lane 2, globin before reaction; lane
3, oxidized globin after reaction; lane 4, dimeric
globin after reaction; lane 5, di- globin before
reaction; lane 6, oxidized di- globin after
reaction.
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Consistent with the results seen with human hemoglobin
A0, the reaction of rHb67 also did not result in
appreciable formation of multimeric hemoglobin (<2%), as evidenced by
native state size exclusion chromatography. In addition, the reaction
of recombinant hemoglobin in the presence of Ni(II)/oxone did not cause
any significant change in the visible absorbance spectrum. These
results indicate that the reaction of rHb67 and oxone in the presence
of nickel(II) produces intramolecularly cross-linked
globin, most
of which does not appear to be disulfide bond mediated, and that just
as in the case of human hemoglobin A0, these cross-links
can be formed without appreciable oxidation of the ferrous hemes.
Characterization of Hemoglobins Oxidatively Modified by Oxone in
the Presence of Nickel(II) Evidences Oxidative Deamination of the
Globin Amino Terminus--
The monomeric and dimeric
globin
fractions separated by reversed phase HPLC from Ni(II)/oxone-reacted
rHb67 were analyzed by peptide mapping using an in-line immobilized
trypsin cartridge method (22) and monitored by UV and LCQTM
mass spectrometry.
globin purified from unreacted rHb67 by reversed
phase HPLC was also analyzed as a control. The peptide maps of these
three fractions are shown in Fig. 3. It can be seen that both fractions
from hemoglobin reacted with Ni(II)/oxone show distinct differences
compared with
globin from the control sample. Since the control
hemoglobin map was well characterized (22), LC-MS mass analysis allowed
us to readily identify most of the major difference peptides produced
in the reaction. These difference peaks included oxidized methionine
and cysteine containing peptides that exhibited mass gains of 16 and 48 Da, respectively. These mass gains are consistent with oxidation of
methionine to methionine sulfoxide and cysteine to cysteic acid, and
these peptide assignments were confirmed by MS/MS fragmentation. Such
side chain oxidations could be expected for the sulfur-containing amino
acids of a protein exposed to these concentrations of the chemical
oxidant oxone (28). More surprising was the finding that the level of NH2-terminal
globin tryptic peptide
1
(Ala1-Lys8), eluting at 34.6 min, was markedly
decreased in the monomeric and dimeric
globin fractions from
Ni(II)/oxone-treated hemoglobin. In both cases a new, asymmetric peak
appeared. This new peak eluted at approximately 41 min, and displayed a
mass exactly 1 Da less than the nominal
1 peptide. MS/MS spectra of
this new peak showed striking commonality of fragment masses with the
nominal
1 peptide (Fig. 3). The 1-Da
mass loss consistently mapped to the B2 ion. Since the mass
of the B1 ion was outside the range of the
LCQTM spectrometer in these MS/MS experiments, further
mapping of the 1-Da mass loss was not possible. This peptide was found
to be blocked to automated Edman protein sequencing. Amino acid
analysis of this peptide yielded the predicted amino acid composition
of the nominal
1 peptide, except for the NH2-terminal
alanine, which was not recovered. Reaction of this new peptide with
2,4-dinitrophenylhydrazine analyzed by LC-MS yielded evidence of
formation of an adduct (two stereoisomers) with characteristic
absorbance at 400 nm and mass of 1102 Da, consistent with hydrazone
formation, indicating the presence a free carbonyl group. No reactivity
with DNP was seen with the nominal
1 peptide. Cyanoborohydride
treatment of the oxidized peptide (41 min peak in trypsin map, Fig. 3)
was seen to yield a doublet of peptides, both exhibiting a mass
increase of exactly 2 Da, (i.e. 1 Da more than the nominal
1 peptide). These findings suggested that the
globin
NH2 terminus had undergone an oxidative deamination in
which the NH2-terminal amino group was replaced with a free
carbonyl group. Reduction with cyanoborohydride created chirality at
the
carbon of the former NH2-terminal alanine producing
two diastereomeric alcohols.

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Fig. 5.
MS/MS fragmentation spectra of peptides
eluting at approximately 34.5 and 41 min, respectively, during HPLC
separation of tryptic digest as shown in Fig. 3. LCQ fragmentation
energy was 22%. Only B and Y type fragmentation ions are annotated.
Peak at 34.5 min is the nominal 1 tryptic peptide. Data shows that
the peak at 41 min is structurally related to the 1 peptide with a
1-Da mass loss located at the N terminus.
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Ni(II)/oxone reacted rHb67 hemoglobin treated with
2,4-dinitrophenylhydrazine prior to globin precipitation and trypsin
mapping also yielded two peaks, which were absent in the control sample (Fig. 6). Masses of both isoforms were
identical and consistent with stereoisomeric hydrazone adducts to
deaminated
1 peptide. No evidence for oxidative deamination produced
by the reaction at any other site on the hemoglobin was found.

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Fig. 6.
HPLC separation of peptides produced by
tryptic mapping of DNP-derivatized recombinant hemoglobin rHb67 after
reaction with Ni(II)/oxone. Chromatography was monitored at 215 nm
for peptides bonds (top trace) and 400 nm for DNP
derivatives (bottom trace). An in-line LCQ mass spectrometer
was used to determine the mass of eluting peptides.
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Surprisingly, although baseline "noise" appeared to be increased,
the map of dimeric
globin did not display any significant difference peaks that would correspond to identifiable peptide cross-links connecting the two globins. We also noted a marked decrease
in the yield of the COOH-terminal peptides
15
(Val133-Lys144) and
15,16
(Val133-His146) in the dimeric
globin
fraction, along with the loss of the
1 peptide.
Histidine at Position 2 of
Globin Is Required for Both
Globin NH2-terminal Deamination and Intramolecular
Globin Dimerization--
The LC-MS data from trypsin maps of
Ni(II)/oxone-oxidized human hemoglobin A0 also showed a
loss of the
1 (Val1-Lys8) tryptic peptide
and a corresponding increase of oxidatively deaminated peptide.
However, there was no indication that the yield of nominal
1
(Val1-Lys7) peptide of
globin was
diminished in the reaction. An extensive query for a peptide(s)
exhibiting 1-Da loss from the nominal
1 peptide failed to yield any
evidence of oxidative deamination of the
globin NH2
terminus. Since valine is the NH2-terminal residue for both
the
and
globin chains of Ao human hemoglobin, we wondered if a
primary sequence difference between
and
globin was conferring
susceptibility to NH2-terminal deamination of valine
specifically to the
globin.
One potentially significant difference between
and
globins
was the presence of histidine at position 2 of
globin. Histidine is
known to form transition metal coordination sites in proteins (29), and
the oxidative deamination of the
globin amino terminus appeared to
require Ni(II). We postulated that this histidine might be required for
deamination of the adjacent amino acid on the
chain. To test this,
we constructed and expressed a recombinant hemoglobin mutant designated
rHb66, which was identical to rHb67 except that the histidine at
position 2 of the
globin was substituted with alanine. rHb66 and
rHb67 were identically prepared and reacted with oxone in the presence
of nickel(II). Trypsin maps of the two reactions revealed that the
globin of rHb66 was not oxidatively deaminated by the reaction (Fig.
7), nor was any cross-linking of the
globins in reacted rHb66 observed by reversed phase HPLC (Fig.
8). These results appeared to confirm
that the histidine at position 2 of
globin is a key component of
both the nickel-dependent oxidative deamination and
intramolecular
globin cross-linking.

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Fig. 7.
Expanded chromatograms showing separation of
tryptic peptides of two variants, rHb66 and rHb67, before (panels
A and C) and after (panels B and
D) reaction with Ni(II)/oxone. Chromatograms show only
the time window in which the NH2-terminal peptides 1 and
their Ni(II)/oxone-modified forms eluted.
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Fig. 8.
C3 reversed phase HPLC separation of globins
from four different variants of recombinant hemoglobin. The
dashed line represents separation of globins before
reaction, and the solid line represents separation after
reaction with Ni(II)/oxone. The following recombinant hemoglobins were
used: A, rHb69; B, rHb68; C, rHb67;
and D, rHb66. The NH2-terminal protein sequence
of the chain of each variant is listed in parentheses. In all
experiments the di- globin sequence was identical.
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To further test this hypothesis, another pair of recombinant hemoglobin
mutants were constructed both with methionine substituted for alanine
at position 1 of the
globin and engineered with either leucine or
histidine at position 2 of the
globin. This pair, designated rHb68
and rHb69, respectively, along with rHb66 and rHb67 were identically
prepared and subjected to the reaction with oxone in the presence of
Ni(II). Reversed phase HPLC analysis of these reactions showed that
only the hemoglobins with histidine at position 2 were observed to form
the peak identified as intramolecularly cross-linked
globin dimer
(Fig. 8). The Met-1 of rHb69 was found to be quantitatively oxidized to
methionine sulfoxide in the reaction. Very little peptide with a mass
consistent with both NH2-terminal deamination and
methionine oxidation was found. Although the His-2 was seen to be
required for cross-link formation, we were unable to find any evidence
that the His-2 was itself modified in the reaction with either rHb67 or
rHb69.
The same four recombinant hemoglobins described above were titrated
with nickel sulfate and absorbance changes at 240 nm were monitored
spectrophotometrically. The two mutants containing histidine at
position 2 of
globin showed a saturable absorbance change indicative of two metal binding sites (as did human hemoglobin A0, data not shown), whereas the two mutants lacking a
histidine at position 2 showed no change at 240 nm, indicating the
absence of similar Ni(II) binding (Fig.
9). This finding suggests the presence of
two unique binding sites for Ni(II) on hemoglobin, which are absent in
mutants lacking
globin His-2.

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Fig. 9.
Stoichiometry of Ni(II)-rHb binding by
titration of pairs of rHbs with NiSO4 monitored by
absorbance change at 240 nm. Within pairs the only difference was
the presence or absence of His residue in position 2 (Table I). A 2:1
Ni(II):rHb ratio was obtained for both rHb69 and rHb67. No Ni(II)
binding was detected in either rHb66 or rHb68 by this method.
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Characterization of
Globin Dimer Suggests Involvement of
Heterogeneous Cross-links between the Amino- and Carboxyl-terminal
Regions of Adjacent
Globins--
Comparative trypsin maps of
oxone-treated human hemoglobin A0 and rHb67 both displayed
significant loss of the
15 (Val133-Lys144) and
15,16 (Val133-His146) tryptic peptides
relative to the controls (data not shown). This was unexpected because
the
15,16 peptide contains no methionines or cysteines, which might
be oxidized by oxone. Comparative pepsin mapping of the reversed phase
HPLC separated monomeric and dimeric fractions of reacted rHb67
globin also showed a substantial decrease in the amount of a
COOH-terminal peptide peak Lys140-His146 in the
dimeric fraction relative to the monomeric fraction (data not shown).
Pepsin mapping also revealed two disulfide cross-linked peptides in the
dimeric fraction. These were identified as a homodimeric
Val111-Glu121 peptide linked by a disulfide
bridge between Cys-112, and a heterodimeric peptide of
Val111-Glu121 and
Ser89-Asn102 linked by a disulfide bond between Cys-112 and Cys-93. These disulfide cross-linked peptides disappeared from the maps when the reaction mixtures were treated with DTT (or
cyanoborohydride) prior to peptide mapping. Because treatment with
these reducing agents did not lower the yield of dimeric
globin
shown by reversed phase HPLC analysis, it appears that these readily
reducible disulfide cross-links are secondary to the oxidative
dimerization of the
globins. No other significant difference
peptides were found in either pepsin or trypsin maps that would
correspond to an oxidative cross-link between two
globins.
The recombinant hemoglobin rHb67 was also subjected to the Ni(II)/oxone
reaction after which sodium cyanoborohydride was added to the reaction
prior to denaturation with acidified acetone. In this case, trypsin
mapping yielded evidence of a low level of cross-linked peptide
containing both the deaminated
1 (923 Da) and the
15,16 (1449.7 Da) tryptic peptides. This peptide exhibited an average mass of 2356.4 Da, which is consistent with the predicted mass (923 + 1449.7
18 + 2 = 2356.7) for a peptide cross-linked by a reduced Schiff's
base between an oxidatively deaminated
1 and the
15,16 tryptic
peptides.
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A* represents deaminated NH2-terminal alanine residue.
Confirmation that this peptide (which eluted as a very minor doublet,
possibly diastereomers) was the product of a reduced Schiff's base
condensation of these two peptides was made using orthogonal analyses.
Protein sequencing of this peptide yielded an unusual double sequence.
In the first cycle only valine was observed, as expected for the
15,16, but with no yield of alanine as would be expected from a
1
peptide. In the next cycles, however, we could clearly read the
expected sequences of both the
15,16 and
1 peptides. In cycle 14, only a residual yield of the expected lysine (Lys-144) was observed,
but cycle 15 showed a normal yield of tyrosine (Tyr-145). In the final
cycle His-146 was not detected, which was inconclusive since
hydrophilic COOH-terminal residues are often poorly recovered. These
sequencing data suggested that the first residue (deaminated
NH2-terminal alanine) was cross-linked to Lys-144 by a
reduced Schiff's base. Reduction would create a secondary amine at the
(former) NH2 terminus of the
1 peptide which could
undergo Edman chemistry, cleavage and then allow further sequencing of
the
1 peptide. This secondary amine would be linked to the side
chain of Lys-144, which in consequence would be absent in cycle 14 of
the Edman sequencing. MS/MS fragmentation spectra of the triply charged
cross-linked peptide (m/z 786.3), although complicated,
also appeared to confirm a cross-link between deaminated
1 and the
15,16 peptide. The spectra showed a series of B ions from the
15,16 peptide with no corresponding Y ions. However, an apparent Y
ion series from the
15,16 peptide cross-linked to oxidatively
deaminated
1 peptide was observed (ions 26-36, Fig.
10 and Table
II). Also, a series of Y ions from the
1 peptide was seen, along with corresponding B ions of a deaminated
1 peptide cross-linked to the
15,16 peptide (ions 12-18, Fig. 10
and Table II). Such an ion series is consistent with fragmentation of
the
1 and
15,16 peptides cross-linked by a secondary amine formed between the deaminated Ala-1 and Lys-144 after cyanoborohydride reduction of their Schiff's base condensation product.

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Fig. 10.
MS/MS fragmentation spectra of cross-linked
globin peptides found during HPLC separation of tryptic digest of
Ni(II)/oxone-oxidized rHb67. LCQ fragmentation energy was 30%.
Cross-link was stabilized by reduction with sodium cyanoborohydride
yielding a mass consistent with a reduced Schiff's base between
NH2- and COOH-terminal peptides. Fragment ions are numbered
according to the designation shown in Table II.
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Table II
Fragment ions of reduced, cross-linked peptides ( 1 + 15,16) formed through Schiff's base condensation between
the free carbonyl at the NH2 terminus of one and
Lys-144 of the second globin
Mass loss of 17 Da corresponds to loss of 1 Da on 1 peptide
due to oxidative deamination, loss of water (18 Da) due to Schiff's
base formation, and gain of 2 Da after cyanoborohydride reduction.
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This reduced Schiff's base cross-link, found only following
cyanoborohydride reduction, was present in an amount too low to account
for most of the cross-linking seen. This observation prompted us to
test whether
globin dimerization was primarily dependent on
Schiff's base formation between deaminated
globin NH2
terminus and lysine 144 on the other
globin. We produced a
hemoglobin mutant, rHb98 (
globin K144A). This mutant formed an
intramolecular
globin dimer under the defined Ni(II)/oxone
conditions to the same extent as rHb67 (Table I), suggesting that
Schiff's base condensation between the newly formed free carbonyl at
the NH2 terminus and Lys-144 from the opposing
globin
was not the predominant mechanism of the cross-linking.
The
globin COOH-terminal region is rich in aromatic residues, which
are potential targets for oxidative cross-linking (5, 30). To test for
specific involvement of these residues in cross-linking, several other
variants of rHb98 were also tested (Table I). Two of these mutants
contained one additional
globin substitution to rHb98, either
H143A, or Y145A, (designated rHb95 and rHb97, respectively). Also
tested was a variant of rHb69 with an additional substitution of Y145H
(the Bethesda mutation), designated rHb96, as well as a variant of
rHb67 with a deletion of the COOH-terminal His-146 residue, designated
rHb80. These mutants were found to form intramolecular
globin
dimers in the Ni(II)/oxone reaction to about the same extent as rHb67,
except for rHb97, which formed less dimer under the standard reaction
conditions. Trypsin mapping of these mutants showed a consistent
decrease in the relative area of the NH2- and COOH-terminal
peptides following reaction with Ni(II)/oxone (an example of maps of
rHb95 is shown in Fig. 11). In the
reaction with rHb95 an additional peak eluting after the
15 peptide
was observed. This peptide displayed a mass gain of 15 Da compared with
the COOH-terminal
15 peptide. MS2 fragmentation analysis
of this peptide identified it as a modified
15 peptide with the
15 ± 0.2-Da mass gain located on the His-146 (data not shown). It
appeared that the substitution Y145A diminished subunit cross-linking
(whereas Y145H did not), which may indicate a special role for aromatic
residues at position 145 in the formation of
-
cross-links.
However, it appears that residues at positions 143-146 can also
participate in the formation of heterogeneous cross-linking.

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Fig. 11.
HPLC separation of peptides produced by
tryptic mapping of recombinant hemoglobin rHb95 before (top
panel) and after reaction with Ni(II)/oxone (bottom
panel). Masses were monitored by in-line LCQ mass
spectrometer to confirm peptide assignments. Loss of NH2-
and COOH-terminal peptides of the globins was observed. Oxidation
of sulfur-containing peptides was also detected.
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Two additional recombinant hemoglobin
globin mutants were
also produced and tested, in which the last 3 or 4 of the COOH-terminal residues were deleted (designated rHb81 and rHb82, respectively). Although the amino termini of rHb81 and rHb82 were found to be oxidatively deaminated during Ni(II)/oxone treatment, rHb81 formed
globin dimer to a significantly lesser extent than rHb67, and rHb82
formed virtually none at all (Fig. 12,
Table I). These findings suggested cross-linking was directed from the
NH2 terminus of one
globin to the aromatic region at
the far COOH terminus of the second
globin.

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Fig. 12.
C3 reversed phase HPLC separation of globins
of recombinant hemoglobin rHb82 containing a 4-amino acid deletion at
the globin COOH terminus ( HYKH). The dashed line
represents separation of globins before reaction, and the solid
line represents separation after reaction with Ni(II)/oxone. No
formation of globin dimer was detected.
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DISCUSSION |
We have shown that a reaction of human hemoglobin
A0 with oxone in the presence of nickel(II) ions produces
intramolecular cross-linking of the
globins and significant
oxidative deamination of the
globin amino termini. Importantly, the
oxidative conditions used did not oxidize the ferrous hemes of the
carbon monoxide-liganded hemoglobin, and no catalytic activity of the
heme centers appears to be involved. We did not observe any evidence
that the
globins are similarly susceptible to
NH2-terminal oxidative deamination under the conditions
used. Examination of different mutants of recombinant hemoglobin shows
that the histidine at position 2 is required for both Ni(II)-mediated
oxidative deamination of the
globin amino terminus and
intramolecular cross-linking of the
globins. Additionally, our
experimental results suggest that Ni(II)-catalyzed oxidative
intramolecular cross-linking of the
globins occurs between the
NH2 terminus of one and the COOH-terminal region of the
other globin. Peptide mapping did not, however, provide identification
of a primary cross-link but rather indicated the heterogeneous
character of cross-links produced by the reaction. Characterization of
a minor doublet of cross-linked peptides found following sodium
cyanoborohydride treatment of the still native state hemoglobin
reaction appears consistent with a Schiff's base reduction, resulting
in a secondary amine bond between the oxidatively deaminated
globin
terminus and the
-amino group of Lys-144 of the opposing
globin
within the same protein molecule. However, the yield of this cross-link
was quite low, and recombinant hemoglobin mutants lacking lysine at
position 144 remain susceptible to
globin dimerization. The
reported structure of R-state hemoglobin indicates close spatial
contact between the amino terminus and the carboxyl terminus of
opposing
globins in the hemoglobin tetramer (Fig.
13) (31). Hemoglobin mutants containing
substitutions: H143A, Y145H, or deletion of His-146 all exhibited
comparable susceptibility to cross-linking, suggesting that no specific
side chain of COOH-terminal residue is strictly required. The
substitution Y145A, however, did appear to both diminish the amount of
dimerization seen in the standard reaction and also displayed an
oxidation of His-146 resulting in a mass gain of 15 Da, which was not
seen in reactions of the other hemoglobin mutants. Two mutants of
recombinant hemoglobin containing 3 or 4 amino acid deletions at the
COOH terminus, showed significantly decreased or virtually no
susceptibility to dimerization, respectively. This may be due to steric
considerations, in that the new COOH-terminal region of these mutants
is likely no longer in close enough proximity with the opposing
globin NH2 terminus for cross-link formation.

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Fig. 13.
Ribbon view representation of the structure
of human carboxy hemoglobin (31). Only globins are shown to
demonstrate the proximity of the NH2 and COOH termini. Side
chains of the first two residues on the NH2 terminus and
the last four residues at the COOH terminus are shown in color.
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From these findings, we postulate that histidine at
globin position
2 confers susceptibility under oxidizing conditions to the
nickel-catalyzed formation of a carbon-centered radical at the
carbon of the
globin amino-terminal residue, analogous to that
proposed by Stadtman (4) on the
carbon in the iron catalyzed
deamination of lysine. The "activated"
carbon can react in one
of three ways, which are depicted schematically (Fig. 14), A, the radical can be
oxidized by the coordinated nickel forming an imino derivative that
spontaneously hydrolyzes resulting in oxidative deamination analogous
to mechanisms proposed by Stadtman (4) and Garrison (32); or
B, the radical can attack a variety of sites on a spatially
adjacent
COOH-terminal region leading to a heterogeneous set of
carbon-carbon and/or carbon-nitrogen bonds. The numerous combinations
of potential products resultant from this pathway may, in part, explain
our difficulty in identifying specific NH2-terminal to
COOH-terminal region cross-linked peptides. A similar type of labile
bond has recently been proposed to form between the
carbon of the
essential tyrosine and a nitrogen of histidine in catalase HPII of
E. coli (33); C, in the case where the
NH2-terminal residue is methionine, the radical can transfer to the side chain sulfur atom leading to the formation of
methionine sulfoxide, effectively "quenching" oxidative
deamination. This finding provides a unique example of methionine
serving as an intrinsic antioxidant in a protein, a role recently
postulated by Levine et al. (34). Recombinant hemoglobins
with NH2-terminal methionine may possess a superior
antioxidative feature over wild type hemoglobin in this respect.

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Fig. 14.
Proposed scheme of oxidative damage
occurring at the NH2 terminus of the chain in
hemoglobins containing a His residue in position 2. Pathway
A results in -ketoamide formation, which can participate in
Schiff's base-mediated cross-linking with Lys-144 of the opposing globin. Pathway B results in heterogeneous intramolecular
cross-linking between the NH2 terminus and the
COOH-terminal region. Pathway C can produce oxidation of the
NH2-terminal methionine.
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The formation of carbonyl derivatives in peptides and proteins by
metal-catalyzed oxidation has been well documented (3, 5, 7, 35-37).
The physiological relevance of such carbonyl formation in
vivo with respect to aging (7, 35, 36) and certain pathological
disorders (1, 3, 38) has been established for a variety of proteins
including carbonic anhydrase isoenzyme III (1), glutamine synthetase
(3), and human fibrinogen A
chain (39). Further study of the
mechanism of the nickel-catalyzed oxidation of hemoglobin may serve as
a useful paradigm, providing insights into metal-catalyzed carbonyl
formation, as well as oxidative cross-linking in proteins with metal
binding sites less proximal to an amino terminus. Oxidative
modification of proteins in vivo, including hemoglobin, is
known to have a variety of physiologically significant consequences,
including increased susceptibility to proteolysis (35, 37, 40). The
physiological relevance of a redox-active nickel binding site on
hemoglobin, as a factor in oxidative damage in vivo, may
merit consideration.
Histidine in position 2 can be readily engineered into recombinant
proteins and peptides to provide a redox-active Ni(II) binding site.
Treatment with an oxidizing agent (e.g. oxone,
peroxyphthalic acid) can be intentionally used to site specifically
introduce a free carbonyl. This terminal
-ketoamide group formed in
the reaction could be an attractive "handle" for reaction with
amines, hydrazides, and alkoxyamines in aqueous solutions to form
Schiff's bases, hydrazones, and oximes respectively. These derivatives are stable under physiological conditions and could be used with a
variety of biophysical or structural probes to stabilize proteins, deliver drugs, control enzymatic activity, and for generation of
semisynthetic proteins (41).
We are grateful to Dr. Robert Woody for
his assistance and helpful suggestions regarding CD spectroscopy, and
to Dr. Doug Lemon for assistance with three-dimensional molecular
modeling.