Stability of L-asparaginase: An enzyme used in leukemia treatment

Article abstract of DOI:10.1016/S0031-6865(99)00009-6

L-asparaginase from Escherichia coli is an important enzyme widely used in leukemia treatment under the trade name Elspar. Up to now, however, the aspects of its stability and storage has not been studied in detail. The aim of this work is to analyze the factors that could interfere in the enzyme's stability. The enzymatic activity was found to be stable in wide pH range (4.5-11.5), showing a slight increase in activity and stability in alkaline pHs, which indicates a more stable conformation of the molecule. The enzyme proved to have a high activity restoration capacity when submitted to temperatures of 65°C, in pH 8.6 buffer and, surprisingly, in physiologic solution. This suggests a positive effect of sodium ions on such restoration capacity. Stability was high in different diluents used as parenteral solutions and in recipients used in medical practice without significant loss of activity for at least 7 days. These results lead us to conclude that the enzyme has a high stability after the lyophilized form has been reconstituted (at least 7 days), since the necessary precautions are taken in terms of sterile manipulation and if it is stored in a suitable parenteral vehicle under low temperature (about 8°C). Copyright (C) 1999 Elsevier Science B.V.

Full text of DOI:10.1016/S0031-6865(99)00009-6

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Pharmaceutica Acta Helvetiae 74 1999 1–9
www.elsevier.comrlocaterpharmactahelv
Stability of L-asparaginase: an enzyme used in leukemia treatment
a
a
b
a,)
A.L. Stecher , P. Morgantetti de Deus , I. Polikarpov , J. Abrahao-Neto
˜
a
Department of Biochemical and Pharmaceutical Technology, Pharmaceutical Sciences School, UniÕersity of Sao Paulo, P.O. Box 66083 CEP
˜
05315-970, Sao Paulo SP, Brazil
˜
b LNLS-Brazilian National Synchrotron Light Source, P.O. Box 6192 CEP 13083-970, Campinas SP, Brazil
Received 15 March 1999; received in revised form 17 May 1999; accepted 25 May 1999
Abstract
L-asparaginase from Escherichia coli is an important enzyme widely used in leukemia treatment under the trade name Elsparw. Up to
now, however, the aspects of its stability and storage has not been studied in detail. The aim of this work is to analyze the factors that
Ž
.
could interfere in the enzyme’s stability. The enzymatic activity was found to be stable in wide pH range 4.5–11.5 , showing a slight
increase in activity and stability in alkaline pHs, which indicates a more stable conformation of the molecule. The enzyme proved to have
a high activity restoration capacity when submitted to temperatures of 658C, in pH 8.6 buffer and, surprisingly, in physiologic solution.
This suggests a positive effect of sodium ions on such restoration capacity. Stability was high in different diluents used as parenteral
solutions and in recipients used in medical practice without significant loss of activity for at least 7 days. These results lead us to conclude
Ž
.
that the enzyme has a high stability after the lyophilized form has been reconstituted at least 7 days , since the necessary precautions are
Ž
.
taken in terms of sterile manipulation and if it is stored in a suitable parenteral vehicle under low temperature about 88C . q 1999
Elsevier Science B.V. All rights reserved.
Keywords: L-asparaginase; Leukemia; Stabilization; Storage
1. Introduction
L-asparaginase was introduced in the therapeutics due to
the fact that in a significant number of patients with acute
leukemia, particularly lymphocytic, the malignant cells are
dependent on a exogenous source of L-asparagine for
survival. Normal cells, however, are able to synthesize
L-asparagine and thus are less affected by its rapid deple-
tion produced by treatment with the enzyme L-asparaginase.
The general medical approach to leukemia therapy is
therefore based on a metabolic defect in L-asparagine
ŽL-asparagine amidohydrolase,
Bacterial L-asparaginases
.
E.C. 3.5.1.1 are enzymes of high therapeutic value due
their use in leukemia treatment. Escherichia coli L-
asparaginase, a high affinity periplasmic enzyme is particu-
larly effective in certain kinds of cancer therapies. A
number of bacteria possess L-asparaginase, although not all
of these enzymes have anti-tumour properties. The varia-
tion in anti-tumour activity has been related to the affinity
of the enzyme for its substrate and the clearance rate of the
particular types of enzymes. Commercially used enzymes
Ž
synthesis of some malignant cells Broome, 1981; Ravin-
.
dranath et al., 1992 . The enzyme also inhibits protein
synthesis by L-asparagine hydrolysis Marlborough et al.,
1975; Ravindranath et al., 1992; Moola et al., 1994 . Its
Ž
Ž
are obtained from E. coli and Erwinia carotoÕora Marl-
borough et al., 1975 .
.
.
action upon DNA and RNA synthesis has not been entirely
elucidated yet, but it is believed to be G1-phase specific
Ž
.
Broome, 1981 .
)
Corresponding author. Fac. Ciencias FarmaceuticasrUSP, Av. Prof.
ˆ
ˆ
The most common therapeutic indications of L-
Lineu Prestes, 580-Bloco 16, 05508-900, Cidade Universitaria, Sao Paulo,
Brazil. Fax: q55-11-815-6386; E-mail: josaneto@usp.br
´
˜
asparaginase are: treatment of Hodgkin disease, treatment
0031-6865r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.
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PII: S0031-6865 99 00009-6

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A.L. Stecher et al.rPharmaceutica Acta HelÕetiae 74 1999 1–9
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of acute lymphocytic leukemia mainly in children , acute
myelocytic leukemia, acute myelomonocytic leukemia and
chronic lymphocytic leukemia, lymphosarcoma treatment,
10 ml with 10 000 UI of the lyophilized enzyme. An
important pharmacological characteristic of Erwinasew
Ž
.
Porton-Down, Salisbury, Wiltshire, UK isolated from
Ž
reticlesarcoma and melanosarcoma Cunningham et al.,
1979; Levine et al., 1983; Capizzi et al., 1984; Ravin-
dranath et al., 1992; Mitchell et al., 1994; Klumper et al.,
Erw. carotoÕora is that this enzyme has no immunological
cross-reaction with preparations derived from E. coli and,
therefore can be used in patients that are hypersensitive to
.
Ž
.
1995; Larson et al., 1995 . The amino acid sequences of
E. coli L-asparaginase Moola et al., 1994 .
several different asparaginases has been reported, includ-
The purpose of the present work is to investigate L-
asparaginase stability and activity changes in different
solutions and buffers in respect to the variations of pH and
temperature in a wide range. The enzyme is normally
supplied in a lyophilized form and, therefore, determina-
tion of its proper storage conditions after reconstitution is
of vital importance for preservation of its high therapeutic
effectiveness and medication.
Ž
.
ing that of E. coli enzyme Wriston, 1985 .
Recent crystallographic studies resulted in an X-ray
Ž
.
structures of native Swain et al., 1993 and T89V mutant
Ž
.
L-asparaginase from E. coli Palm et al., 1996 , of L-
asparaginases from Wolinella succinogenes Lubkowski et
al., 1996 and glutaminase–asparaginase from Pseu-
Ž
.
Ž
.
domonas 7 A Lubkowski et al., 1994 . The protein ap-
peared to be a tetramer of four identical subunits, with
molecular weight of 35 000rsubunit, bound mainly by
Ž
.
non-covalent forces Swain et al., 1993 .
2. Experimental
Structural modifications to improve the clearance and
therapeutic properties have been performed, so that the
modified enzyme has a plasmatic half-life longer than 24 h
2.1. Materials
Ž
Harms et al., 1991; Derst et al., 1992; Wehner et al.,
L-asparaginase was obtained in the form of the concen-
1992; Derst et al., 1994 . The commercially used enzyme
trate Elsparw with the lyophilized enzyme Merck, Sharp
Ž
.
w
Ž
.
.
and Dohme, Pensilvania, E.U.A. in vials of 10 ml with
ˆ
Elspar is isolated from E. coli and supplied in a vial of
Fig. 1. Enzymatic activity and stability of L-asparaginase. The activity was determined by incubation in different buffers varying the pH from 3.5 to 12.5,
Ž
.
3.5 to 5.5 acetate 0.05 M; 6.0 to 8.0 Hepes 0.05 M and 8.5 to 12.5 TrisrHCl 0.05 M , at 378C for 10 min. The stability was determined after incubation
by 1 h in same buffers at 378C. At the end of this time the enzymatic activity was determined as described in Section 2 at pH 8.5.

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A.L. Stecher et al.rPharmaceutica Acta HelÕetiae 74 1999 1–9
3
10 000 UI, through Prodome. Serum bags of polyolefin,
of ammonia andror aspartate. This conductivity is linear
Ž
serum bags of polyethylene Baxter, B. Brawn, obtained
in relation to the time and to the enzymatic concentration
.
Ž
.
from Essenca Distribuidora de Material Hospitalar . L-
and follows Michaelis kinetics Drainas and Drainas, 1985 .
The addition of commercial L-asparaginase in the reaction
mixture that contains the substrate L-asparagine causes a
fast increase in conductivity. This increase is linear to the
enzymatic concentration. This method can detect up to
0.001 U of commercial L-asparaginase. Among the advan-
asparagine, aspartate, protein mixtures and markers of
molecular weight, bovine albumin serum were obtained
from Sigma, St. Louis, E.U.A. All the other chemicals
Ž
.
were from Merck Darmstadt, Germany and were of
analytical degree from Quimitra of Brazil, RJ, Brazil.
Ž .
tages of this method are: a it is faster because it does not
Ž .
require lengthy preparation and a long reaction time; b it
2.2. Determination of the enzymatic actiÕity
Ž .
is very sensitive; c it is reliable due its reproducibility
Ž
and adapted to the enzyme kinetic study Drainas and
L-asparaginase catalyzes the hydrolysis of L-asparagine
producing L-aspartic acid and ammonia. After the adapted
time of incubation, in buffer TrisrHCl 50 mM, pH 8.6
containing 10 mM L-asparagine, the reaction was inter-
rupted with 1.5 M of trichloroacetic acid and the samples,
after centrifugation, were treated with Nessler reagent. The
ammonia concentration produced in the reaction was deter-
mined on the basis of a standard curve previously obtained
with ammonium sulfate as a standard.
.
Ž
.
Drainas, 1985 . An International Unit UI of L-
asparaginase is the amount of enzyme that catalyses the
production of 1 umol of ammonia liberated in 1 min under
Ž
the conditions of the assay Whelan and Wriston, 1969;
Law and Wriston, 1971; Wade and Phillips, 1971; Wris-
.
ton, 1985 .
The lyophilized enzyme contained in the medication
vial was reconstituted with a suitable parenteral vehicle
Ž
The activity of L-asparaginase can also be measured by
using the conductometric method. The method is based on
the increase of conductivity, which is due to the production
proposed for study, stored under different conditions con-
.
ditions and temperatures proposed and the enzymatic
activity was measured at given intervals of time. Results of
Fig. 2. Activity of -asparaginase incubated at different temperatures: 10, 37, 50 and 608C, in buffer TrisrHCl 0.05 M, pHs8.6 with substrate
L
Ž
.
L
-asparagine 100 mM . At the times indicated above, aliquots were removed in order to measure the produced NH3. Technique of measurements is
described in Section 2.

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A.L. Stecher et al.rPharmaceutica Acta HelÕetiae 74 1999 1–9
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enzymatic activity are the average of three experiments
conducted in either a parallel or an independent way.
functional dependence in detail Fig. 2 . In the intervals of
Ž
.
time during which the tests were performed 1 h , if one
2.3. Determination of the protein concentration
The concentration of protein was determined by the
Ž
.
Bradford Method Bradford, 1976 using bovine albumin
serum as standard. Where necessary, the Lowry Method
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.
Lowry et al., 1951 was used.
3. Results and discussion
In order to verify a purity of the product we conducted
Ž
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an electrophoresis in polyacrylamide gel SDS-PAGE
Ž
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Laemmli, 1970 . Analysis of the gel revealed there was
no detectable contamination as it represented just one
distinct band and the molecular weight of the sub-unit of
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about 33 kDa, as expected results not shown .
The analysis of the results of L-ASP activity in different
pHs shows that the enzyme has quite a wide range of
Ž
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activity, between 4.5 and 11.5 Fig. 1 . At pH below 4.0
and above 12, the enzyme totally loses its activity. At a pH
around 4.5 and 11.5, the activity drops to about 50% of its
maximum. After being submitted to these pHs for an hour
and then transferred to pH 8.5, enzyme regains activity
again. This indicates that at these pHs the conformational
changes of the enzyme are still reversible. Also, according
to Fig. 1, the profile of enzyme activity shows that it is not
constant and there is a slight increase in the activity as pH
gets higher. The fact that the activity is maximum in
alkaline pH is probably due to the balance between L-
aspartic acid and L-aspartate. L-aspartic acid in acid pH has
a greater affinity for the active site of the enzyme. Under
Ž
such conditions, it becomes a competitive inhibitor Miller
.
et al., 1993 . In alkaline pH, the balance is shifted toward
the aspartate, which is the form with less affinity to the
active site enabling, in this case, a favorable balance for
Ž
the connection with the substrate L-asparagine Lubkowski
.
et al., 1994 .
Based on the observation of the L-asparaginase, high
stability in diverse pH conditions and the its capacity
toward activity recovering, the activity tests have been
performed at different temperatures in order to study its
Ž
Fig. 3. Thermal stability in different temperatures B — 45; v — 50;
.
Ž
.
Ž .
Ž .
' — 55; % — 60; l — 658C and pH 5.5 A , 8.6 B , and 11.5 C .
After incubation under these conditions, at the indicated times aliquots
were withdrawn and assay of the activity was performed as described in
Section 2.

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A.L. Stecher et al.rPharmaceutica Acta HelÕetiae 74 1999 1–9
5
Ž
.
will take the activity at 378C as a reference 100% , the
enzyme activity at 108C was only 10%. At 508C and 608C,
the enzyme activity was 163% and 186%, respectively,
and, almost constant throughout the interval.
ify a possible similarity between the effect observed in the
Ž
.
treatment with urea Marlborough et al., 1975 and the
effects caused by temperature increase on L-asparaginase
Ž
.
restoration activity Fig. 4 . We observed that in the
studied pHs, L-asparaginase loses its activity totally after
the exposure time has elapsed, and after 60 min, at 378C,
the enzyme sample treated in pH 8.6 recovers 50% of the
initial activity, while the samples treated with pH 5.0 and
11.5 buffers lose their activity completely, beyond recov-
ery. According to Marlborough et al., 1975, L-asparaginase
can be structurally renaturated in alkaline pH but there is
no recovery of the activity. These results demonstrate that
there was an irreversible structure change in pH 5.0 and
11.5 without restoration that prevents it from hydrolyzing
L-asparagine into L-aspartate, while in pH 8.6 there is an
active renaturation. This denaturation can be a conse-
quence of the dissociation of the L-asparaginase sub-units.
It is important to notice that the activity of this enzyme
would be then dependent upon the connections among the
subunits, because if each sub-unit has its own active site,
they could retain activity when dissociated.
Ž
When analyzed under different temperatures 458C,
508C, 558C, 608C and 658C and different pHs 5.0, 8.6
.
Ž
. Ž
.
and 11.5 Fig. 3 , we observe that in pH 5.0 L-asparaginase
is more susceptible to heat if compared to a treatment in
pH 8.6. At 508C, the enzyme submitted to pH 5.0 presents
virtually the same profile as the enzyme submitted to 608C
in pH 8.6. Comparing the profiles of the treated enzymes
in pH 8.6 and 11.5, we see that they are similar at lower
temperatures up to 508C . At 608C, there is a stronger
denaturant effect in pH 11.5 than in pH 8.6. In Fig. 3, one
notices that the enzyme has a higher resistance in higher
Ž
.
Ž
.
pHs 11.5 when compared to acidic pH 5.0 under the
same temperatures. These results suggest that in alkaline
pHs the enzyme not only adapts more active conformation
Ž
.
Fig. 1 , but is also more stable.
It is known that L-asparaginase can completely lose its
activity and, also, recover it partially, depending on the
exposure conditions. We tried to verify the return of the
X-ray studies of L-asparaginase from E. coli show that
the protein is composed of four identical subunits, and this
Ž
.
activity restoration after thermal treatment and thus ver-
Ž .
Fig. 4. -asparaginase activity restoration in different buffers. The enzyme was previously submitted to a thermal treatment in bathing 658Cr20 min .
L
Finished this time, the samples were placed in ice-cold water until 408C and soon after placed in bathing to 378C. At the indicated times aliquots were
withdrawn and assay of the activity was performed as described in Section 2.

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A.L. Stecher et al.rPharmaceutica Acta HelÕetiae 74 1999 1–9
2
˚
tetramer can be considered as a dimer of intimate dimers
13 045.5 A . This indicated that although dimer is struc-
Ž
. Ž
Kraulis, 1991; Swain et al., 1993 see Fig. 5, structural
turally self-sufficient, a tetramer formation leads to the
significant decrease of the free-energy of the enzyme.
Apparently, while ionic forces and hydrogen bonds are the
main responsible factors for the secondary and tertiary
structure, the forces among the subunits in the tetramer are
.
cartoon . Each of the dimers has two active centers each of
them formed by the side chains of the amino acid of both
intimately related subunits Wriston, 1985; Swain et al.,
1993; Lubkowski et al., 1994; Lubkowski et al., 1996 .
Although the dimers contain all the structural elements and
functional groups to create a complete active-site environ-
ment, the active enzyme is always a tetramer. Calculation
of the solvent accessible area of the enzyme PDB entry
code 3eca done with GRASP shows that upon the forma-
Ž
.
Ž
predominantly hydrophobic Cammack et al., 1972; Shifrin
.
et al., 1973 .
These results demonstrate that there is a clear synergism
in terms of denaturation and a possibility of renaturation,
depending on the temperature and pH. L-asparaginase at
pH 8.6 has a good tolerance to heat with return of enzy-
Ž
.
tion of the intimate dimer solvent accessible area decreases
on 1995.4 A , whereas formation of tetramer from two
intimate dimers decreases solvent accessible area on
2
˚
Ž .
matic activity Fig. 4 , while in pH 11.5 it has a relatively
Ž .
high thermal stability Fig. 3 . On the other hand, if the
Ž .
Ž .
Fig. 5. Structural cartoon of the
reaction, aspartates, bound to the active centers of the enzyme is shown as ball-and-stick models.
L-asparaginase tetramer PDB ID code 3eca drawn with MOLSCRIPT 33 . Monomers are color coded. Products of

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A.L. Stecher et al.rPharmaceutica Acta HelÕetiae 74 1999 1–9
7
Ž .
Fig. 6. -asparaginase activity restoration in physiologic solution. The enzyme was previously submitted to a thermal treatment in bathing 658Cr30 min .
L
Finished this time, the samples were placed in ice-cold water until 408C and soon after placed in bathing to 378C. At the indicated times aliquots were
withdrawn and assay of the activity was performed as described in Section 2.
enzyme is submitted to pH 11.5 at 658C, the return of the
activity becomes impossible.
To observe a possible restoration of enzyme activity
after thermal treatment in physiologic solution, it was
Ž . Ž .
Fig. 7. Stability of -asparaginase. The enzyme was stored in refrigerator 88C in bags of physiologic solution ' poly-olefin; % poly-ethylene and
L
Ž
.
Ringer lactate solution B poly-olefin; v poly-ethylene . At the indicated times aliquots were withdrawn and assay of the activity was performed as
described in Section 2.

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A.L. Stecher et al.rPharmaceutica Acta HelÕetiae 74 1999 1–9
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Ž
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exposed for 30 min at 658C in this parenteral solution Fig.
.
Sao Paulo FAPESP and Conselho Nacional de Desen-
˜
Ž
.
6 . Under these treatment conditions, L-asparaginase totally
volvimento Cientıfico e Tecnologico CNPq . We are
grateful to Luıs Augusto Angelotti Meira for his help with
graphical representation of L-asparaginase crystallographic
structure.
´
´
lost the activity after the exposure period. It is remarkable
that within 8 min, the enzyme recovers 50% of the initial
activity, that is, there is a strong activity recovery within
the first 10 min and after this period of time, the recovery
becomes very slow. Comparing the results of activity
restoration obtained from the enzyme treated in physio-
logic solution and in buffers pH: 5.0; 8.6; 11.5 , one
verifies that in the first case, after 8 min, the enzyme
recovers approximately 50% while in the second condition,
recovery is only 30%, that is, the speed of the activity
recovery in physiologic solution is significantly higher
than in Tris buffer pH 8.6 . In addition, considering that
the treatment in physiologic solution was 30 min while in
buffer it was only 20 min, a possible positive effect of
ions, probably sodium, becomes evident in activity recov-
ery. Experiments show that sodium has a protecting effect
on the denaturation of L-asparaginase Ryoyama, 1972 .
As was discussed earlier, the hydrophobic interactions
are important for the maintenance of the quaternary struc-
ture of L-asparaginase. There seems to be a correlation
between the decrease of the enzyme stability and the
presence of chaotropic agents, such as KCNS and urea.
The our results indicate that sodium, present in the physio-
logic solution, also favors the enzymatic renaturation.
To verify the stability of L-asparaginase through time
´
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Ž
.
Ž
..
bags soft plastic , polyethylene bags hard plastic , the
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drop was very small about 8% . Analyzing the enzyme
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Ž
.
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The authors gratefully acknowledge financial support
from the Fundac¸ao de Amparo a Pesquisa do Estado de
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