From the Department of Molecular and Structural
Biology, Science Park Division, University of Aarhus, Gustav Wieds
Vej 10C, 8000 Aarhus C, Denmark, the ¶ Department of Molecular
Biology, University of Odense, 5320 Odense M, Denmark, the
Department of Chemistry, Royal Veterinary and Agricultural
University, Copenhagen, 1871 Frederiksberg C, Denmark, and the
** Department of Immunology and Medicine, Mayo Clinic and Foundation,
Rochester, Minnesota 55905
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ABSTRACT |
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The covalent heme attachment has been extensively
studied by spectroscopic methods in myeloperoxidase and lactoperoxidase (LPO) but not in eosinophil peroxidase (EPO). We show that heme linkage
to the heavy chain is invariably present, whereas heme linkage to the
light chain of EPO is present in less than one-third of EPO molecules.
Mass analysis of isolated heme bispeptides supports the hypothesis of a
heme b linked through two esters to the polypeptide. Mass analysis of
heme monopeptides reveals that >90% have a nonderivatized methyl
group at the position of the light chain linkage. Apparently, an ester
had not been formed during biosynthesis. The light chain linkage could
be formed by incubation with hydrogen peroxide, in accordance with a
recent hypothesis of autocatalytic heme attachment based on studies
with LPO (DePillis, G. D., Ozaki, S., Kuo, J. M., Maltby,
D. A., and Ortiz de Montellano P. R. (1997) J. Biol. Chem. 272, 8857-8860). By sequence analysis of isolated
heme peptides after aminolysis, we unambiguously identified the acidic
residues, Asp-93 of the light chain and Glu-241 of the heavy chain,
that form esters with the heme group. This is the first biochemical support for ester linkage to two specific residues in eosinophil peroxidase. From a parallel study with LPO, we show that Asp-125 and
Glu-275 are engaged in ester linkage. The species with a nonderivatized methyl group was not found among LPO peptides.
Known mammalian peroxidases, including myeloperoxidase
(MPO)1 (1), eosinophil
peroxidase (EPO) (2), lactoperoxidase (LPO) (3), and thyroid peroxidase
(4), show 40-70% identity in pair-wise alignments. All enzymes
contain a heme prosthetic group and use hydrogen peroxide as the
electron acceptor in the catalysis of oxidative reactions. More
distantly related members of this family, such as prostaglandin H
synthase, have also been identified (5, 6).
MPO, EPO, and LPO are primarily found in granules of neutrophil and
eosinophil leukocytes and secretions of exocrine glands, respectively.
Their oxidation of halide and pseudohalide is part of the defense
system against bacteria and parasites. Although several substrates have
been found in vitro, the physiologically relevant substrates
are believed to be chloride (MPO) and thiocyanate (EPO and LPO), which
are oxidized to the toxic products hypochlorite and hypothiocyanite
(7-9). Thyroid peroxidase functions in biosynthesis of thyroid
hormones by oxidation of iodide (10).
LPO and thyroid peroxidase are single chain enzymes of about 75 and 100 kDa, respectively (10, 11). In contrast, the polypeptide chains of MPO
and EPO are cleaved after synthesis to form a heavy chain of
approximately 55 kDa and a light chain of approximately 15 kDa that
remain associated. EPO is a monomer with the expected size of 70 kDa,
whereas MPO is a disulfide-linked dimer of 150 kDa (12-16).
The mammalian peroxidases are distinguished from other heme-containing
peroxidases by tight binding of the heme group to the apoprotein. Early
experiments with LPO demonstrated that the heme group could not be
extracted with acidified acetone and, furthermore, that it was not
linked through thioethers as found in cytochromes. Based on this and
other lines of evidence, heme linkage through ester(s) was proposed
(17). But the heme group has also been suggested to be a thioderivative
disulfide-linked to the protein (18). Results supporting and in
conflict with both of these hypotheses followed (19-24).
Structurally, MPO is the best characterized of the mammalian
peroxidases (5, 25). A 2.28-Å crystal structure has been obtained that
allows evaluation of possible interactions between amino acid side
chains of MPO and the heme group. Based on distances in the crystal
structure, ester linkages from hydroxylated methyl groups on pyrrole
rings A and C to Glu-242 in the heavy chain and to Asp-94 in the light
chain, respectively, were proposed. It was further suggested that the
vinyl groups of pyrrole ring A formed a sulfonium ion linkage with
Met-243 (23, 25, 26). Unlike the two acidic residues, this methionine
has no obvious equivalent in LPO or EPO (11, 25). It has also been
suggested that Asp-94 and Glu-242 exist as protonatable residues
capable of influencing the absorption spectrum of the heme group (27, 28). In short, however, after many years of investigation with mixed
results, linkage of the heme group through two esters seems to be
accepted for LPO and MPO, although for LPO, the residue that
corresponds to Asp-94 in MPO has not been unambiguously identified (24).
Due to its limited availability, EPO is the least studied of the four
peroxidases, and compared with LPO and MPO, insufficient evidence has
been generated to allow generalization within the subfamily. EPO has
primarily been the subject of comparative spectroscopic studies (23,
29-31), and no direct biochemical evidence has been reported.
Biochemical studies on mammalian peroxidases have previously been
carried out on the isolated heme group as released with proteolytic
enzymes, such as Pronase, that also cleaves ester bonds. Exceptions are
recent spectroscopic studies on LPO (20, 24). In this work, we present
data based on analysis of the isolated heme group with covalently bound
EPO peptides. We chemically identify the bonds as esters, and we
identify the residues that are engaged in this linkage. Furthermore, we
demonstrate that in vivo one of the two esters is only
partly formed but that autocatalysis in vitro results in
formation of an enzyme with two esters. LPO was analyzed in parallel.
Proteins--
Human EPO was purified using a previously
published procedure (32) with modifications. Briefly, isolated
eosinophil granules from single donors with marked eosinophilia were
extracted with 0.2 M sodium acetate, pH 4.0, and the
supernatant was loaded onto a Sephadex G-75 SF (Amersham Pharmacia
Biotech) equilibrated and eluted with 50 mM sodium acetate,
pH 4.3. EPO eluted as a reasonably isolated peak. As judged by
SDS-PAGE, the material was more than 90% pure (see Fig. 1). The
original procedure included an additional ion-exchange chromatography
step on CM-Sephadex in 0.1 M CAPS buffer, pH 10.0. In our
hands, polymerization through cysteine residues is minimized to a
negligible level if the pH is not elevated. For this study,
nonpolymerized protein was important, and the purity obtained directly
from the gel filtration column was sufficient. Therefore, the
ion-exchange step was omitted. Different preparations of EPO were used.
The functionality of EPO from patients with marked eosinophilia is the
same as EPO from normal donors (33). Partly purified bovine LPO was
obtained as a gift from Svenska Mejeriernes Riksförening, Sweden.
The lyophilized LPO was further purified before use by ion-exchange
chromatography on a UNO-S12 column (Bio-Rad). The enzyme was dissolved
in 35 mM sodium phosphate, pH 6.0, applied to the column
equilibrated with the same buffer, and eluted by a linear gradient of
0-1000 mM sodium chloride in the equilibration buffer.
Pooled fractions were concentrated by ultrafiltration. The purity of
the protein (>95%), finally in 35 mM sodium phosphate, pH
6.0, was assessed by SDS-PAGE and capillary electrophoresis. The
A412/A280 ratio of the
purified protein was 0.92.
Miscellaneous Procedures--
SDS-PAGE was performed in 16%
Tris-tricine gels (34). Samples (2 µg) were diluted 10-fold in 20 mM sodium phosphate, 150 mM sodium chloride
(phosphate-buffered saline), pH 7.4 (some samples were adjusted as
specified in the text), and prepared for loading by boiling in sample
buffer for 1 min or longer (up to 10 min) with or without 10 mM dithiothreitol. Prior to addition of loading buffer,
some samples were incubated with hydrogen peroxide at different
concentrations for 5 min at room temperature. The concentration of the
30% (v/v) hydrogen peroxide (Merck) stock solution was verified
gravimetrically (expected density of the stock solution is 1.11 kg/liter), and dilutions made in phosphate-buffered saline. Digests
were made of EPO and LPO with Column Chromatography--
Gel filtration on Superose 12 HR
10/30 (Amersham Pharmacia Biotech) was performed in 6 M
guanidine hydrochloride, 20 mM sodium phosphate, pH 7.2. The flow rate was 0.2 ml/min, and the eluate was monitored at 280 and
398 nm via serial connection of variable wavelength monitors. Samples
were loaded after addition of solid guanidine hydrochloride to a final
concentration of 6 M. Peptides were separated by
reversed-phase high pressure liquid chromatography on a 4 × 250-mm Nucleosil C18 column (Macherey-Nagel) eluted with a flow rate of
1 ml/min at 50 °C. A gradient was formed from 0.1% trifluoroacetic
acid (Rathburn) (solvent A) and 90% acetonitrile (Rathburn) containing
0.075% trifluoroacetic acid (solvent B), increasing the amount of
solvent B linearly by 1%/min. The eluate was monitored at 226 and 398 nm via serial connection of variable wavelength monitors. In Figs. 2
and 3, the resulting chromatograms are overlaid and aligned.
Sequence Analysis--
Edman degradation was performed on an
Applied Biosystems 477A sequencer equipped with an on-line high
pressure liquid chromatograph (35). For sample loading, isolated
peptides (20-200 pmol) were pipetted onto polybrene-coated glass
filters, and proteins separated by SDS-PAGE were blotted onto a
ProBlott membrane (Applied Biosystems), Coomassie-stained, and excised.
Mass Spectrometry--
Mass spectra were acquired with a Bruker
BIFLEX matrix-assisted laser desorption ionization-time of flight
instrument (Bruker-Franzen, Bremen, Germany) equipped with a 1-m flight
tube, a reflector, a 337-nm nitrogen laser, and a 500-MHz digitizer.
Thin film matrix surfaces were prepared using the fast evaporation
technique (36) from Preparation and Characterization of Nonpolymerized Human
EPO--
A preparation of human EPO that showed a minimum of
polymerization through cysteine residues in nonreduced SDS-PAGE was
made. In our hands, the traditional final step of ion-exchange
chromatography at pH 10 (32) led to extensive polymerization of the
protein. Because the purity of the protein prior to this
chromatographic step was above 90%, we did not carry out further
purification. Three bands appear in nonreduced SDS-PAGE of the
preparation (Fig. 1, lane 2).
Sequence analysis of protein blotted onto a polyvinylidene difluoride
membrane confirmed the identity of the top band (70 kDa) as two-chained
EPO with equimolar amounts of EPO heavy and light chain, the middle
band (55 kDa) as heavy chain, and the bottom band (15 kDa) as light
chain. As judged from this gel, we estimate that less than one-third of
the EPO molecules have covalently linked light and heavy chains. Native
EPO contains two free sulfhydryl
groups,2 consistent with the
finding that it readily polymerizes at elevated pH. But the partial
separation of the heavy and light chain cannot be explained by
interchange of disulfide bonds, because in the native molecule, the two
cysteine residues of the light chain are paired.2 Thus, it
appears that the EPO exists in two forms. In one, the heavy and light
chains are covalently bound, whereas in the other, the two chains are
noncovalently associated. The length of sample boiling prior to loading
the gel did not affect the extent of chain separation (not shown). When
incubated with reductant, however, the separation of EPO into heavy and
light chain was complete (Fig. 1, lane 1). Incubation at pH
2 with SDS also resulted in complete chain separation (not shown).
The Heme Group Connects the EPO Heavy and Light Chain or Is Bound
to the Heavy Chain Only--
In nondenaturing gel filtration, EPO
elutes as one peak (not shown), but as in SDS-PAGE, the partial chain
separation is evident in denaturing gel filtration (Fig.
2). Furthermore, the recorded absorption
at 398 nm shows that when the heavy and light chains of EPO are
separated by denaturation alone, the heme group is linked to the heavy
chain. Following incubation with reductant, the heme group elutes as a
third peak, decreased in absorption and separate from the heavy and the
light chain peaks (not shown). Although this would be consistent with
heme linkage through disulfides, as proposed earlier for LPO (18),
recent results with different mammalian peroxidases suggests linkage by
ester bonds (20, 23-25). Curiously, incubation of EPO for 30 min at
room temperature with 18 mM dithiothreitol in 6 M guanidine hydrochloride at pH 7.2 decreased the
absorption at 398 nm more than 50% (not shown), suggesting
modification or precipitation of the heme.
Autocatalysis of Covalent Heme Attachment in EPO--
Recently, it
was shown that covalent attachment of the heme group to the single
polypeptide of LPO can occur by an autocatalytic process in the
presence of hydrogen peroxide (22). With EPO, we have independently
confirmed and visualized this process directly by SDS-PAGE (Fig. 1,
lane 3). The intensity of the upper band increased with
increasing concentrations of hydrogen peroxide (not shown). To bring
>90% of the protein to the 70-kDa EPO form, a concentration of 30 µM hydrogen peroxide was required, corresponding to a
10-fold molar excess over EPO (Fig. 1, lane 3). Changing the
pH in the reaction buffer to 6.0 or 8.5 did not affect the result. As
expected, when the modified EPO was incubated with reductant, only
heavy and light chain were seen in SDS-PAGE (not shown).
Isolation and Analysis of EPO Peptides with Covalently Bound
Heme--
Previously, indirect evidence for heme linkage through
esters in the mammalian peroxidases, mainly LPO, was provided through investigations on isolated heme groups and by crystallography of MPO
(25). Recent studies with LPO argue for ester linkage based on studies
with isolated heme peptides, but complete assignment of amino acid
residues could not be made (20, 24). In general, direct peptide
evidence has been lacking for all mammalian peroxidases.
EPO has not previously been investigated at the peptide level. To study
the heme linkage in this protein and to study the basis for the
apparently different attachment to the EPO light and heavy chains,
peptides from a thermolytic digest were separated by reversed-phase
high pressure liquid chromatography (Fig.
3A). The majority of peptides
(90% based on peak heights) that show absorption at both 226 and 398 nm, thus containing the heme group, were further analyzed.
N-terminal sequence analysis revealed that some were heme bispeptides
(E-TL-2, E-TL-3), referred to below as bispeptides, and some were heme
monopeptides (E-TL-1, E-TL-4, E-TL-5), referred to as monopeptides
(Table
I).3
The bispeptides contained equimolar amounts of peptides derived from
two regions in the EPO light and heavy chains, respectively. All
monopeptides contained peptides derived from the heavy chain. This
result, including the relative abundance of bispeptides (about 30%),
is in fine agreement with the results from SDS-PAGE and gel filtration
detailed above. Concordant results were obtained from several other
digests with thermolysin, trypsin, and chymotrypsin alone or in
different combinations (not shown and Table I). We never observed a
monopeptide with polypeptide originating from the EPO light chain.
To substantiate the covalent heme linkages as esters and to identify
the acidic residues putatively engaged herein, peptides E-TCT-101 and
E-TL-3 (Table I) were immobilized on glass filters and incubated in an
atmosphere of ammonia overnight at room temperature to convert the
carboxyl moiety of the putative esters into amides by aminolysis.
Sequence analysis of the peptides after incubation unambiguously
identified the positions of heme linkage (Table II). In cycle 3 of peptide E-TCT-101
(239STETPK), partial conversion of Glu-241 to Gln was
observed. Likewise, in cycle 3 of peptide E-TL-3 (91FIDHD
and 232FLAGDTRSTETPK), partial conversion of Asp-93 to Asn
was observed. Importantly, in cycle 5, no conversion of Asp-95 to Asn
was seen. When the same peptides were subjected to sequencing without
prior incubation with ammonia, very little Gln and Asn was observed in
the respective positions (Table II). We did not see a complete conversion of the expected acidic residues to the corresponding amides;
thus, to some extent, hydrolysis occurred during the incubation. However, the residues of EPO engaged in heme linkage have now been
identified as Asp-93 of the light chain and Glu-241 of the EPO heavy
chain.
Analysis of Heme Peptides Derived from LPO--
Because LPO is a
single-chain protein, partial heme linkage to one of two amino acid
side chains would not be evident from SDS-PAGE. We thus repeated the
peptide isolation procedure with purified LPO. Again, several
heme-containing peptides were found (Fig. 3B) and
subsequently identified by sequence analysis (Table III). In striking contrast to EPO, 80%
of the observed LPO peptides were bispeptides, and the remaining 20%
were monopeptides (Table III). But like EPO, the polypeptides of the
monopeptides were variants of the same peptide stretch. In a sequence
alignment, this region corresponds to the region observed for EPO
monopeptides. When the LPO peptide L-TL-2 (123IVDHD and
273ASEQ) was subjected to sequence analysis following
exposure to ammonia, partial conversion of Asp-125 (but not Asp-127) to
Asn and of Glu-275 to Gln was seen (not shown). Therefore, the residues engaged in ester linkage of the heme group in LPO are Asp-125 and
Glu-275. Fig. 4 shows the heme group as
derivatized with polypeptides from EPO or LPO in heme
bispeptides.4
Two Different Forms of the Heme in the Monopeptide of EPO but Not
LPO--
Mass spectrometry was used to confirm the identity of all
analyzed peptides from EPO and LPO. Regardless of origin, the
contribution of the heme group to the total mass of bispeptides was the
same, 612.6 ± 0.2 Da (Table IV).
The calculated average mass of nonderivatized heme b
(C34H32FeN4O4) is
616.51 Da. The calculated mass of the expected heme contribution to the
total mass in a heme bispeptide with two esters, formally (HC-C-1-heme
b core-C-5-CH) (Fig. 4), is 4.03 mass units lower, i.e.
612.48 Da, in perfect agreement with the observed masses. Hence, this
result further supports that the heme of EPO is heme b and that it is
linked via esters.
In contrast, the contribution of the heme group to the total mass of
monopeptides was not constant, but rather appeared in two groups
separated by 16 mass units (Table IV). A heme monopeptide could arise
from hydrolysis of the ester at C-5 (Fig. 4). Hydrolysis would leave a
hydroxymethyl group on C-5, and the expected heme contribution to the
total mass would be 18 mass units higher than in the bispeptides
(C-5-CH2OH rather than C-5-CH), i.e. 630.51 Da.
This mass was observed for all LPO monopeptides, but rarely for EPO
monopeptides. Of the EPO peptides presented here (Fig. 3A
and Table I), only one, E-TL-1, conformed to this mass. The majority of
peptides, constituting 90% of all EPO monopeptides, had a heme
contribution to the total mass that was 2 mass units higher than for
the bispeptides. This mass is consistent with the presence of a methyl
group at C-5 (C-5-CH3). As discussed below, we suggest that
an ester with the EPO light chain was never formed in the majority of
EPO molecules and that the original methyl group is present at C-5. The
distribution of monopeptides described here was also seen in digests of
other EPO preparations (not shown).
By analysis of intact human EPO and proteolytic peptides thereof,
we show that 1) the heme group is covalently bound to both the light
and heavy chain of the EPO polypeptide in less than one-third of EPO
molecules, 2) the heme group is bound only to the heavy chain in the
majority of EPO molecules, 3) incubation of EPO with excess hydrogen
peroxide attaches the unbound EPO light chain to the heme group in an
autocatalytic reaction, 4) the two acidic residues that are engaged in
binding are Asp-93 and Glu-241, and as they can be converted into the
corresponding amides by aminolysis, linkage by ester bonds is
confirmed, and 5) in molecules where the heme group is attached to the
heavy chain only, the site of possible light chain attachment is a
nonderivatized methyl group that cannot result from ester hydrolysis.
Parallel studies on LPO demonstrate that 6) the two acidic residues in the LPO polypeptide that bind the heme group are Asp-125 and Glu-275, and 7) both of these residues are bound to the heme group in the majority of LPO molecules. Our findings represent the first biochemical data on heme attachmement in EPO, and our data on EPO and LPO are
relevant to a long standing controversy on the nature of the heme
attachment in mammalian peroxidases.
With isolated EPO bispeptides (Table I), we provide evidence for heme
attachment through esters. First, the peptide masses are in perfect
agreement with linkage of a heme b prosthetic group by two esters
(Table IV). Second, the bonds are susceptible to cleavage by ammonia
(Table II). Concordant results were obtained with bispeptides derived
from LPO (Tables III and IV). Previously, based on biophysical methods,
ester bonds have been proposed for the heme linkage in MPO and LPO.
Principally from a high resolution MPO crystal structure (25), and from
spectroscopy of LPO peptides (24), two ester bonds were proposed.
Recent infrared difference spectra of MPO, LPO, and EPO also point
toward ester linkage (23).
The sites of heme attachment in EPO were unambiguously identified by
sequence analysis of peptides converted by aminolysis as Asp-93 of the
light and Glu-241 of the heavy chain (Table II), and in a similar
experiment, the corresponding residues of LPO were identified as
Asp-125 and Glu-275. This is the first biochemical identification of
specific residues engaged in heme linkage for any of the peroxidases.
Prior to this study, only LPO had been studied at the peptide level,
and the peptides 273ASEQIL and
121GQIVDHDLDFAPETEL could be released by alkaline
hydrolysis from an LPO bispeptide (24). The first of those peptides
suggests that Glu-275 is engaged in heme binding; the second leaves
several candidate residues, including the Asp-125 identified here. With MPO, two candidate heme binding acidic residues were pointed out from
the crystallographic structure of this protein, and for the first time
convincing evidence for binding by esters was presented (25). Neither
LPO nor EPO contains a residue equivalent to the methionine of MPO that
is engaged in sulfonium ion linkage of the heme group (25). In
conclusion, two esters are common to heme linkage in MPO, LPO, and
EPO.
Unreduced SDS-PAGE of EPO reveals that the majority of EPO exists in a
form in which the light and heavy chain are not covalently linked (Fig.
1, lane 2). We are not aware of any publication that shows
the result of unreduced SDS-PAGE of purified EPO. However, chain
separation under these conditions was mentioned in one report (16).
After incubation with reductant, the chain separation was complete
(Fig. 1, lane 1), compatible with the earlier hypothesis, now rejected, of heme linkage by disulfide bond(s) (18). In denaturing
gel filtration, the partial chain separation was also evident, and this
experiment further demonstrated that the heme group is never bound to
the EPO light chain alone (Fig. 2). After incubation with reductant,
the protein chains are fully separated, and the heme group elutes
separately (not shown). We conclude that the esters can be broken by
incubation with reductant, possibly in a relatively rapid reaction of
trans-esterification resulting in thioesters. However, the heme group
itself also seems to be modified over time, but less rapidly, because
its absorption at 398 nm decreased gradually in the presence of
reductant. This might also be due to precipitation of the heme group.
Following incubation with hydrogen peroxide the protein appeared intact
with both esters formed in SDS-PAGE (Fig. 1, lane 3). This
experiment was prompted by an elegant, recent study (22) showing that
heme attachment in LPO can occur by an autocatalytic reaction in the
presence of hydrogen peroxide. With the two-chained EPO, this can be
directly visualized in SDS-PAGE. Because the reaction product was still
intact in unreduced SDS-PAGE, even under conditions of prolonged sample
boiling, the chain separation described above was not a result of
sample preparation. As expected, incubation with reductant rapidly
caused complete chain separation (not shown).
A priori, the EPO monopeptides, invariably containing a
peptide fragment derived from the heavy chain, would be expected to result from selective hydrolysis of the ester to the light chain. But
of five thermolytic peptides from one digest (Fig. 3A and Table I), only one (E-TL-1, less than 10% abundance) has the expected
mass of a peptide with a hydroxymethyl group at C-5 of the heme group.
The other EPO monopeptides differ in mass by 16 units, corresponding to
the presence of a nonderivatized methyl group on C-5 (Table IV and Fig.
4). An obvious interpretation of this finding is that the ester to the
light chain of EPO had never been formed during biosynthesis. Because a
methyl group is known to function as a substrate in the autocatalytic
reaction (22), the fact that this process can occur with native EPO
further supports this interpretation.
The likely presence of a methyl group on C-5 seems to be unique to EPO
(Table IV). EPO is also different with regard to the efficiency of the
autocatalytic reaction. Compared with the reaction in LPO, more
hydrogen peroxide was required to drive the reaction to completion.
With LPO, the amount of hydrogen peroxide was four times higher than
theoretically predicted (one equivalent of hydrogen peroxide is
formally required to produce each ester link), demonstrating that the
process is inefficient (22). With EPO, a 10-fold molar excess of
hydrogen peroxide was required to form the remaining esters between the
light chain and the heme group. Thus, it seems that two factors,
efficiency of the reaction and availability of hydrogen peroxide,
determine the extent to which the heme group is attached, and it seems
that the autocatalytic reaction with EPO is even more inefficient than
with LPO. In agreement with this, the majority of heme peptides
isolated from LPO were bispeptides (Table III).
The finding that the EPO heavy chain is always ester-bonded and that
the light chain is bound to a much lower extent indicates that the
formation of an ester at C-1 of the heme group occurs more readily than
at C-5, and possibly that esterification at C-5 requires that the ester
at C-1 is already formed. But that does not explain why the limited
ester hydrolysis seems to occur only at C-5 in both EPO and LPO. We
speculate that the propionic acid at C-6 is able to break the
Asp-esters with the formation of a lactone. The hydrolytic equilibrium
of this seven-member ring would greatly favor the hydroxy acid, which
is the species we observed.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-
(w/w)
thermolysin (Type X protease, Sigma), trypsin (Worthington), and
chymotrypsin (Roche Molecular Biochemicals) alone or in different combinations for 1-4 h at 50 or 37 °C. Prior to addition of
enzymes, the pH was adjusted to 7.2-8.0 with 1 M Tris.
Incubation of isolated heme-containing peptides with ammonia was
performed in a capped 1.5-ml tube containing 50 µl of 25% (w/w)
ammonia. Peptides, immobilized on glass filters (for the Applied
Biosystems 477A sequencer, see below), were inserted into the tube
without making contact to the liquid and incubated for 20 h at
room temperature. After the incubation, traces of ammonia were removed
in an exsiccator.
-cyano-4-hydroxycinnamic acid (Sigma) dissolved
in acetone/water (99:1) to 30 µg/µl. A 0.5-µl volume of the
analyte (0.1-10 pmol/µl) was deposited on the matrix surface and
allowed to dry onto the crystals. Spectra were obtained by averaging
20-50 single-shot spectra. Spectra were calibrated internally by
co-crystallizing small amounts of angiotensin II (Sigma) and
adrenocorticotropic hormone, fragment 18-39 (Sigma), with the analyte
and by using the calibration constants of well known matrix ions.
Nonheme peptides were observed as MH+ species, but heme
peptides were observed as M+ species, with the charge
resulting from the Fe3+ ion of the heme group and the
formal charge of
2 of the four nitrogen atoms. To confirm this,
spectra of hemin
(C34H32FeN4O4, calculated average mass of 616.51 Da) (Sigma) were acquired. Observed masses were 614.37, 616.23 (major peak), 617.28, 618.16, and 619.19, in
agreement with the abundance of iron isotopes (54Fe (53.94 Da), 5.8%;
56Fe (55.93 Da), 91.7%; 57Fe (56.94 Da), 2.2%; and 58Fe (57.93 Da),
0.3%) and with the absence of a 55Fe species. Furthermore, a
cytochrome c heme peptide, with the heme group bound via two
thioethers, was also observed as an M+ species (not shown).
In spectra of EPO and LPO heme peptides, the intact heme peptide was
often observed along with free peptide(s) and the free heme group
resulting from cleavage of the peptide-heme linkage(s) in the
instrument. The free peptides were observed as MH+ species,
and the heme group as an M+ species, but with a mass
reduction of 1 mass unit compared with hemin, because the heme
macrocycle is capable of stabilizing a -CH2 radical.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (78K):
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Fig. 1.
SDS-PAGE of human EPO. Each lane
contains 2 µg of EPO boiled in loading buffer for 2 min with
(lane 1) or without (lane 2) 10 mM
dithiothreitol. The protein loaded in lane 3 was incubated
with 10 µl of 30 µM hydrogen peroxide at pH 7.4 for 5 min at room temperature prior to addition of loading buffer without
dithiothreitol. Protein was visualized by Coomassie staining. The
identity of the three bands in lane 2 was verified by
N-terminal sequence analysis. The bottom band (15 kDa) is
EPO light chain, the middle band (55 kDa) is EPO heavy
chain, and the top band (70 kDa) is intact EPO containing
equimolar amounts of EPO heavy and light chain.
View larger version (17K):
[in a new window]
Fig. 2.
Denaturing gel filtration on Superose 12 HR
10/30 of nonreduced human EPO. Protein (2 mg) dissolved in sodium
acetate buffer, pH 4.3, was made 6 M in guanidine
hydrochloride, loaded onto the column, and eluted with 6 M
guanidine hydrochloride, 20 mM sodium phosphate, pH 7.2, at
0.2 ml/min. The eluate was monitored at 280 nm (thick line)
for eluting proteins and at 398 nm (thin line) for
heme-based absorption. N-terminal sequence analysis revealed that the
peak marked H/EPO contained primarily EPO heavy chain, but
also some light chain. The peak marked L contained EPO light
chain only. The total column volume was 22 ml.
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Fig. 3.
Reversed-phase high pressure liquid
chromatography of thermolytic digests of EPO (A) and
LPO (B). Protein (1 mg) digested with thermolysin
was loaded onto a Nucleosil C18 column, and peptides were eluted with a
linear gradient of increasing (1%/min) acetonitrile concentration. The
eluates were monitored at 226 nm (thick line) for eluting
peptides and at 398 nm (thin line) for eluting heme
peptides. Manually collected fractions containing peptides with
absorption at 398 nm were further analyzed (Tables I and III).
Sequence analysis of heme peptides derived from EPO
Sequence analysis of EPO peptides exposed to ammonia
Sequence analysis of thermolytic heme peptides derived from LPO
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Fig. 4.
Heme structure (see Footnote 4) and heme
linkage to polypeptides of EPO and LPO. Iron-protoporphyrin IX,
heme b, has methyl groups at C-1 and C-5. Here, heme b is shown with
ester linkages through hydroxylated methyl groups at C-1 and C-5, as
proposed for MPO (25). Fragments of the polypeptide chains of EPO and
LPO are shown above and below the heme structure
with indication of the Glu and Asp residues engaged in ester linkages
(Table II). The two heme variants observed in monopeptides, having a
methyl group or a hydroxymethyl group at C-5, are shown in a
smaller font. Residues are numbered from the N termini of
the mature proteins (see Footnote 3). The calculated average mass of
nonderivatized heme b
(C34H32FeN4O4) is
616.51 Da. The calculated mass of the expected heme contribution to the
total mass in heme bispeptides (HC-C-1-heme b core-C-5-CH) is 4.03 mass
units lower, i.e. 612.48 Da. This mass was observed for all
EPO and LPO bispeptides analyzed (Table IV).
Mass analysis of selected EPO and LPO heme mono- and bispeptides
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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David Loegering and James Checkel are thanked for preparation of eosinophil granules and for EPO purification. Lene Kristensen is also thanked for excellent technical assistance.
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FOOTNOTES |
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* This work was supported by grants from the Danish Natural Science Research Council, the Danish Biotechnology Program, and the Novo Nordisk Foundation.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. Tel.: 45-8942-5060; Fax: 45-8612-3178; E-mail: co{at}mbio.aau.dk.
2 A. R. Thomsen., C. Oxvig., and L. Sottrup-Jensen, unpublished observation.
3 The residues of EPO and LPO are numbered from the N termini of the mature proteins. The light and heavy chains of EPO are not separately numbered; the numbers correspond to those prior to cleavage between Gly-111 and Val-112.
4 Recent IUPAC recommendation for numbers of the heme b macrocycle differ from the numbering used in this study. In the recent IUPAC numbering (37), propionic acids are at positions 13 and 17; methyl groups at positions 2, 7, 12, and 18; vinyl groups at positions 3 and 8; and hydrogen atoms at positions 5, 10, 15, and 20. The orientation of the heme group is according to the orientation of the heme group in the x-ray structure of MPO (25).
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ABBREVIATIONS |
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The abbreviations used are: MPO, myeloperoxidase; EPO, eosinophil peroxidase; LPO, lactoperoxidase; PAGE, polyacrylamide gel electrophoresis; CAPS, 3-(cyclohexylamino)-1-propanesulfonic acid.
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REFERENCES |
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