Relationship between the hydration degree of poly-N-isopropylacrylamide gel and activity of immobilized α-chymotrypsin

Article abstract of DOI:10.1023/A:1014398516690

The influence of the pH, temperature, and dimethyl sulfoxide concentration on the hydration degree of the poly-N-isopropylacrylamide gel and the activity of α-chymotrypsin immobilized into the polymer was studied. The behavior of more hydrophilic preparat

Full text of DOI:10.1023/A:1014398516690

Russian Chemical Bulletin, International Edition, Vol. 50, No. 10, pp. 1891—1895, October, 2001
1891
Relationship between the hydration degree
of poly-N-isopropylacrylamide gel and
activity of immobilized α-chymotrypsin
N. L. Eremeev« and N. F. Kazanskaya
Department of Chemistry, M. V. Lomonosov Moscow State University,
Leninskie Gory, 119899 Moscow, Russian Federation.
Fax: +7 (095) 939 5417. E-mail: eremeev@enzyme.chem.msu.ru
The influence of the pH, temperature, and dimethyl sulfoxide concentration on the
hydration degree of the poly-N-isopropylacrylamide gel and the activity of α-chymotrypsin
immobilized into the polymer was studied. The behavior of more hydrophilic preparations
based on polyacrylamide and copolymer of acrylamide and acrylic acid was studied for
comparison. An increase in both the temperature and dimethyl sulfoxide content decreases the
hydration of the poly-N-isopropylacrylamide, which correlates with a decrease in the activity
of the immobilized enzyme. The use of substrates with different structures and an irreversible
inhibitor proves that the change in the properties of α-chymotrypsin immobilized into the
poly-N-isopropylacrylamide gel is related to the change in the rate constants of enzymatic
reactions. Comparison of all experimental data obtained suggested an opportunity of local
interactions between the protein globule and polymeric chains with a change in the hydration
degree of poly-N-isopropylacrylamide during its phase transition.
Key words: poly-N-isopropylacrylamide, phase transition, hydration degree of gel,
α-chymotrypsin, enzyme activity.
It is known that the activity of a biocatalyst often
depends on the state of its microenvironment: a change
in this state changes the enzyme activity. This assertion
can be illustrated by examples of enzyme-containing
systems both in vivo1—3 and in vitro.4 The main route of
regulation of the activity of membrane-immobilized
enzymes in living nature is the phase transition
"solid—liquid crystal" (melting) of phospholipid mem-
branes (supports of a biocatalyst) with temperature chang-
ing.5 Thus, the lipid membrane (matrix-support) acts as
a unique "antenna," i.e., it receives the temperature
signal, changes its state, and regulates the enzyme activ-
ity of immobilized enzymes through this changed state.
It is remarkable that similar phase transitions are revers-
ible. This implies a possibility of reversible regulation of
the enzyme activity with changing the temperature
around the melting point of the membrane-support. Is it
possible to model artificially the natural scheme of
regulation of the biocatalyst activity?
For the class of the so-called stimulus-sensitive poly-
mers,6 the energy parameters of chain interaction change
as a response to some physical (temperature, light) or
chemical (ðÍ, concentration of an organic solvent)
effect (stimuli). As a result, solvent molecules are dis-
placed from the solvate shell of the polymer by the
polymer units (this is the phase transition from the
viewpoint of physicochemistry7). For linear water-soluble
polymers, the phase transition, exfoliation of the true
solution, occurs usually in a very narrow interval of
changing the external signal. For three-dimensional poly-
meric networks (hydrogels), the phase transition is ex-
pressed as a change in the hydration degree (compres-
sion), i.e., a decrease in the linear sizes and weight
proportionally to the amount of water "extruded" from
the sample.8 This process depends on many factors
(nature and concentration of the monomer, concentra-
tion of the cross-linking agent, etc.) and can occur in a
rather wide interval of the external effect.9 Tempera-
ture-sensitive hydrogels are representatives of the de-
scribed above family of materials. Note that the phase
transition in these polymers is reversible, as in the case
of natural supports of biocatalysts (lipid membranes).
The first studies devoted to the activity of asparginase
immobilized into the thermosensitive poly-N-isopropyl-
acrylamide gel showed an interesting result: breaks and
even regions with the apparent negative activation en-
ergy of the reaction appeared in the plots of the loga-
rithm of the enzymatic reaction rate vs. inverse tem-
perature.10,11 Later similar data were demonstrated for
α-chymotrypsin,12 alkaline phosphatase,13 urease,14 etc.,
and the change in the activity of the immobilized en-
zyme was manifested in the region of temperatures
corresponding to those of the phase transition of the
polymeric matrix.10,11 Pronounced analogies in the tem-
perature behavior of enzymes incorporated into mem-
branes and immobilized into thermoreversible gels al-
lowed the assumption about the resembling character of
the influence of the phase transition of the matrix on
the activity of the immobilized biocatalyst. However, it
remains unclear whether the activity of the immobilized
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 10, pp. 1806—1810, October, 2001.
1066-5285/01/5010-1891 $25.00 © 2001 Plenum Publishing Corporation

1892
Russ.Chem.Bull., Int.Ed., Vol. 50, No. 10, October, 2001
Eremeev and Kazanskaya
of the N-acetyl-L-tyrosine that formed with an alkali; the ionic
strength being created by a salt solution (0.2 Ì NaCl + 0.02 Ì
CaCl2). In the case of BTNA, the activity of preparations in a
0.05 Ì borate buffer was measured on a UV-265FW spectro-
photometer (Shimadzu, Germany); the extinction coefficient
of the reaction product (p-nitroanilide) was determined in an
independent experiment for each used concentration of an
organic solvent. The concentrations of the substrate and DMSO,
temperature, and pH of the solution were varied.
In the first approximation, the mechanism of enzymatic
hydrolysis of substrates with α-chymotrypsin is close to that of
the effect of a heterogeneous catalyst16: the equilibrium stage of
enzyme-substrate complex formation followed by the kinetic
stage of hydrolysis to form the reaction products and active
enzyme regeneration
enzyme is really regulated by the precisely phase transi-
tion of the matrix-support (and the corresponding change
in its hydration degree) or this phenomenon is related to
an increase in the density of the polymeric network
regardless of the mechanism of its compression (for
example, due to the compensation of the electrostatic
repulsion of the likely charged groups in the polymer
composition). The search for the answer to this question
is the subject of this study.
The interrelation between the hydration degree of
the poly-N-isopropylacrylamide gel that undergoes the
phase transition and the activity of immobilized
α-chymotrypsin. Unlike methods described in litera-
ture, the compression of this matrix was induced by the
temperature increase and also in an increase in the
concentration of dimethyl sulfoxide in the reaction mix-
ture. In this case, substrates were used for which the
native enzyme in water—DMSO mixtures demonstrates
different kinetic behavior. Comparative kinetic analysis
of the activities was performed using both native
α-chymotrypsin and that immobilized in the non-
compressible polyacrylamide gel. The influence of the
density of the polymeric network on the enzyme activ-
ity was studied using the pH-sensitive preparation based
in the copolymer of acrylamide and acrylic acid, which
does not undergoes the phase transition. In this case,
the gel compression occurs due to the compensation of
the electrostatic repulsion between the acidic groups in
the polymer composition with a decrease in the pH.
E + S
ES
E + ΣP,
The plot of the observed reaction rate vs. concentration of the
reactants is described by the Michaelis—Menten equation
V = Vmax[S]0/(Km + [S]0),
where [S]0 is the substrate concentration; Vmax = kcat[E]0
([E]0 is the enzyme concentration; kcat is the turnover number
of the catalyst per unit time, vz., the maximum reaction rate at
the complete saturation of the enzyme with the substrate at
[S]0 >> Km; and Km is the Michaelis constant numerically equal
to the substrate concentration at which a half of the maximum
reaction rate is achieved. The Vmax and Km parameters for the
hydrolysis of the substrates with various preparations of
α-chymotrypsin were determined from the plots of the initial
hydrolysis rates vs. substrate concentration in the double in-
verse Lineweaver—Burk coordinates (1/V — 1/([S]0).
Hydration degree of gels. In order to measure the hydration
degree of gels, the block-copolymer samples were cut to plates
of 10½2½5 mm. The samples were equilibrated under specified
conditions for 3 h. The temperature, concentration of DMSO
in the mixture, and pH of the solution (0.05 Ì acetate-
ammonia, 0.05 Ì Na-phosphate, and 0.05 Ì borate buffers for
pH intervals of 4—6, 5.5—8, and 7.5—10.5, respectively) were
varied. The hydration degree of gels was determined as a ratio
of the gel weight under certain conditions (m) to the gel weight
under the conditions of maximum swelling of the sample (m0)
and expressed in %.
Experimental
α-Chymotrypsin (A trade mark, OAO "Samson," Russia),
acrylamide (ÀÀm), acrylic acid (AAc), N,N´-methylenebis-
acrylamide (MBA), ammonium persulfate, N,N,N´,N´-tetra-
methylethylenediamine (TEMED) (Reanal, Hungary), N-acetyl-
L-tyrosine ethyl ester (ATEE), and N-benzoyl-L-tyrosine p-nitro-
anilide (BTNA) (BDH, England), phenylmethylsulfonyl fluoride
(Sigma, USA), dimethyl sulfoxide (OAO "Khimreaktivkomplekt"
plant, Russia), and acryloyl chloride (Merck, Germany) were
used. N-Isopropylacrylamide (NIPAA) was synthesized by a
known procedure.15 Salts and components of buffer solutions
used were not worse than analytical grade (Reakhim, CIS).
Immobilized preparations of α-chymotrypsin. The monomer
(AA) (200 g, NIPAA, AAm/AAc 85/15) and cross-linking agent
Results and Discussion
Influence of the ðÍ on the hydration degree of
gels and activity of α-chymotrypsin
(MBA; [MBA]/[monomer] 1/750 (mol L–1)/(mol L–1) were
added to a solution (1 mL) of the enzyme (5 mg mL
–1
)
The plots of the hydration degree of the obtained
gels vs. pH of the medium are presented in Fig. 1. It is
seen that the nonionized polyacrylamide (1) and poly-
N-isopropylacrylamide (2) preparations are insensitive
to this parameter. Only the charged gel of poly(acryl-
amide/acrylic acid) (3) changes its hydration degree
when the pH decreases from 8 to 5. Recall that, in this
case, the collapse mechanism is determined by blocking
of the Coulomb repulsion of the carboxyl groups in the
gel rather than the phase transition of the polymer.
The pH plots of the maximum reaction rate of
ATEE hydrolysis by native α-chymotrypsin and its im-
mobilized preparations are presented in Fig. 2. As can
be seen, the immobilization of the enzyme shifts the
preliminarily modified by acryloyl chloride.12 The reaction was
initiated by the addition of an 0.78 Ì aqueous solution (0.02 mL)
of ammonium persulfate and TEMED (0.01 mL), and block
copolymerization was carried out at ∼20 °C.
Determination of the activity of α-chymotrypsin. In order
to measure the activity of preparations, the obtained block-
copolymer was crushed in a homogenizing reactor to particles
of 20—150 µm in size and washed with a salt solution (0.2 Ì
NaCl + 0.02 Ì CaCl2) to the complete absence of activity in
washing water. The activity of α-chymotrypsin preparations was
determined from the initial rates of hydrolysis of the corre-
sponding substrate at the pH-optimum of effect of the specified
sample. In the case of ATEE, the potentiometric method was
used: the hydrolysis rate was measured in a thermostatted cell
of an RTS-822 pH-stat (Radiometer, Denmark) by the titration

Hydration degree of gel and enzyme activity
Russ.Chem.Bull., Int.Ed., Vol. 50, No. 10, October, 2001
1893
n (%)
without changing its phase state) has no effect on the
kinetic characteristics of the immobilized enzyme.
100
Temperature influence of the hydration degree
of gels and activity of α-chymotrypsin
80
1
60
The temperature plots of the hydration degrees of
the gel preparations are presented in Fig. 3. It is seen
that both polyacrylamide and polyacrylamide/acrylic
acid are not sensitive to a change in this parameter.
Only the thermosensitive gel of poly-N-isopropylacryl-
amide undergoes the phase transition accompanied by
the compression of the polymeric network in a tempera-
ture interval of 30—50 °C. What does occur with the
activity of immobilized α-chymotrypsin in this case?
As can be seen in Fig. 4, enzyme immobilization
increases the temperature coefficient of the enzymatic
reaction of N-acetyl-L-tyrosine ethyl ester (4) hydroly-
sis. This value is the same for all immobilized prepara-
tions, including 2, in the 18—30 °C interval (i.e., under
the conditions where the hydration degrees of the gels
are equal). However, in the case of preparation 2, with
the further temperature increase the region (35—45 °C)
with the observed "negative activation energy" appears.
This demonstrates clearly that this is precisely the phase
transition in the polymeric matrix (and the phase transi-
2
3
40
4
5
6
7
8
9
10
pH
Fig. 1. Plots of the hydration degree (n) of hydrogels 1 (1),
2 (2), and 3 (3) vs. pH of the solution.
vmax/vmax (pH-optimum) (%)
100
80
1
2
3
4
60
40
5
6
7
8
9
10 pH
Fig. 2. ðÍ-Plots of the relative maximum reaction rates of
hydrolysis of N-acetyl-L-tyrosine ethyl ester with native
α-chymotrypsin (1) and its immobilized preparations (2—4):
1 (2), 2 (3), and 3 (4). The activity of the preparation at the
pH-optimum of its effect was taken as 100%.
n (%)
100
80
1
60
pH-optimum of the reaction, which depends on the
nature of the matrix. The effect of the pH-optimum
shift for immobilized hydrolases is always observed and
imaginary because it is related to a change in the local
pH value inside the polymeric network.17 At the same
time, for different immobilized preparations, the maxi-
mum reaction rates at the pH-optima are the same and
independent of the nature of the matrix. The values of
the Michaelis constants during immobilization increase
compared to that of the native enzyme, however, at the
pH-optima they are also equal for all matrices (the Km
values for native α-chymotrypsin and its immobilized
preparations at the pH-optimum of their effect are
2
3
40
20
20
30
40
50
T/°C
Fig. 3. Temperature plots of the hydration degree (n) of
hydrogels 1 (1), 2 (2), and 3 (3).
ln(vmax)/mol L–1 s
–1
100
–4
–1
6•10 and 4•10–3 mol L , respectively). In addition,
it is evident that the pH profiles of the maximum
reaction rates have virtually identical shapes. The shift
of the ðÍ-optimum by 1 or 1.5 ðÍ units depends on the
hydrogel composition rather than the hydration degree
of the matrix (cf., Figs. 1 and 2). Thus, we may con-
clude that the similar procedure of enzyme immobiliza-
tion into polymeric gels with similar spatial three-di-
mensional structures gives preparations with almost iden-
tical kinetic properties of α-chymotrypsin at the
pH-optimum of its effect. The second important con-
clusion: the pH-induced compression of the matrix of 3
(an increase in the density of the polymeric network
1
2
3
4
80
60
40
–1
–1
3.10 3.15 3.20 3.25 3.30 3.35
T
•103/K
Fig. 4. Temperature plots of the maximum reaction rates of
hydrolysis of N-acetyl-L-tyrosine ethyl ester at the pH-opti-
mum of the effect of native α-chymotrypsin (1) and its immo-
bilized preparations 1 (2), 2 (3), and 3 (4) in the Arrhenius
coordinates.

1894
Russ.Chem.Bull., Int.Ed., Vol. 50, No. 10, October, 2001
Eremeev and Kazanskaya
n (%)
tion-induced change in the hydration degree of the gel)
which results the anomalous temperature behavior of the
matrix-immobilized biocatalyst (cf. Figs. 3 and 4). It is
also of interest that the temperature run of the Michaelis
constant is the same for all immobilized preparations,
including α-chymotrypsin immobilized in gel 2: the plot
of the logarithm of the Michaelis constant vs. inverse
temperature is linear and have no breaks (the data are
not presented). This implies that the Michaelis constant
does not "feel" the phase transition of the matrix
(nonsensitivity to the phase transition of the support for
constants of equilibrium stages of reactions of enzymes
immobilized into thermosensitive gels have also been
observed previously13,18). An important moment is the
reversibility of the enzyme behavior: when the tempera-
ture is multiply increased—decreased, the activity of the
preparation is not determined by its thermal prehistory
(i.e., the number of cycles of temperature change) but it
is determined only by the temperature at which a certain
measurement of the activity was performed.13
100
80
1
2
60
10
20
30
[DMSO] (vol.%)
Fig. 5. Plots of the hydration degree (n) of hydrogels 1 (1) and
2 (2) vs. dimethyl sulfoxide concentration in the medium.
are presented in Fig. 5. In this case, the phase transition
of gel 2 (and the corresponding change in its hydration
degree) is observed in an interval of DMSO concentra-
tions of 25—50 vol.%. Since below 20 vol.% DMSO in
the mixture the hydration degrees of both preparations
are virtually the same, it was reasonable to expect the
resemblance in the kinetic behavior of α-chymotrypsin
immobilized into 1 and 2 in this range of DMSO
concentrations, which, however, they are not.
Thus, the observed effect of decreasing the enzyme
activity of preparation 2 with temperature is related to
precisely the change in the kinetic parameters, i.e., the
maximum reaction rate. This fact finds two possible
explanations: the compression of the matrix either re-
sults in lowering of the catalytic rate constant of sub-
strate hydrolysis or creates steric hindrances for the
enzymatic reaction, i.e., "shields" some active sites of
the enzyme. To answer this question, we performed the
experiment on the inactivation of α-chymotrypsin im-
mobilized into the poly-N-isopropylacrylamide gel with
benzylsulfonyl fluoride at 50 °C. It has been shown that
the all enzyme was bound to the inactivator within at
most 10 min. It is substantial that the enzyme activity
did not increase with the temperature decrease contra-
dicting the assumption about shielding of some active
sites of the enzyme.18 Thus, even under the conditions
of complete compression of the matrix where the rates
of the enzymatic reaction are close to zero (cf., Figs. 3
and 4) all active sites of immobilized α-chymotrypsin
are accessible for the interaction with molecules compa-
rable in size with the inactivator molecule (including
with the substrate used by us). It follows from this that
the specific influence of poly-N-isopropylacrylamide on
immobilized α-chymotrypsin during the phase transition
of the polymeric matrix results in the precisely de-
crease (!) in the catalytic constant of the enzyme with an
increase (!) in the temperature. Since this fact contra-
dicts the laws of thermodynamics, it should be assumed
that some interactions between the biocatalyst and poly-
mer occur in this system and result in the experimentally
observed decrease in the catalytic constant.
The different kinetic behaviors of native α-chymot-
rypsin during the hydrolysis of various substrates in
water—DMSO mixtures have previously been de-
scribed19—21 (cf. data on the hydrolysis of 4 and
p-nitroanilide-N-benzoyl-L-tyrosine (5) with the native
enzyme, Fig. 6). This phenomenon was explained by
local conformational changes in the vicinity of the
active site of the enzyme due to the specific interaction
of DMSO with the protein globule, presumably, in the
substrate-binding protein pocket. This conclusion was
drawn from comparison of the data for native α-chy-
motrypsin and that immobilized into gel 2. The immo-
bilization into gel 2 protects the enzyme, from the one
hand, from the denaturating effect of DMSO and, on
the other hand, removes the influence of the substrate
structure on the kinetic behavior of immobilized prepa-
vmax/vmax (H2O) (%)
1.25
1.00
1
0.75
2
3
0.50
4
5
0.25
6
0
10
20
30
[DMSO] (vol.%)
Fig. 6. Plots of the relative maximum reaction rates of hydroly-
sis of N-acetyl-L-tyrosine ethyl ester (1—3) and p-nitroanilide-
N-benzoyl-L-tyrosine (4—6) at the pH-optimum of the effect
of native α-chymotrypsin (1, 5) and its immobilized prepara-
tions 1 (3, 4) and 2 (2, 6) gels vs. dimethyl sulfoxide concentra-
tion in the medium.
Influence of dimethyl sulfoxide on the hydration
degree of gels and activity of α-chymotrypsin
The plots of the hydration degrees of preparations 1
and 2 vs. DMSO concentration in the reaction medium

Hydration degree of gel and enzyme activity
Russ.Chem.Bull., Int.Ed., Vol. 50, No. 10, October, 2001
1895
rations (Fig. 6). In this study, we showed that the
kinetic behavior of α-chymotrypsin immobilized into
gel 2 does not virtually differ from that of the native
enzyme for which the detected activity is determined by
the substrate nature. The values of the Michaelis con-
stants for preparations 1 and 2 (the data are not pre-
sented) coincide in the whole interval of the used
concentrations of an organic solvent, i.e., neither the
type of the matrix, nor its state affect the en-
zyme—substrate equilibrium. Similar facts suggest that
specific interactions of the protein globule of α-chymot-
rypsin with both DMSO and the matrix of 2, which
undergoes the phase transition, are similar and result in
local conformation changes near the active site of the
enzyme (cf. Figs. 5 and 6).
In fact, the driving force of gel 2 compression is an
enhancement of the hydrophobic interactions in the
polymer with both the temperature increase and an
increase in the concentration of an organic solvent in
the mixture. If the interactions of α-chymotrypsin with
2 become more energetically favorable than those with
water with an increase in the polymer hydrophobicity,
we can assume that the polymer (similarly to DMSO)
results in local conformational changes in the active site
of the enzyme by the interaction with the substrate-
binding protein pocket. The same mechanism (the in-
teraction of 2 with the protein with an increase in the
polymer hydrophobicity) takes place, most likely, in the
case of the temperature compression of the poly-N-iso-
propylacrylamide matrix.
biocatalysts — phospholipid membranes) during the phase
transition act as unique "antennas," which perceive the
external stimulus and, specifically interacting with the
protein globule, regulates the activity of the biocatalyst.
Especially note the complete reversibility of the ob-
served kinetic effects, which provides a possibility to
create catalyst preparations purposefully regulated by
various external effects.
This work was financially supported in part by the
Russian Foundation for Basic Research (Project No.
98-03-32204à).
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Thus, the compression of the gel due to blocking of
the electrostatic repulsion between the likely charged
groups of the support, i.e., a simple increase in the
density of the polymeric network, has no effect on the
observed activity of immobilized α-chymotrypsin. At
the same time, the specificity of the interaction of the
protein globule with the matrix is observed for the
enzyme immobilized in the poly-N-isopropylacrylamide
gel in which the compression of the matrix (and the
corresponding change in its hydration degree) is in-
duced by the phase transition of the polymer. Analysis
of the data on the behavior of α-chymotrypsin immobi-
lized into the poly-N-isopropylacrylamide gel during the
compression of the matrix in water/dimethyl sulfoxide
mixtures suggested the local interactions of polymeric
chains of the stimulus-sensitive support with the protein
globule. The data presented indicate that the stimulus-
sensitive hydrogels (as well as natural supports of
Received March 5, 2001;
in revised form September 7, 2001