From the
Commercial low molecular mass heparin accelerates the inhibition
of neutrophil elastase by mucus proteinase inhibitor, the predominant
antielastase of lung secretions (Faller, B., Mély, Y.,
Gérard, D., and Bieth, J. G.(1992) Biochemistry 31,
8285-8290). To study the kinetic mechanism of this rate
enhancement, we have isolated a 4.5-kDa heparin fragment from
commercial heparin. This compound is fairly monodisperse as shown by
analytical ultracentrifugation. It binds elastase and inhibitor with a
1:1 stoichiometry and an equilibrium dissociation constant of 3 and 210
nM, respectively. It also forms a tight complex with
EI. Flow calorimetry shows that the inhibitor-heparin
interaction is characterized by a large negative enthalpy change
(
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
where, k
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
where K
Neutrophil elastase (NE)
The concentration of NE in the azurophil
granules of polymorphonuclear leukocytes is thought to be in the
millimolar range
(6) . An efficient anti-NE control system must
therefore be present at inflammatory sites where neutrophils are
recruited and activated or where they die in order to prevent
undesirable degradation of the aforementioned proteins. Healthy
individuals have efficient NE inhibitors including
The 53-kDa glycoprotein
MPI is the most abundant
physiologic NE inhibitor of the upper respiratory tract, where it
occurs in concentrations as high as 5
µM(10, 11) . It is an 11.7-kDa basic
protein whose structure is stabilized by eight disulfide bonds. It is
formed of a single chain of 107 amino acid residues of known
sequence
(12, 13) and is composed of two domains of
similar size and architecture
(14) . The x-ray diffraction study
on the MPI-chymotrypsin complex has identified Leu
The inhibition of NE by both
In our previous work, we used a
heparin preparation qualified as ``5.1-kDa heparin'' by the
manufacturer. Such a material, known as low molecular mass heparin, is
obtained by controlled chemical depolymerization of standard heparin
and is erroneously believed to be less polydisperse than standard
heparin. We therefore decided to use the 5.1-kDa heparin to purify a
polymer with a low degree of dispersity. This material was then used to
assess the kinetic mechanism of the activation of MPI by heparin.
The source of NE, MPI, and commercial 5.1-kDa heparin was the
same as before
(19) . Suc-Ala
On-line formulae not verified for accuracy where p
The effect of
heparin on the stoichiometry and the equilibrium dissociation constant
K
The rate
of inhibition of NE by MPI was measured using the progress curve
method
(27, 28) . NE with or without heparin was added to
a buffered solution of MPI, 0.3 mM
MeO-Suc-Ala
On-line formulae not verified for accuracy where a = rate in the presence of MPI/rate in its
absence and K
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
H
= -45.2 kJ
mol
) and a small entropy change (
S = -23.7 J K
mol
). Stopped-flow kinetics run under
pseudo-first-order conditions ([I
]
[E
]) show that in the absence of heparin
the inhibition conforms to a simple bimolecular reaction,
= 3.1
10
M
s
,
k
= 10
s
, and K
= 33
pM, whereas in the presence of heparin, E and I react
via a two-step mechanism,
* =
86 nM, k
= 2.2
s
, k
=
10
s
, and
K
= 37 pM. Thus, heparin
increases both the rate of inhibition by promoting the formation of a
high affinity EI* intermediate and the rate of EI
dissociation. Since the dissociation is negligible in bronchial
secretions where the inhibitor concentration is much higher than
K
, it may be concluded that heparin
significantly potentiates the inhibitor's antielastase potential
in vivo.
(
)
is an essential
component of the phagocytic machinery of polymorphonuclear leukocytes.
This 30-kDa serine proteinase is a cationic glycoprotein formed of 218
amino acid residues with 19 arginines but no lysines
(1) . X-ray
crystallographic studies show that the arginine residues are all
located in patches at the surface of the protein
(2, 3) .
NE has a number of physiologic functions including proteolysis of
phagocytosed proteins, neutrophil migration, and tissue remodelling
following injury (4). It also cleaves extracellular matrix proteins
such as elastin, interstitial and type IV collagens, proteoglycans,
laminin, and fibronectin as well as plasma proteins such as
antithrombin, immunoglobulins, fibrinogen, and components of the
complement system (5).
-proteinase inhibitor,
-macroglobulin, mucus proteinase inhibitor (MPI),
(5) and elafin
(7) . The two former proteins are present
in plasma and are able to diffuse in the interstitial fluid while the
latter are found in airway or genital tract secretions and in skin (for
review see Refs. 5 and 7).
-proteinase inhibitor is the most important NE
inhibitor on a molar basis. In addition, it inhibits NE irreversibly
with a very high association rate constant, k
> 10
M
s
(8, 9) . It therefore acts
in vivo as an extremely fast and efficient NE inhibitor. Its
biological NE inhibitory function is best illustrated by the fact that
its hereditary deficiency frequently leads to pulmonary emphysema, a
disease characterized by lung elastin and collagen fiber disruption
with subsequent airflow limitation.
as the
P1 residue of the inhibitor's active site. NE binds at the same
site as chymotrypsin
(15) .
-proteinase inhibitor and MPI can be modulated by
heparin, an anionic glycosaminoglycan synthesized by mast cells. This
polymer mostly consists of trisulfated disaccharide units formed of
L-iduronic acid-2-sulfate linked to
D-glucosamine-N,3,6-sulfate
(16) . It modulates
the activity of numerous biological systems
(17) . Among others,
it binds and activates a number of protein proteinase inhibitors that
belong to the serpin superfamily, namely antithrombin, heparin
cofactor, protease nexin, protein C, and plasminogen activator
inhibitor
(17, 18) . Heparin does not bind
-proteinase inhibitor, another serpin, but strongly
decreases the rate of NE inhibition by this inhibitor as a result of
its strong binding with the enzyme. In contrast, the polymer strongly
binds MPI, a non-serpin inhibitor and significantly increases its rate
of reaction with NE. It also increases the intrinsic fluorescence of
the inhibitor, blue-shifts its maximum emission wavelength, and
increases its fluorescence lifetime, suggesting that it buries
Trp
, the single tryptophan residue of the protein in a
very hydrophobic environment (19).
-pNA and
MeO-Suc-Ala
-Pro-Val-pNA were from Bachem,
Switzerland, while MeO-Suc-Ala
-Pro-Ala-thiobenzylester came
from Enzyme System Products (Livermore, CA). Stock solutions of most
substrates and of 4,4`-dithiodipyridine (Sigma) were made in
dimethylformamide, while Suc-Ala
-pNA was dissolved
in N-methylpyrrolidone. The final concentration of organic
solvent was 2% (v/v) throughout. Unless otherwise stated, all
experiments were performed at 25 °C in 50 mM Hepes, 100
mM NaCl, pH 7.4, a solution that will be referred to as the
``buffer.'' The buffer was filtered through a 0.45-µm
microporous membrane and degassed prior to use. Except for the
stopped-flow experiments, all regression analyses were done using the
Enzfitter software.
Purification of Heparin
One gram of commercial 5.1-kDa
heparin was chromatographed on a 5 190-cm column of Sephadex
G-50 Superfine (Pharmacia Biotech Inc.) eluted with a 200 mM
NaCl solution at a flow rate of 22 ml/h. The collected fractions (11
ml) were tested for the presence of heparin using the azure A blue
(Sigma) assay
(20) . Heparin eluted as a broad peak, which was
divided into seven portions, each corresponding to an elution volume of
about 80 ml. These fractions were lyophilized, dissolved in a minimal
volume of deionized water, and desalted on a Sephadex G-10 column. The
salt-free fractions were concentrated by lyophilization and
rechromatographed on Sephadex G-50 as described above. The tubes
containing the highest concentrations of heparin were pooled, desalted,
and lyophilized. The material issued from the last fraction of the
initial chromatography is referred to as ``heparin'' in the
text and was used throughout the present investigation.
Ultracentrifugation
Ultracentrifugation of heparin
was performed at 20 °C using a Beckman Model E ultracentrifuge
equipped with a Schlieren optical system and a camera. One hundred
forty µl of a 1 mg/ml heparin solution in 200 mM NaCl were
injected in one compartment of a double sector cell while 160 µl of
200 mM NaCl were injected in the other compartment. The
ultracentrifuge was run at 5,000 rpm (diffusion coefficient) or 50,000
rpm (sedimentation coefficient). A number of photographs of the
Schlieren patterns were taken at regular intervals of time over a
period of 35 min. The sedimentation and diffusion coefficients were
determined by graphical methods based on classical equations. The
molecular mass was calculated using Svedberg's equation, a
partial specific volume of 0.42 ml g(21) ,
and a solution density of 1.01.
High Performance Liquid Chromatography
The
molecular mass of heparin was also determined by chromatography on a 60
0.75-cm TSK 3000 SW column (Touzart et Matignon, France) eluted
with 200 mM NaCl at a flow rate of 0.5 ml/min. The eluted
heparin was detected using a differential refractometer from Knauer
(Berlin). The column was calibrated with 2.6-9.5-kDa heparin
standards (a gift from Dr. Petitou, Sanofi, France) whose molecular
masses had been determined by ultracentrifugation
(22) .
The Heparin-Protein Binding Stoichiometry
The
heparin-protein binding stoichiometry was determined using the ability
of a heparin-binding dye to displace heparin from its complex with a
heparin-binding protein
(23) . The dye was azure A blue
(20) instead of proflavine
(23) because the latter has a
poor color yield with our heparin preparation. With 150 µM
azure A blue the absorbance at 595 nm decreased linearly when the
heparin concentration increased from 0.07 µM to 3
µM, thus making the stoichiometry determination possible.
Constant concentrations of heparin (2 µM) were
preincubated for 5 min at room temperature with variable concentrations
of NE, MPI, or NEMPI complex in the buffer. Fifty µl of these
mixtures were then added to a cuvette containing 950 µl of a
solution of 150 µM azure A blue dissolved in the buffer.
Absorbances were measured at 595 nm against a 150 µM azure
A blue control solution.
Flow Calorimetry
Flow calorimetry was used to
measure the K of the MPI-heparin complex.
Equal volumes of MPI and heparin buffered solutions were injected at a
flow rate of 0.198 ml min
into a ThermoMetric 2277
Thermal Activity Monitor. The power was recorded until the thermal
equilibrium was reached (about 20 min). The equilibrium power p was measured at 25 °C using constant concentrations of MPI (2
µM) and variable concentrations of heparin (0.5-10
µM). The data were analyzed by nonlinear regression based
on the equation,
is the extrapolated power at
infinite heparin concentration, and [I]
and
[H]
are the total MPI and heparin concentrations,
respectively.
Enzymatic Methods
The kinetic constants
k and K
were
measured for the various substrates of NE in the presence and absence
of heparin as described previously
(24) .
of the NE
MPI complex was
determined by reacting constant concentrations of NE with increasing
concentrations of MPI in the absence or presence of a constant
concentration of heparin for a given time and measuring the residual
enzymatic activity with a synthetic substrate. The stoichiometry was
determined using 0.2 µM NE with or without 40
µM heparin, an incubation time of 5 min at 25 °C, and
1 mM Suc-Ala
-pNA
(25) . The
K
was measured with 20 nM NE
with or without 40 µM heparin, an incubation time of 15
min at 25 °C, and 4 mM
MeO-Suc-Ala
-Pro-Val-pNA
(26) .
-Pro-Ala-thiobenzylester
(29) , and 3
mM 4,4`-dithiodipyridine
(30) . The reactions were
followed using the same stopped-flow apparatus as
previously
(9) .
Molecular and Protein-binding Properties of
Heparin
Fig. 1
shows that heparin yields symmetrical and
gaussian Schlieren patterns during low speed ultracentrifugation,
indicating that it is fairly monodisperse. Its M was 4500 ± 500 and 5200 ± 800 as determined by
ultracentrifugation and high performance liquid chromatography,
respectively. Heparin bound free NE and MPI with a 1:1 stoichiometry
and the NE
MPI complex with a 2:1 stoichiometry (Fig. 2).
The K
of the MPI-heparin complex,
determined by microcalorimetry, was found to be 210 ± 30
nM, which corresponds to
G
=
-38.2 kJ mol
. On the other hand,
H
= -45.2 kJ mol
as derived from p
(Equation 3), this
leading to
S
= -23.7 J
K
mol
. The
K
of the NE-heparin complex, determined
as described previously
(24) , was found to be 3 ± 0.4
nM. A published program for computing distribution diagrams
for multiple equilibria
(31) was used to calculate the degree of
saturation of NE and MPI by heparin in mixtures of the three partners.
It was found that for any experimental condition described in this
paper, 40 µM heparin saturates both NE and MPI to at least
99%.
Figure 1:
Schlieren patterns photographed during the
determination of the diffusion coefficient of heparin using low speed
ultracentrifugation.
Figure 2:
Stoichiometry of binding of heparin to NE
(), MPI (
), and the NE
MPI complex (
). Increasing
concentrations of proteins were mixed with a constant concentration of
heparin (2 µM), and the mixtures were reacted with azure A
blue and read at 595 nm against a dye control. Relativeabsorbance, absorbance of the heparin + protein
+ azure A blue/absorbance of the free heparin + azure A
blue.
Effect of Heparin on the Stoichiometry and the
K
MPI is a
reversible competitive inhibitor of NE (32). Use was made of this
property to infer the stoichiometry and the K of NE
MPI Complex
of the NE
MPI complex from enzyme:inhibitor titration
curves
(33) . The binding stoichiometry was determined using a
relatively high enzyme concentration (0.2 µM) to favor the
enzyme-inhibitor association and a relatively low substrate
concentration ([Suc-Ala
-pNA] = 1
mM, K
= 0.55 mM
and 3.6 mM in the absence and presence of heparin,
respectively) to avoid excessive complex dissociation. Under these
conditions straight inhibition curves were obtained in the absence and
presence of heparin (see Fig. 3, insets). These curves
indicate that the NE:MPI binding stoichiometry is 1:1 whether heparin
is present or not. The K
was determined
under conditions that disfavored enzyme-inhibitor binding
([NE] = 20 nM) but favored complex
dissociation, i.e. [S]
/K
1
([MeO-Suc-Ala
-Pro-Val-pNA] = 4
mM, K
= 0.085 mM
and 0.123 mM in the absence and presence of heparin,
respectively). Now concave titration curves were obtained
(Fig. 3). The inhibition data were fit to Equation 4
(33) by nonlinear regression analysis.
=
K
(1 +
[S]
/K
). The concave
inhibition curves shown in Fig. 3are theoretical and have been
calculated using Equation 4 and the best estimates of
K
(app). There is a good fit between these
curves and the experimental points. The K
values were 33 ± 9 pM in the absence of heparin
and 37 ± 7 pM in the presence of heparin. The polymer,
therefore, does not significantly alter the enzyme-inhibitor affinity.
Figure 3:
Effect
of heparin on the stoichiometry () and the equilibrium
dissociation constant K of NE
MPI complex (
) at pH
7.4 and 25 °C. The experiments were done as described under
``Experimental Procedures.'' Relativeactivity, rate in the presence of MPI/rate in its
absence.
Effect of Heparin on the Rate of Inhibition of NE by
MPI
Fig. 4
shows typical progress curves recorded upon
addition of NE to a mixture of MPI and substrate in the absence of
heparin and in the presence of a saturating concentration of the
polymer. These curves were analyzed by nonlinear regression
(19) to calculate k, the apparent first-order rate
constant of inhibition. This constant was found to be 0.05 and 0.40
s in the absence and presence of heparin,
respectively.
Figure 4:
Progress curves for the inhibition of 30
nM NE by 300 nM MPI at pH 7.4 and 25 °C in the
absence or presence of 40 µM heparin.
The reversible competitive inhibition process may be
described by Fig. SIor II
(34) , where E stands
for NE or the NE-hep