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INTRODUCTION |
Oxidation of methionine residues to methionine sulfoxide has been
shown to affect the activity of a number of biologically active
proteins, such as
1-protease inhibitor, chymotrypsin, phosphoglucomutase, ribonuclease, and subtilisin, as well as several peptide hormones (1). Such oxidation can occur in vivo in
response to oxidants released by neutrophils (e.g.
superoxide, hydroxyl radical) or in vitro when proteins are
exposed to oxidizing agents such as hydrogen peroxide
(H2O2), periodate, dimethyl sulfoxide, chloramine-T, N-chlorosuccinamide (2, 3), or
t-butyl hydroperoxide (4). In the majority of cases studied,
oxidation of methionine residues has been shown to cause a decrease in
the biological activity of the protein. Early studies demonstrated a
loss of activity in chymotrypsin upon oxidation of a methionine three residues from the active-site serine (5). More recently, it was shown
that oxidation of human growth hormone had no effect on binding to
lactogenic receptors, whereas oxidation of the closely related human
chorionic somatomammotropin on nonhomologous methionines caused a loss
of binding to these receptors (6). Methionine oxidation may also lead
to an increase in biological activity, as in the case of the C5
component of complement (7), or to no apparent change in structure or
activity, such as with
2-plasmin inhibitor (8).
One of the most thoroughly studied examples of methionine oxidation and
its effect on activity involves the
1-protease inhibitor (
1-PI).1 This
protein can be oxidized on two of its eight methionine residues, and
oxidation of one of these residues (Met358) causes an
almost complete loss of inhibitory activity of
1-PI toward its primary biological target, elastase (9). As in the case of
chymotrypsin, the relevant methionine in
1-PI is
proximal to the active site for binding of the target protease, in this case the P1 residue within the reactive loop of the protease cleavage site. Oxidation of this methionine in vivo has been
suggested to contribute to the development of emphysema in cigarette
smokers through a change in the equilibrium between lung elastase and its inhibitor, ultimately resulting in increased elastase activity (10,
11). Such an imbalance is known to promote emphysema in cases of
inherited
1-PI deficiency, particularly among those who
smoke (12, 13). It has been proposed that methionine oxidation may
actually be a general means of physiological inactivation of plasma
protease inhibitors (1).
Here we have examined the susceptibility to oxidation of a plasma
protease inhibitor closely related to
1-PI, namely
antithrombin (AT). Like
1-PI and other serpins, AT has a
reactive loop structure that binds to and then is cleaved by the target
protease (for a review, see Ref. 14). In the case of AT, however, the
reactive loop is partially inserted into the protein, based on x-ray
crystal structure (15, 16). It has been proposed that the binding of
heparin causes a conformational change that results in activation of AT
as an inhibitor (17). This conformational shift also changes the
environment of tryptophan residues within AT, causing an increase in
fluorescence (18, 19).
There has been one report of oxidative inactivation of AT (20);
however, the sites(s) and extent of oxidation were not characterized in
this study. AT contains 12 methionines, five of which are relatively exposed on the surface of the protein. Within the three-dimensional structure, two of these methionine residues (Met314 and
Met315) are positioned adjacent to the reactive loop of AT,
which is cleaved by thrombin between Arg393 and
Ser394. Another two of the exposed methionines
(Met17 and Met20) border on the heparin binding
site of AT. The three-dimensional structure of AT is quite similar to
1-PI, which, as discussed above, is inactivated by
oxidation of a methionine within its reactive loop. If, as suggested by
Swaim and Pizzo (1), methionine oxidation serves as a general means of
plasma protease inhibitor inactivation, it might be expected that
oxidation of Met314 and/or Met315 in AT could
be a means of physiologically down-regulating its activity. We report
here on the relative sensitivity of these methionine residues to
oxidation and the resulting effects on AT in terms of both
thrombin-inhibitory activity and heparin binding.
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EXPERIMENTAL PROCEDURES |
Antithrombin--
Antithrombin used in the forced oxidation
experiments was recombinant human AT purified from the milk of
transgenic goats as described in Ref. 21. Commercially available
preparations of human plasma derived AT were obtained from Behringwerke
(Kybernin) and Miles (Thrombate III). Nonoxidized AT refers to samples
that have not been treated with hydrogen peroxide but may contain some oxidized material. The term "unoxidized" is used here to refer to
protein (or peptide) that has been shown to contain no oxidation, for
example the A form in Fig. 1.
Hydrogen Peroxide-induced Oxidation of AT--
Purified
recombinant AT was oxidized to varying degrees using hydrogen peroxide
(Sigma catalog no. H-1009) essentially as described by Teh et
al. (6). Each reaction contained 200 µg of AT in a total volume
of 150 µl (23 µM AT) in 100 mM
NH4HCO3, 0.5 mM EDTA, pH 8.0, with
varying concentrations of hydrogen peroxide as indicated. All reactions
were carried out for 30 min on ice with two exceptions. The samples
referred to as 400/60/ice and 400/60/RT were incubated for 60 min in
400 mM peroxide either on ice or at room temperature,
respectively. The reaction was stopped by the addition of immobilized
catalase (Sigma catalog no. C-9284), which was subsequently removed by
filtration (Rainin Microfilterfuge no. 7016-022).
Separation of Oxidized Forms by High Performance Liquid
Chromatography (HPLC)--
Samples of AT oxidation reactions were
applied to a C4 HPLC column (2.1 × 250 mm; 5 µm; narrow bore;
Vydac catalog no. 214TP52) equilibrated in 75% solvent A (0.1%
trifluoroacetic acid), 25% solvent B (acetonitrile in 0.08%
trifluoroacetic acid) at a flow rate of 0.3 ml/min and eluted with
solvent B using a series of linear gradients as follows: 0-5 min
25-40% solvent B, 5-20 min 40-45% solvent B, 20-25 min 45-80%
solvent B.
The eluted protein was monitored by absorbance at 215 and 275 nm. For
preparative runs involving isolation of the A, B, and C forms of
oxidized AT, solvent B was held constant at 45% for 5 min (20-25 min)
followed by the 45-80% increase. Fractions were collected
automatically on a Gilson FC203B fraction collector.
Endoproteinase Lys-C Mapping of Oxidized Forms of Recombinant
AT--
Samples of oxidized AT (50 µg) were reduced and
pyridylethylated in denaturing buffer (0.25 M Tris, 6.0 M guanidine HCl, pH 8.6) containing
-mercaptoethanol at
a ratio of 1000:1 (
-mercaptoethanol:cysteine) and 4-vinylpyridine at
a ratio of 1005:1 (4-vinylpyridine:cysteine). Modified protein was
desalted by reverse phase HPLC on a C4 guard cartridge into 75%
acetonitrile in 0.08% trifluoroacetic acid, partially dried down on a
Speed-vac, and then digested with endoproteinase Lys-C (sequencing
grade; Boehringer Mannheim) in 0.25 M Tris-HCl, 0.01 M EDTA, pH 8.5, at an enzyme:substrate ratio of 1:50 (w/w) for 18 h at 37 °C. The resultant digests were quenched and
mapped by reverse phase HPLC on a C8 column (2.1 × 150 mm; 5 µm; narrow bore; Vydac catalog no. 208TP5215), equilibrated in 100%
solvent A (0.1% trifluoroacetic acid), 0% solvent B (acetonitrile in
0.08% trifluoroacetic acid) at a flow rate of 0.3 ml/min, and eluted with solvent B using a series of linear gradients as follows: 0-3 min
0% solvent B, 3-43 min 0-20% solvent B, 43-73 min 20-30% solvent
B, 73-103 min 30-45% solvent B, 103-131 min 45-90% solvent B. Peptide elution was monitored by absorbance at 215 nm.
LC/MS Analysis of Proteolytic Digests--
LC/MS was carried out
using a Finnigan TSQ 700 triple quadrupole mass spectrometer (San Jose,
CA) interfaced through a Finnigan electrospray ionization source with a
Michrom BioResources UMA HPLC system (Auburn, CA). Peptide mapping was
carried out on 0.5-1.0-nmol aliquots of AT Lys-C digests, which were
separated using a Michrom column (1.0 × 100 mm) packed with Vydac
C8 resin. The flow rate was 50 µl/min, and peptides were eluted with
a gradient of 0-46% acetonitrile in 0.1% trifluoroacetic acid. HPLC
eluant from the UV detector was mixed with methoxyethanol
(Pierce)/methanol (Burdick & Jackson, Muskegon, MI) (3:1) at 25 µl/min through a "T" union before entering the electrospray
interface. Centroid mass spectra were acquired over an
m/z range of 200-3000 at a 3-s scan rate.
Analysis of Oxidation Sites on the K30 Lys-C
Peptide--
Unoxidized, singly oxidized, and doubly oxidized forms of
the K30 Lys-C peptide were collected from Lys-C maps of AT that had
been partially oxidized using 50 mM hydrogen peroxide. For this map, peptides were fractionated using an abbreviated gradient similar to that described above but with the second and third segments
shortened to 12 and 8 min, respectively. The isolated peptides (1-2
nmol) were dried, reconstituted in 70% formic acid, and digested with
CNBr (CNBr:Met molar ratio of 200:1) for 18 h in the dark at room
temperature with a nitrogen overlay. Reactions were then dried under
nitrogen, reconstituted in 0.1% trifluoroacetic acid, and analyzed by
LC/MS as described above except that a Vydac C18 column (1.0 × 100 mm) was used with a gradient of 10-60% acetonitrile in 0.1%
trifluoroacetic acid over 30 min.
AT Activity Assay (Thrombin Inhibition)--
AT activity was
assayed as described previously (21) by measuring inhibition of
thrombin (Calbiochem catalog no. 605190; 243 milliunits/ml in the
assay)-catalyzed cleavage of the thrombin-specific chromogenic
substrate S2238 (Kabi; 337 µM in the assay). A saturating concentration of heparin (55.5 nM; porcine; Sigma catalog
no. H-3393) was used except where noted. In calculations of the molar concentration of heparin, the molecular mass was assumed to be 13,500 daltons for this heparin preparation.
Fluorescence Assay of Heparin Binding--
Binding of heparin to
AT was analyzed by fluorescence enhancement as described by Fan
et al. (22). Samples of AT were diluted to 20 nM
in 4 ml of 20 mM sodium phosphate, 100 mM NaCl,
100 µM EDTA, 0.1% polyethylene glycol, pH 7.4. Fluorescence was measured using an excitation wavelength of 280 nM (5-nm bandwidth) and an emission wavelength of 340 nM (10-nm bandwidth). Heparin (porcine; Sigma catalog no.
H-3393) was added sequentially in 1-µl aliquots (up to 14 µl total
added volume) and allowed to mix for 20 s prior to measuring the
resulting increase in fluorescence.
Heparin Affinity Chromatography--
Heparin binding affinity of
the various samples of oxidized AT was also compared by relative
retention on a heparin affinity column (Toso-Haas TSK-Gel Heparin-5PW;
5-mm inner diameter × 50 mm) as described by Fan et
al. (22). Samples of AT were applied to the column equilibrated in
50 mM Tris-Cl, 10 mM sodium citrate, pH 7.4 (HAC buffer) at a flow rate of 0.5 ml/min. Protein was eluted from the
column using a linear gradient of 0-3 M NaCl in HAC buffer
over 20 min while monitoring absorbance at 215 nm.
Circular Dichroism--
CD measurements for the oxidized and
nonoxidized AT samples were carried out on a JASCO 720 spectrophotometer at room temperature. Oxidized samples were prepared
as outlined above. All samples were in ammonium bicarbonate reaction
buffer, pH 8.0. Oxidized samples used for the CD measurements were as
follows: (i) 50 mM hydrogen peroxide incubated for 30 min
on ice, (ii) 400 mM hydrogen peroxide incubated for 60 min
on ice (400/60/ice), and (iii) 400 mM hydrogen peroxide
incubated for 60 min at room temperature (400/60/RT). Measurements were
made both in the far and near ultraviolet region using 0.01 cm (far UV)
and 1 cm (near UV) cuvettes. Spectra were scanned 45 times, averaged,
smoothed, and base line-corrected. The concentration of AT samples
varied between 0.92 and 0.99 mg/ml. For heparin binding studies,
heparin was added at saturating concentration to AT samples (123 µM). To eliminate the contribution of heparin to the CD
signal, the same amount of heparin was also added to the buffer, and
the buffer-heparin CD was subtracted from the sample.
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RESULTS |
Shift in Reverse Phase Mobility of AT with
Oxidation--
Incubation of AT with hydrogen peroxide resulted in a
concentration-dependent shift in the reverse phase HPLC
mobility of the protein to shorter retention times. We used this forced
oxidation as a means of characterizing the sites on AT that are most
sensitive to oxidation and to study the effect of oxidation on protein
function. We refer here to the predominant peak prior to oxidation as
the A form of the protein and to the two earlier eluting peaks
that increase with oxidation as the B and C forms, as shown in Fig. 1. The discrete shift to less hydrophobic
forms with increasing oxidation suggests that each peak represents a
distinct oxidized form of the protein. Quantitation of the peaks in
Fig. 1 by integration of absorbance (inset) illustrates the
transition from the A to the B to the C forms with increasing peroxide
concentration. Other relatively minor peaks eluting even earlier than
the C form were apparent following treatment with the highest
concentrations of peroxide.

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Fig. 1.
Oxidation of AT with hydrogen peroxide.
A series of oxidation reactions of purified recombinant AT was
performed as described under "Experimental Procedures" by
incubating 23 µM AT with various concentrations of
hydrogen peroxide for 30 min on ice. The samples labeled 400/60/ice and
400/60/RT were incubated for 60 min in 400 mM peroxide on
ice or at room temperature, respectively. An aliquot containing 3.6 µg of AT from each reaction was subjected to reverse phase HPLC for
separation of the oxidized forms as described under "Experimental
Procedures." Integration was performed (inset) on peaks A
( ), B ( ), and C (×) for each peroxide concentration.
mAU, milliabsorbance units.
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Identification of Oxidation Sites on AT--
Samples of oxidized
AT from the reactions represented in Fig. 1 were reduced, alkylated,
and digested with Lys-C endoproteinase. The resulting digests were
fractionated by HPLC, and these peptide maps are shown in Fig.
2A. The only two peptides
significantly affected by oxidation were the K2 peptide (residues
Pro12-Lys28 in AT) and the K30 peptide
(residues Glu298-Lys332 in AT). The peaks
corresponding to these peptides and their oxidized counterparts were
identified by LC/MS analysis of the digests as shown for the K30
peptide in Fig. 2B. There was a substantial decrease in the
amount of unoxidized K30 peptide in the digest as the peroxide
concentration was increased, and there was a corresponding increase in
peaks having somewhat shorter retention times. LC/MS analysis of the
digests (Fig. 2B) revealed that the three peaks eluting at
94-96 min in the oxidized samples correspond to K30 plus 16 mass units
(singly oxidized K30), and the overlapping peaks at ~88 min
correspond to K30 plus 32 mass units (doubly oxidized K30). There is a
similar change in the K2 peptide, although at higher peroxide
concentrations, and peaks corresponding to singly and doubly oxidized
K2 were again shown to be present using mass spectrometry. From these
experiments, it appears that the K30 region of AT is the most sensitive
to oxidation, followed by the K2 region.

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Fig. 2.
Regions of AT most susceptible to
oxidation. A, endoproteinase Lys-C peptide maps of
50-µg aliquots of AT from the oxidation reactions in Fig. 1. The K2
peptide corresponds to amino acids Pro12-Lys28
from the AT sequence, while the K30 peptide corresponds to amino acids
Glu298-Lys332. Peaks designated 1 Ox and 2 Ox represent the singly and doubly oxidized
forms of the given peptide as determined by LC mass spectrometry. Shown
in B are the complete relative ion current trace from the
mass spectrometry data as well as selected ion monitoring traces for
K30, K30 + 16, and K30 + 32. The m/z values used
for the selected ion monitoring analyses were as follows: K30, 1421.3 and 1066.25; K30 + 16, 1426.7 and 1070.25; K30 + 32, 1432.0 and
1074.25. mAU, milliabsorbance units.
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The K30 peptide from AT contains three methionines and one tryptophan
as possible sites of oxidation. One of the methionines (Met320) is buried within the core of the intact protein
and is therefore an unlikely target of oxidation. The other two
methionine residues (Met314 and Met315) are
exposed at the surface of the protein and are in fact adjacent to the
active site loop that is cleaved during serpin inhibition, based on the
three-dimensional structure of AT (Fig.
3). The tryptophan residue in K30
(Trp307) is partially exposed in the intact protein (not
seen in Fig. 3, since it is on the opposite side of the protein) and
therefore could be oxidized; however, tryptophan oxidation is less
likely under the conditions used here. The K2 peptide contains two
methionines (Met17 and Met20), which are both
exposed at the surface of the intact protein and are at one end of the
binding site for heparin, which stretches along helix D (Fig. 3).

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Fig. 3.
Position of the K2 and K30 methionines within
AT. Three-dimensional structure of AT based on the model of the I
form of Carrell et al. (15). Positions of the K2 methionines
(Met17 and Met20) and the two adjacent
methionines of the K30 peptide (Met314 and
Met315) are shown in yellow. Also highlighted
are the reactive loop (green;
Ala383-Val400) containing the thrombin
cleavage site (blue; Arg393-Ser394)
and the region of the protein thought to interact with heparin
(red; Arg13-Arg24 and
Lys107-Lys139).
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To determine the specific sites of oxidation on K30, the three singly
oxidized peaks and one doubly oxidized peak of the K30 peptide were
collected along with unoxidized K30 from Lys-C peptide maps of
partially oxidized AT as shown in Fig.
4A. The three singly oxidized
peaks were designated x, y, and z for the purposes of this analysis.
Each of the isolated peptides was subjected to CNBr digestion, which
would be expected to result in four peptide fragments in the case of
unoxidized K30 (M1-M4; Fig. 4B). These digests were
analyzed by LC/MS using a short reverse phase HPLC gradient, and the
observed masses for each digest are listed in Table
I. Oxidation of a given methionine
residue should not only result in an increase of 16 mass units for
peptides containing that methionine, it should also prevent cleavage at
that site and therefore result in the absence of specific peptides in
the digest (23). CNBr digestion was not complete in all cases as evidenced by the presence of M(2-3), M(3-4), and M(2-4) in the digest of unoxidized K30. Also, M2 is a single amino acid and is
therefore too small to be detectable by this analysis. Nevertheless, the absence of M3 in the singly oxidized (x) digest, along
with the fact that M1 and M4 were present, indicates that cleavage did
not occur at Met315 and therefore that this methionine was
oxidized. This is supported by the fact that M(2-3) and M(2-4)
displayed an increase of 16 mass units in this digest. Similarly, M1
was absent in the singly oxidized y and z
digests, while M3 and M4 were present, indicating that oxidation of
Met314 blocked cleavage between M1 and M2. Once again this
was supported by a 16-mass unit increase in M(1-2) and M(1-3). The
digest of the doubly oxidized K30 peptide contained only M(1-3) (plus
32 mass units) and M4, indicating that both Met314 and
Met315 are oxidized in this peptide. The absence of an M1 + 16 mass units peptide in any of the oxidized K30 peaks demonstrates
that Trp307 is not oxidized under these conditions.

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Fig. 4.
Identification of specific sites of oxidation
on K30. A, reverse phase HPLC of AT that had been
oxidized with 50 mM peroxide and then reduced, alkylated,
and digested with Lys-C. Peaks corresponding to unoxidized K30 as well
as its singly oxidized (K30 + 16; peaks x, y, and z) and doubly
oxidized (K30 + 32) forms were collected and subjected to CNBr
digestion, which would be expected to result in three peptides and a
methionine residue for the unoxidized peptide (B). Cleavage
would be blocked at oxidized methionine residues. Digests were analyzed
by LC/MS in order to determine sites of oxidation (Table I).
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Table I
Mass analysis of oxidized K30 peptides digested with CNBr
Underlined numbers are peptide masses that display an increase of +16
or +32, indicating that the peptide is either singly or doubly
oxidized, respectively.
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As shown in Table I, the y and z forms of K30
resulted in identical sets of peptides in the CNBr digest but were
clearly separated on the HPLC of the original peptide map (Fig.
4A). The most likely explanation for this is that these two
peptides are stereoisomers with respect to the added oxygen atom on the
chiral sulfur of Met314. Such separation of stereoisomers
of oxidized peptides by HPLC has been reported previously (24). In some
maps, peak splitting was also seen for oxidation at Met315
(peak x), although this was not as distinct as for the peptide oxidized
at Met314. Peak splitting of doubly oxidized K30 was
consistently observed in the maps (see Figs. 2 and 4A),
presumably for the same reason.
Presence of Oxidized Forms in Commercial Preparations of
AT--
Two different commercially available preparations of AT
(Kybernin from Behringwerke and Thrombate III from Miles) as well as
transgenic recombinant AT (Genzyme Transgenics Corp.) were tested for
the presence of oxidized forms (Fig. 5)
employing the reverse phase HPLC method used in Fig. 1. Each of these
preparations contained the B, and to a lesser extent C, oxidized forms
of AT. The A, B, and C forms of AT were isolated in this manner from both a peroxide-oxidized sample of recombinant AT (50 mM
peroxide) and one that had not been treated with peroxide. They were
each then Lys-C-mapped as described above. The peptide maps for the three isolated forms from the untreated sample (Fig.
6) were comparable with those for the
three forms isolated from the peroxide-treated sample (data not shown),
with oxidation again occurring primarily in the K2 and K30 regions of
the protein. The fact that oxidation was consistently seen in AT
isolated from both plasma and milk suggested that oxidation may occur
in vivo as a means of controlling the activity of this
protein.

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Fig. 5.
Oxidized forms are present in commercial
preparations of AT. C4 reverse phase HPLC analysis of recombinant
human AT derived from transgenic goats and two commercially available
AT preparations derived from human plasma (Behringwerke Kybernin and
Miles Thrombate III). Peak A is unoxidized AT, while peaks B and C
represent oxidized forms. AU, absorbance units.
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Fig. 6.
Analysis of isolated oxidized forms of
AT. Lys-C peptide maps of isolated A, B, and C peaks from C4
reverse phase HPLC of untreated, inherently oxidized AT. Peaks
corresponding to the unoxidized (No Ox), singly oxidized
(1 Ox), and doubly oxidized (2 Ox) forms of the
K2 and K30 peptides were quantitated by integration of the 215-nm
absorbance, and each was expressed as a percentage of the total area
for each peptide in the accompanying bar graph. mAU,
milliabsorbance units.
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Peaks corresponding to unoxidized, singly oxidized, and doubly oxidized
K2 and K30 in Fig. 6 were integrated for each of the three isolated
forms of intact AT, and the results are shown in the bar
graphs to the right. The amount of each oxidized
form for each peptide is plotted relative to the total amount of that peptide. These data clearly show that the B and C forms correlate well
with singly and doubly oxidized K30, respectively, indicating that it
is oxidation of K30 that is primarily responsible for the shift in
retention time seen for intact AT with oxidation (Fig. 1).
Effect of Oxidation on Antithrombin Activity--
As mentioned
above, the K30 region of AT contains two adjacent methionines on the
surface of the protein (Fig. 3), which are in close proximity to the
reactive loop of AT that binds to thrombin and is subsequently cleaved.
It is, therefore, reasonable to expect that oxidation of one or both of
these residues might affect the ability of AT to bind and inhibit thrombin.
Samples of untreated (nonoxidized) antithrombin and AT oxidized to
various levels were assayed as described under "Experimental Procedures" at multiple concentrations of AT in the presence of saturating concentrations of heparin (Fig.
7A). In the sample of AT that
was oxidized using 50 mM peroxide for 30 min on ice, ~80% of the protein was oxidized to at least some degree, based on
the data of Fig. 1, yet there was no difference in its ability to
inhibit thrombin in this assay. The sample of AT that was oxidized with
400 mM peroxide for 60 min on ice (400/60/ice) was >95%
oxidized, but even this preparation was able to inhibit thrombin nearly as effectively as nonoxidized AT in this assay.

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Fig. 7.
Effect of oxidation on AT activity.
Shown is thrombin-inhibitory activity of nonoxidized AT ( ) and 50 mM peroxide ( ), 400/60/ice ( ), and 400/60/RT ( )
samples of AT at various AT concentrations with a saturating
concentration of heparin (55.5 nM) (A) and at a
single AT concentration (3 nM) with varying concentrations
of heparin (B).
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The activity assays in Fig. 7A were done in the presence of
excess heparin. At less than saturating concentrations of heparin, however, high levels of oxidation on AT were found to result in decreased thrombin-inhibitory activity relative to the nonoxidized control (Fig. 7B). Nevertheless, the preparation of AT that
was ~80% oxidized (50 mM peroxide) exhibited little if
any difference in its requirement for heparin in this assay. At the
higher levels of oxidation, there was a clear effect on the requirement
for heparin that increased with increasing oxidation. The half-maximal heparin concentrations for thrombin inhibition are 0.49 nM
for highly oxidized AT (400/60/RT) and 0.034 nM for the
control, based on this assay.
Effect of Oxidation on Heparin Affinity--
The data of Fig.
7B suggested that the binding of heparin to AT might be
affected by oxidation. We therefore studied this more directly using
the heparin binding assay of AT based on fluorescence. The inherent
fluorescence of AT at 340 nm increases with increasing heparin
concentration due to a conformational change in the protein that occurs
with the binding of heparin and alters the exposure of one or more
buried tryptophan residues (19). This property can be exploited to
measure the binding affinity of AT for heparin (22). Less oxidized
samples of AT (peroxide concentration
50 mM) displayed no
significant change in heparin binding relative to the nonoxidized
material (Fig. 8A). Since the
AT in the 50 mM oxidation was ~80% oxidized within the
K30 peptide (Fig. 1; inset), oxidation of the K30
methionines does not appear to affect heparin binding. This is
consistent with the fact that the K30 methionines are far removed from
the heparin binding site. At higher levels of oxidation (peroxide
concentration >50 mM), where increasing oxidation of K2
methionines is observed, there was a shift in the binding curve toward
lower maximal fluorescence, and this shift increased with increasing
oxidation. This suggests that a high level of oxidation either results
in a difference in the heparin-induced conformational change in AT or
that it generates a subpopulation of AT that does not bind heparin and, therefore, lowers the overall measured change. The fact that
thrombin-inhibitory activity (in the presence of heparin) is relatively
unaffected by even high levels of K2 and K30 oxidation indicates that
the first alternative is most likely the case.

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Fig. 8.
Effect of oxidation on heparin binding.
Heparin binding assays of nonoxidized AT ( ) and samples of oxidized
AT designated 50 mM peroxide ( ), 100 mM
peroxide (×), 200 mM peroxide ( ), 400 mM
peroxide ( ), 400/60/ice ( ), and 400/60/RT ( ). Heparin binding
was measured by both fluorescence assay (A; 20 nM AT with increasing heparin) and heparin affinity
chromatography (B; 95 µg of AT/run). In A, a
least squares fit using the equation described in Ref. 27 was performed
on the fluorescence data to give the curves shown. The linear salt
gradient for the heparin affinity column in B is indicated
by the short dashed line.
mAU, milliabsorbance units.
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To further assess changes in heparin binding in these samples of AT, a
heparin affinity column was used. Nonoxidized and oxidized samples were
bound to the column and then eluted using a linear salt gradient (Fig.
8B). Both preparations of AT eluted from the column as
multiple peaks due to heterogeneity both in the AT itself and in the
heparin on the column. However, there was a significant shift in the
binding of the oxidized preparation toward lower affinity (elution at
decreased salt concentration). It is clear that high levels of
oxidation caused a decrease in the ability of AT to bind to
heparin. As discussed previously, the K2 region of AT contains two
exposed methionine residues that can become oxidized and are near the
binding site for heparin on AT (Fig. 3). It is, therefore, likely that
oxidation of one or both of these methionines was responsible for this
decrease in heparin affinity. The K2 region of AT is considerably more
difficult to oxidize than is the K30 region (Fig. 6), and this is
consistent with the fact that changes in heparin affinity are only seen
at relatively high levels of oxidation.
Effect of Oxidation on Structure/Conformational
Changes--
Structural changes induced by different levels of
methionine oxidation in AT and their influence on the conformational
changes that occur upon heparin binding were investigated by far and
near UV CD spectroscopy. Fig.
9A shows the far UV CD spectra
for AT in its native form and in three different oxidized forms. For AT
treated with 50 mM peroxide, the negative bands at 210 and 220 nm show an increase in intensity (see inset), while the
positive band at 196 nm shows a marginal decrease. In the sample
treated with 400 mM peroxide for 60 min on ice
(400/60/ice), the band at 220 nm becomes less intense when compared
with the 50 mM oxidized form. For the sample treated with
400 mM peroxide for 60 min at room temperature (400/60/RT),
there is a further reduction in the magnitude of the positive band at
196 nm. The spectra also indicate that the positive to negative
cross-over point is right-shifted upon oxidation of AT.

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Fig. 9.
Circular dichroism analysis of oxidized
AT. Shown are the circular dichroism spectra of nonoxidized AT
(solid line) and various samples of oxidized AT,
including 50 mM peroxide (dotted
line), 400/60/ice (dotted and
dashed line), and 400/60/RT (dashed
line). Spectra were measured in the absence of heparin for
both far UV (A) and near UV (B). A portion of the
far UV spectrum has been enlarged to clarify local differences
(A, inset). Molar ellipticity (Mol.
Ellip.) is expressed in degrees × cm2 × dmol 1.
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Changes are even more pronounced in the near UV region of the spectra
(Fig. 9B). For the 50 mM peroxide sample, the
changes in the spectra are marginal except for a minor variation in the peak observed at 275 nm. However, for the more oxidized 400/60/ice sample, there is a significant upward shift for all peaks and valleys
between 255 and 280 nm, indicating a change in the environment around
phenylalanine and/or tyrosine residues. It is also interesting to note
that the peaks at 284, 288, 292, and 300 nm are not affected in this
sample. Since the peaks for these latter wavelengths usually arise from
tryptophan residues, these data would suggest that methionine oxidation
does not perturb tryptophan even at relatively high levels of
oxidation. In the most highly oxidized sample, 400/60/RT, all peaks are
increased somewhat, including those at 292 and 300 nm.
Changes in heparin binding affinity as a result of oxidation were also
monitored by both near and far UV spectroscopy. As described under
"Experimental Procedures," a saturating concentration of heparin
was added to preparations of AT for these experiments. Changes in the
far UV spectra of highly oxidized AT (400/60/RT) with the addition of a
saturated level of heparin are virtually the same as those for the
nonoxidized control, with only marginal variation except a left shift
of the positive peak (around 196 nm) by 1.5 nm when compared with that
in the absence of heparin (data not shown).
Near UV spectra of AT-heparin complex on the other hand shows a
dramatic upward shift for all bands upon adding heparin to AT. The
spectra depicted in Fig. 10 represent
the difference in CD between AT alone and AT-heparin complex for the
nonoxidized (left panel), moderately oxidized (50 mM peroxide; center panel), and
highly oxidized (400/60/RT; right panel) forms.
It is clear from these spectra that the upward shift is not affected in
the moderately oxidized form but is substantially less in the highly oxidized form, particularly in the 280-300 nm region, which reflects changes in the environment of tryptophan residues. This inverse relationship between the level of oxidation and the relative change in
tryptophan band intensity upon binding of heparin is consistent with
the fluorescence data presented in Fig. 8A.

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Fig. 10.
Effect of oxidation on heparin-induced
conformational change. Near UV circular dichroism spectra of
nonoxidized AT (A), 50 mM peroxide-oxidized AT
(B), and 400/60/RT-oxidized AT (C) in the absence
(solid line) and presence (dashed
line) of a saturating concentration of heparin (123 µM). Molar ellipticity is expressed in degrees × cm2 × dmol 1.
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To determine which oxidation site(s) might be responsible for the
reduced response to heparin binding, the crystal structure of AT (15)
was analyzed using INSIGHT graphics software (Molecular Simulations)
with particular attention to the residues surrounding the potential
oxidized methionine sites. Methionines located at 17, 20, 314, and 315 have been shown here to be the primary sites of oxidation in the
protein. Since treatment with 50 mM hydrogen peroxide
oxidizes primarily Met314 and Met315 and there
is very little change in the heparin response for this sample of
oxidized AT, it is unlikely that oxidation of these sites affects the
heparin response. Methionines at 17 and 20 become much more
oxidized at higher hydrogen peroxide concentrations, at which point the
reduced response to heparin is seen in the CD and fluorescence data.
However, these two residues are not very close to any aromatic
residues; hence, oxidation of these two methionines may not have any
direct influence on changes in the spectra due to aromatics.
Nevertheless, any change in the conformation of the segment that
contains these two methionines could affect the orientation of
neighboring aromatic residues. Oxidation of Met20 is
unlikely to cause such conformational changes, since its sulfur is well
exposed and pointing away from the peptide backbone (from examination
of the three-dimensional structure; not shown). The sulfur of
Met17, on the other hand, is close to the backbone; thus,
oxidation could change the backbone orientation. Since the N-terminal
segment containing Met17 borders on the heparin binding
site of AT, our analysis strongly suggests that oxidation of
Met17 is the major cause for the change in response to
heparin binding.
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DISCUSSION |
It is now well established that oxidation of methionine at the
elastase cleavage site serves as a means of down-regulating the
activity of the serpin
1-PI in the lung. Antithrombin is a serpin closely related in structure to
1-PI but with
an Arg-Ser bond at the P1-P1' cleavage site rather than a Met-Ser bond
as in the case of
1-PI. Although AT does not contain an
oxidizable methionine at this site, it does contain a pair of
methionine residues (Met314 and Met315) that
are quite close to the Arg-Ser cleavage site in the three-dimensional structure of the protein. Also, AT and other serpins have been reported
to be inactivated by oxidation (20), suggesting that oxidation might be
a general mechanism for down-regulating serpin activity.
Here we have shown that the two methionines at positions 314 and 315 are the most sensitive to oxidation in AT. Oxidation of either
methionine alone causes a discrete shift in retention time on reverse
phase HPLC to a form that is easily resolved from the unoxidized
protein. Oxidation of the second of these two residues causes a further
shift to lower retention time, again to a clearly resolved form.
Oxidation of other methionines in AT does occur, particularly in the K2
region of the protein, however less readily than on the K30 methionines.
Based on the data presented here for AT incubated with 50 mM peroxide, oxidation of Met314 and
Met315 does not appear to affect either the ability of AT
to bind heparin or to inhibit thrombin. However, further oxidation of
AT does affect heparin binding to some degree, as evidenced by the
heparin affinity column data presented in Fig. 8B.
Nevertheless, even at very high levels of oxidation,
thrombin-inhibitory activity is unaffected in the presence of high
heparin concentrations. This would appear to conflict with the earlier
study in which AT was inactivated by oxidation (20); however, in this
study, the oxidation was carried out with chloramine T in the presence of detergent, and the extent of oxidation was not characterized.
The results of the fluorescence assay presented here (Fig.
8A), in which the maximal change in fluorescence was
decreased, suggest that oxidation interferes with the ability of the
protein to undergo the conformational change that occurs as a result of heparin binding. This is supported by structural data from CD, which
also shows a reduced response to saturating heparin in the highly
oxidized sample, particularly in the portion of the spectrum representing changes in tryptophan. Other factors have been shown to
influence the ability of AT to bind heparin. In particular, the
presence of an oligosaccharide chain on Asn135 decreases
heparin affinity significantly (25). The form lacking glycosylation at
this site (known as the
form) and having a higher heparin affinity
represents ~10% of total AT in plasma. Recently, it was proposed
that glycosylation of this site decreases heparin affinity by
interfering with the conformational change in AT that occurs as a
consequence of the initial binding of heparin (26). It is thought that
this conformational change results in a much tighter association
between heparin and AT. It is possible that the decreased heparin
affinity seen here with relatively high levels of oxidation also occurs
as a result of interference with this conformational shift. The two
methionines within the K2 peptide (Met17 and
Met20) border on the binding site for heparin (Fig. 3) but
are at the opposite end of the D helix from the Asn135
glycosylation site. This helix contains multiple basic residues that
participate in the binding of heparin and is thought to be involved in
the conformational change that leads to tighter binding. It is likely
that the oxidation of one or both of these residues, which occurs only
at higher levels of oxidation, interferes with the conformational
change and, therefore, the ability of AT to adopt the high affinity
conformation. Based on the structure of AT, neither of these two
residues is close enough to any tryptophan residue to be directly
responsible for the reduced response to heparin resulting from
oxidation. Changes in the conformation of the segment containing these
two methionines could affect the orientation of neighboring tryptophan
residues. However, oxidation of Met20 is unlikely to cause
such conformational changes, since its sulfur is directed away from the
peptide backbone. The sulfur of Met17, on the other hand,
is close to the backbone, and thus oxidation could change the backbone
orientation. It is therefore likely that oxidation of Met17
is the major cause for the change in response to heparin binding.
As mentioned above, it has been proposed that methionine oxidation
might be a general means for regulating the activity of proteins. It
has clearly been demonstrated that oxidation of an active site
methionine inactivates
1-PI and that this is probably a
physiologically important means of regulating this inhibitor. However,
we have shown here that for antithrombin, oxidation of the two most
susceptible methionines, those on K30 near the active site, does not
inactivate the protein, nor does it affect the ability of AT to bind
heparin. Higher levels of oxidation do affect heparin binding. However,
it is unlikely that such high levels would occur physiologically, as
indicated by the low level of oxidation seen in preparations of AT from
plasma in this study. We conclude that oxidation is not a critical
factor in the regulation of AT activity.