968 Biotechnol. Prog.

2004, 20, 968−974

NOTES
Reduction of Active Elastase Concentration by Means of
Immobilized Inhibitors: A Novel Therapeutic Approach
Valentina Grano,†,‡ Gianluca Tasco,§ Rita Casadio,§ Nadia Diano,†,‡
Marianna Portaccio,† Sergio Rossi,‡ Umberto Bencivenga,‡ Mario Compiani,|
Anna De Maio,† and Damiano Gustavo Mita*,†,‡
Department of Experimental Medicine, Second University of Naples, Via S. M. di Costantinopoli 16,
80138 Naples, Italy, Institute of Genetics and Biophysics “A. Buzzati Traverso” of CNR, Via G. Marconi 12,
80125 Naples, Italy, Laboratory of Biocomputing, Centro Interdipartimentale per le Ricerca Biotecnologica
(CIRB), Bologna, Italy, and Department of Chemical Sciences, University of Camerino, Camerino, Italy

The inhibitory power of three different active Nylon membranes, separately loaded
with three different protease inhibitors, was studied with the aim of reducing the
increased elastase concentration occurring during hemodialysis or extracorporeal blood
circulation in patients undergoing cardiopulmonary bypass. Chemical grafting was
carried out to make the inert Nylon membrane suitable for the immobilization of the
inhibitors. The behavior of immobilized R1-antitrypsin, bovine pancreatic trypsin
inhibitor (BPTI), or elastatinal was separately studied. R1-Antitrypsin and BPTI were
covalently immobilized by means of a diazotization process, whereas elastatinal was
covalently attached via a condensation process mediated by glutaraldehyde. The
inhibitory power of each membrane type was studied as a function of the amount of
immobilized inhibitor and temperature. All active membranes have shown good
inhibitory power. The most efficient membrane was that loaded with R1-antitrypsin,
the less efficient that with BPTI.

1. Introduction synergistic role in pulmonary injury. Therefore, in some

clinical conditions it is important to reduce the protease
The knowledge of the interactions between proteases concentrations in blood.
and antiproteases and the understanding at a molecular
More recently (4, 5) it has been demonstrated that it
level of the pathologies related to these interactions are
is possible to reduce the concentration of active elastase
of fundamental relevance in clinical investigations (1, 2).
in an aqueous solution interacting with a modified
It is well-known, indeed, that several acute diseases occur
polyethersulfone membrane loaded with R1-antitrypsin.
when the natural balance between proteases and anti-
Elastase was chosen as protease model, because it
proteases is altered in blood. Hemodialysis (HE) or
represents one of the main proteolytic enzymes released
extracorporeal blood circulation (EBC) during cardio-
during HE or CPB (6, 7). R1-Antitrypsin from human
pulmonary bypass (CPB) are recognized among the main
plasma was chosen as protease inhibitor, because its
causes responsible for the alteration of this physiological
primary function in vivo is to control the activity of
equilibrium. To give an example, it has been recently
neutrophil elastase (8). In refs 4 and 5 it was demon-
found (3) that in patients undergoing CPB, the concen-
strated that the reduction of elastase concentration was
tration of plasma matrix metalloproteases-9 (MMP-9)
dependent (a) on the initial elastase concentration, when
changed from the initial value of 28.3 mg/mL to the value
the amount of immobilized inhibitor was kept constant,
of 44.3 mg/mL after 5 min from the beginning of the
or (b) on the amount of immobilized R1-antitrypsin, when
operation, to reach the value of 191 mg/mL at the end of
the initial elastase concentration was kept constant.
the operation. Increased plasma levels of neutrophil
elastase (NE) have also been found in patients undergo- This paper concerns the results obtained with three
ing CPB. Moreover, it is well documented that the Nylon porous membranes, grafted with glycidyl meth-
increased concentration of NE and MMP-9 have a acrylate, on which three different protease inhibitors
were separately immobilized. Nylon was chosen in place
of the polyethersulfone owing to its better biocompatibil-
* To whom correspondence should be addressed. Tel/Fax: 0039- ity and its availability in different forms, such as beads,
081-2395887. E-mail address: mita@iigb.na.cnr.it. hollow-fibers, etc. R1-Antitrypsin, bovine pancreatic trypsin
† Second University of Naples.
‡ Institute of Genetics and Biophysics “A. Buzzati Traverso”of inhibitor (BPTI), and elastatinal were the inhibitors. The
CNR. inhibitory power of each membrane type was studied as
§ Centro Interdipartimentale per le Ricerca Biotecnologica. a function of the amount of immobilized inhibitor and
| University of Camerino. temperature. Results are discussed in view of the valida-
10.1021/bp034304b CCC: $27.50 © 2004 American Chemical Society and American Institute of Chemical Engineers
Published on Web 02/11/2004
Biotechnol. Prog., 2004, Vol. 20, No. 3
Scheme 1. Structural Formula of Elastatinal
tion of this technology for the reduction of the increase
of the protease concentrations during HE or CPB.
2. Materials and Methods
2.1. Materials. Hydrolon Nylon membranes from Pall
(Pall Italia, Milano, Italy) were used as solid support to
be grafted with glycidyl methacrylate (GMA). 1,4-Phen-
ylenediamine (PDA) or hexamethylenediamine (HMDA)
were separately employed as spacers between the grafted
membrane and the protease inhibitor, according to the
immobilization method used. Glutaraldehyde (GA) was
employed as bifunctional agent coupling elastatinal to
the graft membrane.
Elastase (EC 3.4.21.36) from porcine pancreas was
purchased from Roche (Roche Diagnostic GmbH, Man-
nheim, Germany). To measure the elastase activity
N-succinyl-Ala-Ala-Ala-p-nitroanilide was used as sub-
strate.
R1-Antitrypsin, BPTI, and elastatinal were separately
used as inhibitors. All of these inhibitors are irreversible
inhibitors. R1-Antitrypsin and BPTI are proteic molecules
of large molecular mass, whereas elastatinal is a small
peptide aldehyde, as can see in Scheme 1.
2.2. Methods. 2.2.1. Preparation of Active Mem-
branes. Preparation of active membranes was carried
out by means of two steps: (a) grafting copolymerization
and (b) inhibitor immobilization.
(a) Grafting Copolymerization. Grafting copolymeriza-
tion (Figure 1a) was carried out by using K2S2O8/Na2S2O3
as initiating system. Membranes were immersed, for 70
min at 40 °C, in a reaction vessel filled with a 1/1 water/
ethanol solution containing 4% (v/v) GMA, 0.012 M
K2S2O8, and 0.008 M Na2S2O3 and in the presence of 0.5%
(w/v) CuCl2. Membranes were then washed with double
distilled water to remove the unbound homopolymer and
left to dry until a constant weight was measured. At this
point, a Hydrolon/polyGMA membrane was obtained. For
the measures of percent degree of grafting we adopted
the classical definition for this parameter. The grafting
degree (X, %) was determined by the difference between
the membrane masses before, GB, and after, GA, the
grafting process according to the formula
GA - GB
X (%) ) × 100
GB
With membranes used in this experimentation, average
grafting percentage was 26 ( 2%.
(b.1) Inhibitor Immobilization via Diazotization. R1-
Antitrypsin and BPTI were immobilized through a dia-
zotization process (Figure 1b) involving the phenolic
group of tyrosine residues. PDA was used to obtain
aminoaryl derivatives on the Nylon/polyGMA mem-
branes. Grafted membranes were treated for 90 min at
room temperature, with a 2% (w/v) PDA solution in 0.1
M sodium carbonate buffer, pH 9.0. Once washed with
water, the aminoaryl derivatives were treated for 40 min
at 0 °C with an aqueous solution containing 2 M HCl and
4% NaNO2. At the end of this treatment, membranes
were washed at room temperature with double distilled
water and 0.1 M Tris/HCl buffer solution, pH 7.4 and
969
then treated for 16 h at 4 °C with the same buffer solution
containing R1-antitrypsin or BPTI at the requested
concentration. At the end of this step, membranes were
washed with the Tris/HCl buffer solution to remove the
unbound inhibitor.
(b.2) Inhibitor Immobilization via Condensation. The
only way to immobilize the elastatinal was to use
glutaraldehyde in a condensation process (Figure 1c)
involving the NH2 group of glutamine. To this aim HMDA
was used as a spacer. Nylon-poly(GMA)-HMDA mem-
branes were obtained by immersing the GMA grafted
membranes in a 1% (v/v) HMDA aqueous solution for 15
min at room temperature. After this step, the membranes
were washed with water to remove the unreacted amines
and then treated for 1 h at 30 °C with a 2.5% (v/v) GA
aqueous solution. After further washings with double
distilled water and 0.1 M Tris/HCl buffer solution, pH
7.4, the membranes were treated for 16 h at 4 °C with
the same buffer solution containing elastatinal at the
requested concentration. At the end of this step, mem-
branes were washed with the Tris/HCl buffer solution
to remove the unbound inhibitor.
2.2.2. Measurements of the Amount of Immobilized
Inhibitor. The amount of the immobilized inhibitor was
measured by the difference in the inhibitor concentration
in the solution used for the immobilization before and
after the immobilization process. Protein concentration
was measured according to the Lowry method (8).
2.2.3. Membrane Efficiency Measurements. Mem-
brane efficiency, i.e., its inhibitory power toward elastase
activity, was determined by measuring as a function of
time the elastase activity in the solution in contact with
the activated membrane by using N-succinyl-Ala-Ala-Ala-
p-nitroanilide as substrate. Elastase hydrolyses this
substrate to N-succinyl-Ala-Ala-Ala and p-nitroaniline.
The reaction rate, expressed as mM/min, is obtained by
spectrophotometrically measuring at 410 nm the p-
nitroaniline production as a function of time. The cata-
lytic reaction starts by adding directly 20 µL of elastase
solution into a cuvette containing the reaction mixture
constituted by 900 µL of 0.1 M Tris/HCl buffer solution,
pH 7.4, plus 80 µL of 3 mM N-succinyl-Ala-Ala-Ala-p-
nitroanilide solution in the same buffer solution. The
volume of elastase solution in contact with the membrane
loaded with the inhibitor was 10 mL. From this volume
samples of 20 µL were withdrawn at regular time
intervals and processed according to the described meth-
odology to assess the concentration of active elastase. The
elastase solution contained a 10% (v/v) glycerol concen-
tration, since in the absence of glycerol we have found
some loss of elastase activity. All experiments were
carried out at room temperature, i.e., at 23 ( 0.5 °C, or
at 37 ( 0.5 °C, i.e., the human body temperature.
The experimental apparatus is represented in Figure
2. It is constituted by a cylindrical silicone module, 5.5
cm in length and 0.8 cm in diameter, containing the
membrane, in series with a hydraulic circuit (T) starting
and ending in a common reservoir R. The hydraulic
circuit is driven by a peristaltic pump (PP) and is
constituted by silicone tubes (34 cm in length and 0.4 cm
in diameter), usually used for blood circulation during
hemodialysis. The solution is kept at the requested
temperature by means of a thermostatic bath. Every
experimental point reported in the figures represents the
average value of five experiments performed under the
same conditions. Each experiment lasted 300 min. Ex-
perimental errors did not exceed 4%. Only in the mea-
surements of the amount of immobilized inhibitor the
error was higher, but within 6%.
970
Figure 1. Schematic representation of the preparation of active membranes: (a) grafting copolymerization; (b) diazotization process;
(c) condensation process.
3. Results and Discussions
3.1. Study of Elastase-Inhibitor Interactions. We
first take advantage of a computational approach in order
to determine the ligand-protein interactions and to
highlight the residues that could be used to immobilize
the inhibitor.
In Figure 3, the 3D structure of elastase, taken from
the Protein Data Bank (PDB ID 1BOE) and visualized
Biotechnol. Prog., 2004, Vol. 20, No. 3
using RASMOL (9), is presented. The amino acidic
residues of the catalytic triad are highlighted with a
spacefill representation and colored as follows: aspartic
acid 102 in violet, histidine 57 in blue, and serine 195 in
red.
In Figure 4a the 3D structure of R1-antitrypsin/elastase
complex is reported. The elastase is in orange, and the
R1-antitrypsin is in gray. Methionyl residue 358 of R1-
Biotechnol. Prog., 2004, Vol. 20, No. 3
Figure 2. Schematic (not to scale) representation of the
experimental apparatus: T, hydraulic circuit; PP, peristaltic
pump; R, reservoir; W, working volume; B, thermostatic bath.
Figure 3. 3D structure of elastase. The catalytic triad is shown
under spacefill form and colored as follows: serine 195 in red;
aspartic acid 102 in violet; histidine 57 in blue.
antitrypsin, involved in the interaction with elastase, is
highlighted with a spacefill representation and colored
in yellow. The tyrosine residues of R1-antitrypsin, in-
volved in the link with the activated support, are green.
The tyrosine residues are far from the 358 methionyl
active residue, thus preserving the integrity of the
binding site (henceforth the ligand binding interaction)
despite the immobilization process. The protein-ligand
complex was obtained by considering that elastase is
similar to trypsin (sequence identity is 34%) and that the
latter molecule has been solved with R1-antitrypsin (PDB
ID: 1EZX). By superimposing elastase to trypsin we
computed a 3D model of the R1-antitrypsin/elastase
complex.
In Figure 4b the BPTI/elastase complex is reported.
BPTI is in gray, and elastase in orange. The 15 lysil
residue involved in the interaction with elastase is
highlighted with a spacefill representation and colored
in yellow. The tyrosine residues involved in the attach-
971
Figure 4. 3D structures of the elastase/inhibitor complexes.
The elastase is in orange and the inhibitors are in gray. The
catalytic triad of elastase is represented as in Figure 3. (a)
Elastase/R1-antitrypsin complex. The interaction residue of the
inhibitor, methionine 358, is evidenced in spacefill form and
colored in yellow, and tyrosine residues, employed in the
attachment to the membrane, are highlighted in green. (b)
Elastase/BPTI complex. The interaction residue of the inhibitor,
lysine 15, is evidenced in spacefill form and colored in yellow,
and tyrosine residues are highlighted in green. (c) Elastase/
elastatinal complex. The inhibitor is represented in CPK
nomenclature. The green arrow indicates the NH2 group of
glutamine involved in the interaction with the carrier, and the
cyan arrow indicates the aldeydic group involved in the inter-
action with elastase.
ment to the activated support are represented in green
and are far from the 15 lysine. The 3D structure of the
complex was computed with a strategy similar to that
described for the R1-antitrypsin/elastase. We started with
the trypsin-BPTI complex (PDB ID 3BTD) and super-
imposed elastase to trypsin.
In Figure 4c the elastatinal/elastase complex is re-
ported. Elastatinal is represented in CPK nomenclature,
and the amino acidic residues of the elastase catalytic
triad are represented as in Figure 3. The green arrow
indicates the NH2 group of glutaminyl attached to the
carrier, and the blue arrow indicates the aldeydic group
972 Biotechnol. Prog., 2004, Vol. 20, No. 3

Table 1. Elastase Activity after 30 Minutes of Interaction

with the Different Inhibitors in Soluble Forma
inhibitor activity (µM/min) T (°C)
R1-antitrypsin 0 23
R1-antitrypsin 0 37
elastatinal 0 23
elastatinal 0 37
BPTI 8.86 23
BPTI 9.32 37
a Inhibitor concentration was 1 mg/mL, and the elastase

concentration was 0.1 mg/mL. The solution was Tris/HCl buffer,

pH 7.4.

involved in the interaction with elastase. It is evident

that the aldehyde moiety of elastatinal is close to serine
195, while the amino group is free and available for the
interaction with the membrane. For elastatinal no solved
structure was available. Therefore we started with build-
ing the molecule with Molden (10) and then allowing the
molecule to dock in flexible fashion to the putative active
site of elastase, also involving the catalytic triad.
The above results clearly indicate that all of the
inhibitors have free groups, exposed to the solvent and
noninteracting with the binding site of the inhibitor. For
these reasons these molecules can be bound to the
synthetic membrane without hampering their inhibitory
power.
3.2. Inhibitory Power of Activated Membranes.
It is well-known that the immobilization process affects
the activity of an immobilized enzyme since (a) the 3D
structure of the immobilized macromolecule can be
modified; (b) the electrostatic or hydrophobic interactions
between the carrier and the solution can alter the
concentrations of solute species (also H+ and OH-) in the
microenvironment around the enzyme; and (c) the pres-
ence of diffusion limitations reduces the movement of the
substrate molecules toward the catalytic site. Each of the
three causes has been studied by us (11-14) with graft
Teflon or Nylon membranes.
Now we have immobilized the “substrate”, but it is
reasonable to suppose that also in this case the presence
of the support may alter the interactions between the
immobilized inhibitor and the elastase. For this reason
as a blank we have performed the following experiments,
keeping in mind that the initial elastase activity in the
absence of inhibitor was 0.20 or 0.24 mM/min, at 23 or
37 °C, respectively. An elastase solution (0.1 mg/mL in
Tris/HCl buffer, pH 7.4) was allowed to react for 30 min
with a concentration of soluble inhibitor equal to 1 mg/
mL. At the end of the experiment the elastase activity
was assayed (Table 1). Data in the table indicate that at
the end of the experiment no active elastase was present
in the solutions containing R1-antitrypsin or elastatinal,
whereas a residual elastase activity was present in the
solution containing BPTI. The presence of a higher
elastase activity at 37 °C than at 23 °C, anyway, does
not mean that the concentration of active elastase is
higher at 37 °C, in so much from calibration curves of
elastase activity as a function of elastase concentration,
keeping constant the substrate concentration, we derived
that the active elastase concentration is 4.2 µg/mL at 23
°C and 3.9 µg/mL at 37 °C. The indication from this set
of experiments is that, by using the same amount of
soluble inhibitors, BPTI has the smallest inhibitory
power toward elastase.
Let us examine the behavior of immobilized inhibitors.
The first lot of experiments (from Figures 5a-7) refers
to the R1-antitrypsin/elastase interaction and it is indica-
Figure 5. (a) Elastase activity as a function of contact time
with the active membranes loaded with different amount of R1-
antitrypsin. T ) 37 ( 0.5 °C. Symbols: (O) 0.250 mg im-
mobilized; (b) 0.766 mg immobilized; (4) 1.231 mg immobilized;
(2) 1.681 mg immobilized; (0) 2.590 mg immobilized. (b)
Elastase activity after 30 min of interaction with the membrane
loaded with R1-antitrypsin as a function of the amount of
immobilized inhibitor.
tive of the procedure followed also for the study of the
interactions BPTI/elastase and elastatinal/elastase. In
Figure 5a is reported, as a function of the contact time,
the elastase activity in 10 mL of a solution (Tris/HCl
buffer, pH 7.4) of 0.1 mg/mL elastase interacting with a
series of Nylon membranes (24 cm2 in surface area)
loaded with different amounts of R1-antitrypsin. The
temperature was 37 °C. The curve parameter is the
amount of immobilized R1-antitrypsin. The results of
Figure 5a show that the rate of the decrease of the
elastase activity and the saturation level associated with
the inhibitory process are functions of the amount of
immobilized inhibitor.
When the values of elastase activity after 30 min of
interaction with the membrane loaded with R1-anti-
trypsin are plotted as a function of the amount of
immobilized inhibitor, one obtains the results reported
in Figure 5b. These results clearly indicate that the
reduction of elastase activity, i.e., the residual elastase
activity at fixed time, is linearly proportional to the
amount of immobilized R1-antitrypsin. It is interesting
to observe that after 30 min of interaction under the same
experimental conditions (0.1 mg/mL of elastase and 1 mg/
mL of R1-antitrypsin) the residual elastase activity is
about 0.17 mM/min with the immobilized inhibitor,
whereas by using soluble R1-antitrypsin it is zero (see in
Table 1). This means that the immobilization procedure
reduces the number of active R1-antitrypsin molecules,
so that same amounts of inhibitor exhibit a higher
inhibitory power when used in soluble form than when
immobilized. At the same time one concludes that the
Biotechnol. Prog., 2004, Vol. 20, No. 3
Figure 6. (a) Relative elastase activity as a function of contact
time with the active membranes loaded with different amount
of R1-antitrypsin. T ) 37 ( 0.5 °C. Symbols: (O) 0.250 mg
immobilized; (b) 0.766 mg immobilized; (4) 1.231 mg im-
mobilized; (2) 1.681 mg immobilized; (0) 2.590 mg immobilized.
(b) Elastase activity as a function of the contact time (during
the first 30 min) with the active membrane loaded with
R1-antitrypsin. T ) 37 ( 0.5 °C. Symbols: (O) 0.250 mg
immobilized; (b) 0.766 mg immobilized; (4) 1.231 mg im-
mobilized; (2) 1.681 mg immobilized; (0) 2.590 mg immobilized.
Figure 7. Initial percentage reductions of elastase activity
(I.I.P.) as a function of the amount of immobilized R1-antitrypsin.
diffusion limitations affect the interaction R1-antitrypsin/
elastase, as well as the changes in the affinity between
the two macromolecules introduced by the immobilization
process.
In Figure 6a the data of Figure 5a are reported in the
form of relative residual elastase activity as a function
of contact time. The value of elastase activity at zero time
is taken as 100%. Data in Figure 6a confirm that the rate
of elastase activity decrease (and hence the decrease of
the active elastase concentration) is proportional to the
amount of immobilized inhibitor. Figure 6a, in addition,
gives direct information of the inhibitory power of each
active membrane during time.
973
Figure 8. Absolute inhibitory power (A.I.P.) of each of the three
active membranes as a function of the amount of immobilized
inhibitor. (a) T ) 37 ( 0.5 °C. Symbols: (9) R1-antitrypsin; (2)
elastatinal; (b) BPTI. (b) T ) 23 ( 0.5 °C. Symbols: (0) R1-
antitrypsin; (4) elastatinal; (O) BPTI.
To know the initial rate of elastase activity decrease
we have plotted in Figure 6b the values of elastase
activity during the first 30 min. The experimental points
have been taken from Figure 5a. The slope of each
straight line, divided by the initial value of elastase
activity and multiplied for 30 min and by 100, give the
value of the initial percentage reduction of the elastase
activity, (I.I.P. ) initial inhibitory power), according to
the equation
At)30 - At)0
I.I.P. (%) ) | |‚100
At)0
The initial percentage reductions of elastase activity
in the case of the experiments of Figure 5a are reported
in Figure 7. Data in this figure clearly show that the
initial reduction of elastase activity exhibits a saturation
trend with the increase of the amount of immobilized
inhibitor.
To know the absolute inhibitory power (A.I.P.) of our
active membranes toward elastase it is appropriate to
determine this value when the process is at saturation.
In our case we have taken 300 min as saturation time.
The A.I.P., therefore, is defined as
At)300 - At)0
A.I.P. (%) ) | |‚100
At)0
In Figure 8a the A.I.P. values relative to the experi-
ments of Figure 5a are reported with the symbol (9).
When experiments similar to the ones discussed in
Figures 5a-7 are carried out with 0.1 mg/mL elastase
solutions in contact with Nylon membranes (24 cm2 in
974
surface area) loaded with the different amounts of BPTI
or elastinal immobilized, one obtains the results reported
in Figure 8a and indicated by symbols (2) and (b) for
elastatinal and BPTI, respectively. The temperature also
in this case was 37 °C and the pH of the Tris/HCl buffer
containing the elastase was 7.4. Results in Figure 8a
show that the absolute reduction of elastase activity by
means of immobilized R1-antitrypsin or BPTI or elasta-
tinal exhibits a saturation trend with the increase of the
amount of immobilized inhibitors. Data in the figure also
show that the immobilized BPTI, among the three
inhibitors, exhibits the smallest inhibitory power toward
elastase. This result agrees with that reported in Table
1.
In Figure 8b we have reported with open symbols the
values of the absolute inhibiting power of each membrane
type when experiments, similar to the ones performed
at 37 °C, were carried out at 23 °C. Results at 23 °C when
compared with those at 37 °C show (a) for each mem-
brane type the same saturation trend with the increase
of the amount of immobilized inhibitors; (b) a lower
inhibitory power of each membrane at lower temperature;
(c) the same relative inhibitory power, i.e., A.I.P.R1-antitrypsin
> A.I.P.elastatinal > A.I.P.BPTI.
4. Conclusions
Before concluding, some general remarks relative to
the molecular mechanism underlying the interactions of
each inhibitor type with elastase are necessary. The
amino acidic residue of R1-antitrypsin involved in the
interaction with elastase is the 358 methionyl residue,
which has a polarizable side chain, assuming a positive
charge in accommodating the active site of trypsin-like
enzymes or to fit equally well into the hydrophobic
centers of both chymotrypsin and elastase. The inhibitory
reactive site forms an exposed loop with the P1-P1′
residues acting as a bait for the target protease. The
interaction with the protease leads to the formation of
an irreversible R1-antitrypsin-enzyme complex, which is
further cleaved at the P1-P1′ peptide bond. The stoichi-
ometry of the interaction between elastase and R1-
antitrypsin/elastase is 1:1 (15).
The inhibition of elastase by BPTI is a competitive
inhibition mediated by the lysine 15 at the P1 position
of the inhibitor (16). BPTI forms a loose complex with
serine proteases, blocking their active centers. Also in
this case the stoichiometry of the interaction is 1:1.
The molecular mechanism responsible for the elastase
inhibition by elastatinal consists of the formation of a
hemiacetal adduct between the aldehyde group of the
inhibitor and the active serine of the protease (17).
Coming now to the results reported in the present
paper, all results above-reported clearly indicate that it
is possible to reduce the active elastase concentration in
a liquid solution through the interaction with an “active”
membrane loaded with specific inhibitors. Among the
three inhibitors used, the most efficient was R1-antit-
rypsin, while the less efficient was BPTI. The inhibitory
power of each active membrane was an increasing
function of the amount of immobilized inhibitor and
temperature. Further studies are in progress in our
laboratories to test the biocompatibility of our “active”
membranes.
Acknowledgment
This work was partially supported by MIUR, through
a Research Programme of National Interest (PRIN)-
funds 2002.
Biotechnol. Prog., 2004, Vol. 20, No. 3
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Accepted for publication January 7, 2004.
BP034304B