Oxidized elafin and trappin poorly inhibit the elastolytic
activity of neutrophil elastase and proteinase 3
Shila M. Nobar
1
, Marie-Louise Zani
2
, Christian Boudier
1
, Thierry Moreau
2
and Joseph G. Bieth
1
1 Laboratoire d’Enzymologie, INSERM U392, Universite
´
Louis Pasteur de Strasbourg, Illkirch, France
2 INSERM U618, Universite
´
Franc¸ois Rabelais, Tours, France
Many amino acid residues of proteins are susceptible to
oxidation by reactive oxygen species. Methionine, the
most sensitive of amino acids to oxidation, is readily
transformed into a mixture of the S- and R-epimers of
methionine sulfoxide. The latter may be recycled by
methionine sulfoxide reductases in the presence of thio-
redoxin, which itself may be regenerated by thioredoxin
reductase in an NADPH-dependent reaction. Excessive
methionine sulfoxide production and ⁄ or a defect in its
recycling is believed to be involved in age-related
diseases and in shortening of the maximum life span [1].
Oxidative processes also take place in lung infection
and inflammation, where they are used, in conjunction

E-mail:
(Received 20 May 2005, revised 24 August
2005, accepted 22 September 2005)
doi:10.1111/j.1742-4658.2005.04988.x
Neutrophil proteinase-mediated lung tissue destruction is prevented by
inhibitors, including elafin and its precursor, trappin. We wanted to estab-
lish whether neutrophil-derived oxidants might impair the inhibitory func-
tion of these molecules. Myeloperoxidase ⁄ H
2
O
2
and N-chlorosuccinimide
oxidation of the inhibitors was checked by mass spectrometry and enzy-
matic methods. Oxidation significantly lowers the affinities of the two
inhibitors for neutrophil elastase (NE) and proteinase 3 (Pr3). This
decrease in affinity is essentially caused by an increase in the rate of inhibi-
tory complex dissociation. Oxidized elafin and trappin have, however, rea-
sonable affinities for NE (K
i
¼ 4.0–9.2 · 10
)9
m) and for Pr3 (K
i
¼ 2.5–
5.0 · 10
)8
m). These affinities are theoretically sufficient to allow the oxi-
dized inhibitors to form tight binding complexes with NE and Pr3 in lung
secretions where their physiological concentrations are in the micromolar
range. Yet, they are unable to efficiently inhibit the elastolytic activity of

(also called secretory leukoprotease inhibitor, or SLPI;
an 11.7-kDa protein that inhibits NE [5] and cathepsin
G, but not Pr3 [3]); and elafin and its precursor trap-
pin-2 (also called pre-elafin and referred to as trappin
throughout this article; that inhibit NE and Pr3 [6],
but not cathepsin G [3]). The two former proteins are
mainly synthesized in the liver and reach the lung via
the blood circulation. They are irreversible inhibitors
that belong to the serpin family. Their interaction with
proteinases is characterized by a single constant – the
association rate constant (E þ I À!
k
ass
EI) [7]. The two lat-
ter molecules are synthesized in the lung and belong to
the canonical type of inhibitors that interact reversibly
with their target enzymes, the reaction being described
by an association and a dissociation rate constant
and E þ I Ð
k
ass
k
diss
EI an equilibrium dissociation constant
K
i
¼ k
diss
⁄ k
ass

i
in the 10
)4
Æs
)1
and 10
)10
m range, respectively [15].
In inflammatory lung diseases, activated or lysed
neutrophils do not only release proteinases but also
the aforementioned oxidants. The present article
reports the kinetic consequences of inhibitor elafin and
trappin oxidation on their interaction with NE and
Pr3. It also examines the effect of insoluble elastin on
the inhibitory properties of the native and oxidized
inhibitors.
Results
Oxidation decreases the affinity of elafin and
trappin for NE and Pr3
We oxidized elafin and trappin using either N-chloro-
succinimide, a classical reagent for surface-exposed
methionine residues [16] or with the myelopero xidase ⁄
H
2
O
2
⁄ halide system, the neutrophil’s oxidation device
[17]. Figure 1 shows the effect of increasing concen-
trations of native and oxidized elafin and trappin on
the activity of a constant concentration of NE and

lyzed using Eqn (1):
a ¼ 1 À
ð½E
0
þ½I
0
þ KÞÀ
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ð½E
0
þ½I
0
þ KÞ
2
À 4½E
0
½I
0
q
2½E
0
ð1Þ
where a is the relative enzyme activity (rate in the
presence of inhibitor ⁄ rate in its absence), [E]
0
and [I]
0
are the total enzyme and inhibitor concentrations,
respectively, and K ¼ K
i

nificantly increases the K
i
(decreases the affinity) for
its complexes with NE and Pr3. Oxidation by N-chloro-
succinimide or myeloperoxidase yields inhibitors
whose K
i
values are not significantly different from
each other.
Oxidized elafin and trappin form unstable
complexes with NE and Pr3
Is the above-observed increase in K
i
caused by an
increase in the dissociation rate constant, k
diss
,a
decrease in the association rate constant, k
ass
,oran
effect on both parameters (K
i
¼ k
diss
⁄ k
ass
)? To answer
this question, we measured k
diss
by extensively diluting

(M) k
ass
(M
)1
Æs
)1
) k
diss
(s
)1
)
NE Elafin None 8.0 ± 0.5 · 10
)11
3.7 ± 0.1 · 10
6
3.2 ± 0.1 · 10
)4
NCS 5.7 ± 0.6 · 10
)9
1.1 ± 0.3 · 10
6
6.3 ± 0.6 · 10
)3
MPO 4.0 ± 0.6 · 10
)9
ND ND
NE Trappin None 3.0 ± 1.0 · 10
)11
3.6 ± 0.5 · 10
6

NCS 5.0 ± 2.0 · 10
)8
ND ‡ 0.1
a
MPO 3.5 ± 0.5 · 10
)8
ND ND
a
Calculated assuming that dissociation is terminated in 30 s or less, which corresponds to a t
½
£ 6s.
S. M. Nobar et al. Inhibition of elastase and proteinase 3 by elafin
FEBS Journal 272 (2005) 5883–5893 ª 2005 FEBS 5885
Quantitative calculation of k
diss
confirms this
(Table 1). The complexes formed of Pr3 and oxidized
elafin and trappin were found to dissociate within the
mixing time because no presteady state was visible
(Fig. 2, curves 1 and 2). Hence, k
diss
could not be cal-
culated for these systems but is estimated to be greater
than 0.1 s
)1
(Table 1 legend). Thus, the oxidation of
elafin and trappin leads to a > 250-fold increase of
k
diss
of their complexes with Pr3. We conclude that the

residues of the inhibitors’ active centers. Mass spectro-
metry of the two proteins oxidized by N-chlorosuccini-
mide or myeloperoxidase showed that oxidation
increased the m ⁄ z by 32 Da, indicating that their two
methionine residues had been converted into methio-
nine sulfoxide (Fig. 3).
To establish which methionine residue leads to a
decrease in inhibitory activity upon oxidation, M25L
elafin and M63L trappin (two variants with a nonoxi-
dizable leucine residue at P
1
¢) were prepared. These
variants inhibited NE and porcine pancreatic elastase,
but did not react with Pr3. In addition, their affinity
for NE was lower than that observed with the wild-
type inhibitors (Table 2). Oxidation of the two vari-
ants with N-chlorosuccinimide and myeloperoxidase
increased their m ⁄ z value by 15 Da, indicating oxida-
tion of M51 and M89 of M25L elafin and M63L trap-
pin, respectively. On the other hand, oxidation of
M25L elafin and M63L trappin did not significantly
affect their K
i
for NE (Table 2). We therefore conclude
that the oxidant-promoted alteration of the K
i
of elafin
and trappin is caused by the oxidation of their P
1
¢

reagents and stirring. After 1 min, 70% of the enzymes
were adsorbed. Adsorption was complete after 10 min.
The affinity of elastin for NE or Pr3 was assessed by
adding a constant concentration of enzyme to increas-
ing concentrations of elastin, stirring for 10 min,
centrifugating the suspensions and measuring the
concentration of unbound enzyme using a synthetic
substrate. Both NE and Pr3 gave hyperbolic saturation
curves, as shown in Fig. 4A. Double reciprocal plots
of the data (not shown) were linear, indicating that
saturation conformed to classical reversible receptor–
ligand binding, that is R+L Ð RL (where R repre-
sents elastin and L represents NE or Pr3). The binding
curves may therefore be described by the following
equation:
[L]
bound
=[L]
total
¼ [R]
0
=ð[R]
0
þ [R]
0:5
Þð2Þ
where [R]
0
is the total concentration of elastin and
[R]

excess of inhibitor over enzyme. Figure 5 shows the
results of competition experiments carried out with
Table 2. Effect of M25L elafin and M63L trappin oxidation by
N-chlorosuccinimide on their affinity for neutrophil elastase (NE).
Inhibitor K
i
(M)
Elafin
Wild-type
a
0.8 ± 0.05 · 10
)10
M25L mutant 0.8 ± 0.2 · 10
)9
Oxidized M25L mutant 1.0 ± 0.1 · 10
)9
Trappin
Wild-type
a
0.3 ± 0.1 · 10
)10
M63L mutant 0.9 ± 0.2 · 10
)9
Oxidized M63L mutant 1.3 ± 0.2 · 10
)9
a
From Zani et al. [15].
Fig. 4. (A) Binding of constant concentrations of neutrophil elastase
(NE) (s) and proteinase 3 (Pr3) (h) to different concentrations of
insoluble elastin. The curves are theoretical and were generated

0.5
) to calculate the percentage
of inhibition that would have been observed if the
system behaved like classical competitive inhibition.
Table 3 compares this theoretical inhibition with the
observed inhibition derived from the progress curves
shown, for example, in Fig. 5. It was found that (a)
the observed inhibition is lower than that with the
theoretical inhibitor, regardless of the enzyme, the
inhibitor and the state of oxidation of the latter, indi-
cating that elastin does not simply act as a competing
substrate but also hinders the inhibition process, (b)
Pr3 is much more resistant to inhibition by native ela-
fin than NE, although the two enzyme–inhibitor sys-
tems have similar kinetic constants (Table 1) and (c)
oxidized elafin and trappin are very poor inhibitors of
NE and Pr3.
Discussion
The active site of serine proteinase inhibitors is com-
posed of about eight surface-exposed amino acid resi-
dues, labeled P
5
to P
3
¢, which interact with subsites S
5
to S
3
¢ of the proteinase’s active center. S
1

M; [remazol-Brilliant Blue–elastin] ([RBB–
elastin]) ¼ 3mgÆmL
)1
. The theoretical percentage of inhibition
was calculated using Eqn (1) (competitive inhibition) with
K ¼ K
i
(1 + [R]
0
⁄ [R]
0.5
). K
i
values are from Table 1, [R]
0
is the total
concentration of elastin (3 mgÆmL
)1
) and [R]
0.5
is the elastin con-
centration at which 50% of enzyme is bound ([R]
0.5
¼ 0.77 and
1.12 mgÆmL
)1
for NE and Pr3, respectively). The observed percent-
age of inhibition is that resulting from competition experiments,
such as those shown in Fig. 5.
Enzyme Inhibitor

complexes are so unstable that they relax ‘instantane-
ously’ when diluted into a substrate solution. This
means that their half-lifes are not longer than a few
seconds. The reason why oxidation renders the inhibi-
tory complexes so unstable is not clear. Methionine
sulfoxide is bulkier than methionine. Perhaps steric
hindrance prevents easy binding of the methionine
sulfoxide residue at the S
1
¢ subsite of the active centers
of NE and Pr3. The fact that the S
1
¢ subsite of Pr3 is
significantly smaller than that of NE [21] might then
explain why (a) Pr3 is more sensitive to inhibitor oxi-
dation than NE and (b) Pr3 does not react with the
Met fi Leu mutants.
Lung secretions also contain mucus proteinase inhib-
itor (SLPI), an 11.7 kDa NE inhibitor that shows
some homology with elafin [5] and whose P
1
and P
1
¢
residues are Leu and Met, respectively [22]. Oxidation
of SLPI also reduces its NE inhibitory capacity [23] as
a result of methionine sulfoxide formation [8]. Table 4
compares the kinetic properties of native and oxidized
elafin and SLPI. It can be seen that the two native
inhibitors have very close K

[16].
Oxidation does not fully abolish the inhibitory prop-
erties of elafin and trappin. This raises the following
question: are the oxidized inhibitors still sufficiently
potent to inhibit NE and Pr3 in lung inflammation?
The in vivo potency of a proteinase inhibitor depends
upon its in vivo concentration ([I]
vivo
) and the kinetic
constants describing its inhibition of the target protein-
ase [24]. The absolute concentration of a protein in
lung secretions is difficult to measure because this pro-
tein is collected by bronchoalveolar lavage, which
dilutes it to an undefined extent. According to the rea-
soning of Ying & Simon [25], the elafin concentration
in bronchial secretions would be 1.5–4.5 lm.Ifwe
assume that an inflammatory lung secretion contains
3 lm oxidized elafin and £ 3 lm NE + Pr3 and that
there are no competing biological substrates present,
we may calculate the percentage of free enzyme using
Eqn (1) with, say, [E]
0
¼ 0.3 lm, [I]
0
¼ 3 lm and K ¼
K
i
from Table 1. This calculation shows that there is
only 0.2% free NE and 1% free Pr3 in this lung secre-
tion, indicating that, in the absence of competing sub-

(s
)1
)
Native SLPI
a
9.2 ± 2.5 · 10
)11
4.9 ± 0.5 · 10
6
4.5 ± 0.8 · 10
)4
Oxidized SLPI
a
1.1 ± 0.3 · 10
)8
2.6 ± 0.3 · 10
5
2.9 ± 0.5 · 10
)3
Native elafin
b
8.0 ± 0.5 · 10
)11
3.7 ± 0.1 · 10
6
3.2 ± 0.1 · 10
)4
Oxidized elafin
b
5.7 ± 0.6 · 10

mental one, again confirming the above hypothesis.
The most important differences were found for the
inhibition of Pr3 by native and oxidized elafin. The
experiments were carried out with 1.5 lm elafin, which
is within the physiological concentration range [25]. In
an equimolar mixture of enzyme and oxidized elafin,
NE and Pr3 are inhibited to the extent of 25% and
10%, respectively. This clearly shows that oxidized ela-
fin is a poor inhibitor of the elastolytic activity of these
two enzymes. Oxidized trappin is somewhat more
potent because it inhibits the two proteinases to the
extent of 30 and 19%, respectively. It may be anticipa-
ted that the oxidized inhibitors will also poorly protect
other insoluble extracellular matrix proteins from pro-
teolysis.
Inhibitor-based therapy of inflammatory lung dis-
eases has been proposed in the last decade. For
instance, aerosol-delivered a
1
-antitrypsin [26] and SLPI
[27] have been shown to augment the anti-NE capacity
of lung secretions. As elafin and trappin inhibit both
NE and Pr3, they might be potential drugs in cystic
fibrosis where enormous amounts of free NE and Pr3
are found in lung secretions [28]. However, the sensi-
tivity to biological oxidation of the wild-type inhibitors
prohibits their therapeutic use: oxidation-resistant vari-
ants must be designed. The Met ⁄ Leu variants des-
cribed here can obviously not be used because they do
not inhibit Pr3. The preparation of variants with less

M63L–trappin
Using the elafin cDNA cloned into pGE-SKA-B ⁄ K (20 ng)
as a template [15], PCR amplification was perforrmed
according to the standard procedure of Higuchi et al. [30]
to obtain cDNAs encoding M25L–elafin and M63L–trap-
pin. For this purpose, forward primers 5¢-CGACTCGA
GAAAAGAGCGCAAGAGCCAGTCAA-3¢ and 5¢-CGAC
TCGAGAAAAGAGCTGTCACGGGAGTTCCT-3¢ were
used for amplification of the elafin and the trappin cDNA
5¢ end, respectively, and reverse primer 5¢CGAGCGGCCG
CCCCTCTCACTGGGGAAC-3¢ was used for the common
3¢ end of elafin and trappin. Oligonucleotides 5¢-GGTGCG
CCTTGTTGAATCC-3¢ (forward) and 5¢ -GGATTCAACA
AGGCGCACC-3¢ (reverse) were used to introduce the
Met ⁄ Leu substitution (Leu codon: TTG). Amplified frag-
ments were cloned into the pPIC9 vector and electroporat-
ed into P. pastoris yeast strain GS115 (his4) competent cells
(Invitrogen, Carlsbad, CA, USA).
Both recombinant inhibitors were produced and purified
by ion exchange chromatography, as described previously
for wild-type elafin and trappin [15]. Each of the molecules
migrated as a single band at 7 kDa (M25L–elafin) and
12 kDa (M63L–trappin) in a reducing SDS ⁄ PAGE gel,
indicating homogeneity of each preparation.
Oxidation of inhibitors
We used either N-chlorosuccinimide [16] or the myeloper-
oxidase ⁄ H
2
O
2

gen laser (k ¼ 337 nm). The samples were mixed with 1 lL
of a matrix formed of a saturated solution of a-cyano-
4-hydroxycinnamic acid in H
2
O ⁄ acetonitrile (1 : 1, v ⁄ v).
After vacuo dessication, measurements were performed in
the positive linear mode. Calibration was carried out with
insulin (m ⁄ z ¼ 5734.4) and horse heart myoglobin (m ⁄ z ¼
16952.5).
Enzymatic methods
All kinetic measurements were carried out in 50 mm Hepes,
150 mm NaCl, pH 7.4, a solution called the buffer.
The rate of solubilization of fibrous elastin was measured
using 3 mgÆmL
)1
RBB–elastin (particle size: 200–400 mesh)
(Elastin Products Co., Owensville, MO, USA) suspended in
the buffer at 37 °C. The suspension was stirred for 15 min
before the addition of enzyme, inhibitor or complex. While
stirring was continued, 500 lL samples of suspension were
withdrawn at given time-points, mixed with 500 lLof
0.75 m acetate buffer, pH 4.0, centrifuged at 10 000 g for
10 min and read at 595 nm against a blank prepared from
a reaction mixture where enzyme and inhibitor were absent.
Full solubilization of 3 mgÆmL
)1
RBB–elastin corresponds
to an absorbance at 595 nm of 1.55.
The kinetics of adsorption of NE or Pr3 to RBB–elastin
was measured by adding enzyme (final concentration

Eqn (1) [18] by nonlinear regression analysis.
The dissociation rate constant, k
diss
, of the enzyme-oxi-
dized inhibitor complexes was measured by dissociating the
complexes by both high dilution (100-fold) and high sub-
strate concentration (13.4 K
m
). A 1 lm enzyme concentra-
tion was mixed with 1 lm inhibitor in the buffer. After
30 min at 25 °C, 10 lL of this mixture was added to
990 lL of a buffered substrate solution contained in a
thermostated spectrophotometer cuvette. The substrate was
1.5 mm MeOSuc-Ala
2
-Pro-Val-pNA for the NE–inhibitor
complexes and 0.1 mm MeOSuc-Lys-(pico)-Ala-Pro-Val-
thiobenzylester [31] for the Pr3–inhibitor complexes. The
latter reaction medium also contained 3 mm 4,4¢-dithiodi-
pyridine (Sigma Aldrich), which reacts with benzylthiol to
form a complex that absorbs at 324 nm [32]. The hydro-
lysis of substrate was recorded until the absorbance varied
linearly with time, indicating that the enzyme ⁄ inhib-
itor ⁄ substrate system had reached a steady state. These
data were used to calculate the derivative curve represent-
ing the time-dependent release of free enzyme from the
inhibitory complex. The dissociation rate constant, k
diss
,
could then be calculated from this curve, as described pre-

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