Full text of DOI:10.1002/1097-0126(200007)49:7<699::AID-PI441>3.0.CO;2-P

Polymer International
Polym Int 49:699±702 (2000)
Bioelectret state in amylase: a study by the
thermally stimulated discharge current
technique
Vinay Mishra and R Nath*
Department of Physics, Dr H S Gour Vishwavidyalaya, Sagar, Madhya Pradesh, India
Abstract: The bioelectret state has been proposed to be a universal property of enzymes and to play an
important role for the catalytic action of enzymes. In the present investigation the bioelectret state in
an enzyme amylase (EC 3.2.2.1) has been studied using the thermally stimulated discharge current
(TSDC) technique. The enzyme has been found to be able to manifest the bioelectret state; the TSDC
spectrum is characterized by four peaks at temperatures around 170K, 220K, 265K and 300K. The
265K peak is attributed to rotation of some polar amino acid residues, whereas bound water molecules
are identi®ed as the source of polarization storage for all other peaks. The 170K peak is ascribed to
bound water molecules directly attached to the polypeptide backbone; the 220K peak is ascribed to a
second layer of multiple hydrogen-bonded water molecules, proposed to be a chain-like structure
around the polypeptide backbone, and the 300K peak is attributed to the bound water molecules
adsorbed on the surface of the enzyme matrix. The existence of several relaxation polarizations
È
supports the Frohlich model of longitudinal polarization waves that are proposed to play an important
role for the catalytic action of the enzyme.
# 2000 Society of Chemical Industry
Keywords: bioelectret state; enzyme action; bound water; amylase
INTRODUCTION
the present investigation we have employed the
thermally stimulated discharge current (TSDC) tech-
nique to study the polarization storage in amylase
under varied hydration. The TSDC technique, ap-
plied to a variety of natural and synthetic biopolymers,
has provided a valuable contribution to the under-
standing of complex behaviour of water±biomolecule
systems.7 TSDC is specially suited to our objective
because: (a) it is highly sensitive, able to detect effects
due to bound water at hydration levels as low as 10mg
The study of physical properties of enzymes is of
special importance as it lies on the borderline where
the biological and physical sciences meet. From the
point of view of physics, the enormous ef®ciency of
enzymes as bio-catalysts poses the question whether
this involves certain general physical properties com-
mon to all the enzymes. The importance of electrical
polarization properties of enzymes for their catalytic
action has been pointed out by Frohlich1±3 and Green4
who suggested that there are strong connections
between charge storage and conformational properties
of enzymes. Change in polarization storage in enzyme
macromolecules induces changes in their conforma-
tion that are of paramount importance for their
catalytic action.
water per gram of enzyme, and (b) it can detect
1
processes with very long relaxation times (10
103s).
to
MATERIALS AND METHODS
The state of persistent polarization, ie the bioelectret
state, has been proposed to be a universal property of
enzymes,5 and continuous experimental evidence is
needed in a variety of enzymes. It is in this context that
a systematic study of the polarization properties of the
enzyme amylase (EC 3.2.2.1) has been undertaken in
our laboratory. The results of our studies on
frequency/temperature dependence of dielectric con-
stant/loss factor and a c conduction6 have demon-
strated the great in¯uence of bound water molecules
on the dielectric behaviour of amylase. Therefore, in
The enzyme used in the present investigation was
procured from Central Drug House (CDH), India, in
powder form. The purity of the material was estab-
lished by thin layer chromatography and column
chromatography. The powder was compressed to
make pellets of diameter 13mm and thickness ranging
between 0.5 and 2mm. The pellets were provided with
non-injecting silver electrodes8 and samples with the
desired degree of hydration (7±20%) were obtained
using a standard procedure.9
The TSDC technique consists of studying the
* Correspondence to: R Nath, Department of Physics, Dr H S Gour Vishwavidyalaya, Sagar, Madhya Pradesh, India
Contract/grant sponsor: CSIR-India
Contract/grant sponsor: UGC-India
(Received 10 January 2000; accepted 10 February 2000)
# 2000 Society of Chemical Industry. Polym Int 0959±8103/2000/$30.00
699

V Mishra, R Nath
thermally activated release of stored dielectric polar-
ization. In this technique, the specimen is ®rst
polarized at room temperature (poling temperature,
Tp) by applying a d c electric ®eld (poling ®eld, Ep) and
then with ®eld kept on, it is cooled to low temperature
(usually to 100K), so that the polarization is frozen.
Now the ®eld is removed, the sample is short circuited
and is heated at a linear rate; depolarization takes
place, giving rise to a discharge current in the external
circuit. The general form of TSDC is
T
Z
IꢀT† ˆ A exp‰ E=kT
B
expꢀE=kT†dTŠ
T0
where I(T) is the externally measured discharge
current as a function of absolute temperature T. T0 is
the starting temperature of TSDC, E is the activation
energy of dipole-rotation or trap depth of the charge
carriers, k is the Boltzmann constant, while A and B
are constants depending on the type of polarization
and heating rate.
Figure 1. TSDC spectrum of amylase (Ep =2kV, Tp=300K, h =9%).
To study the nature of these peaks and hence to
identify the possible sources of polarization storage,
the analysis of the TSDC spectrum is attempted by
systematically varying the poling parameters and the
degree of hydration as discussed below.
A number of expressions have been suggested for
calculating activation energy E.10 Knowing the activa-
tion energy, the relaxation time t can be calculated as
The TSDC spectra were recorded for four poling
®eld strengths (1, 2, 3 and 4kVcm 1) keeping all other
parameters the same. It is observed that with the
increasing poling ®eld the 170K, 220K and 265K
peaks show a linear increase in peak magnitude and
area under the curve: however, a saturation effect is
observed for the 300K peak, as can be inferred from
Fig 2. The TSDC spectra recorded for various poling
temperatures show that the change in poling tempera-
ture has no effect on peak magnitude or peak
temperature for the 170K, 200K and 265K peaks,
but an increase in peak magnitude and a shift in peak
temperature towards the lower side is observed for the
300K peak, as shown in Fig 3.
t ˆ t0 expꢀE=kT†
where the pre-exponential factor t0, for peak tempera-
ture Tm and heating rate b, is given by
t0 ˆ ‰kT2=bEŠ expꢀ E=kT†
The discharge current is measured with the help of a
sensitive electrometer, and when plotted as a function
of temperature shows a number of peaks characteristic
of the mechanism by which the polarization storage
takes place. An analysis of the TSDC spectrum can be
made by varying various poling parameters, and hence
the sources for polarization storage and related
phenomena may be studied. A standard experimental
apparatus11 was used in the present investigation to
measure the TSD currents in the temperature range
100±320K. The measurements were made in the
range of poling ®eld 1±4kVcm 1, poling temperature
295±320K, thickness of the sample 0.5±2mm, and
degree of hydration (de®ned as the ratio of the weight
of the sorbed water to the weight of the dried sample)
7±20%.
The hydration has a pronounced effect on the
TSDC spectrum of amylase. An increase in peak
magnitude and a shift in peak temperature towards the
lower side are observed for the 170K, 220K and 300K
peaks, as is evident from Figs 4±6. This observation
suggests that the polarization corresponding to 170K,
220K and 300K peaks is directly related to bound
water present in the system. The 265K peak does not
shift with increasing hydration; however, a small
increase in peak magnitude is observed. This peak is
masked by 300K at higher hydration.
RESULTS
A representative TSDC spectrum of amylase is shown
in Fig 1. The various poling parameters were as
follows: (i) poling ®eld 2kVcm 1, (ii) poling tempera-
ture 300K, (iii) heating rate 2.5Æ0.3Kmin 1, (iv)
sample thickness 1mm, and (v) degree of hydration
9%. The spectrum is characterized by peaks at
temperatures around 170K, 220K and 265K, and a
very sharp peak of large magnitude at around 300K.
The `activation energy' and `relaxation-time' values
corresponding to these peaks are tabulated in Table 1.
Table 1. Activation energy and relaxation time corresponding to various
TSDC peaks of amylase
Peak temperature
(K)
Activation
energy (eV)
t0
t (s)
12
18
19
33
170
220
265
300
0.4
0.8
1.0
2.0
7.50Â10
3.21Â10
7.92Â10
3.35Â10
2.5605
2.1444
2.4888
1.5948
700
Polym Int 49:699±702 (2000)

Bioelectret state in amylase
Figure 2. Effect of poling field on peak magnitude and stored polarization.
(Series A, B, C: peak magnitude (I) for 170K, 220K and 265K peaks,
respectively. Series E, F, G: stored polarization (P) for 170K, 220K and
265K peaks, respectively. Series D, H: peak magnitude (I) and stored
polarization (P) for 300K peak, on Y2 axis.).
Figure 4. Effect of hydration on peak magnitude of 170K peak.
The 220K peak has also been observed in many
hydrated biomaterials,10,17±19 and could be attributed
to a second layer of bound water molecules. A peak at
180K (along with the 160K peak) has been observed
in the TSDC spectrum of pure water.17 In fact, water
in the macromolecules may be found in several
states.16,17,25,26 The peak occurring at around 220K
may be attributed to the multiple hydrogen-bonded
water molecules, the dipoles of which can rotate only
by breaking several bonds. This is supported by the
observed higher value of activation energy (around
0.8eV), which is double the activation energy value for
the 170K peak.
DISCUSSION
The observed effects of poling ®eld and poling
temperature on the TSDC spectrum suggest that the
170K, 200K and 265K peaks are due to dipolar
reorientation, while the 300K peak is due to space
charge.10,12±14 The 170K peak has been observed in
almost all the hydrated biomaterials studied so far, and
has been attributed to the reorientation of bound water
molecules, probably attached to polar residues or to
the peptide bonds.10,15±20 These bound water mol-
ecules correspond to `structured' water deeply located
inside the enzyme matrix and are able to present
dipolar rotation. The activation energy is found to be
around 0.4eV. For pure water, a peak at about 160K is
reported16 and the corresponding activation energy is
found to be around 0.3eV. The higher value of
activation energy in the case of amylase agrees with
the suggestion that the peak is due to water molecules
that are bound structurally in the enzyme matrix.
Similar results have been observed in some other
hydrated systems.21±24
In the case of the 265K peak, the source of
polarization storage may be the rotation of some polar
side-groups of amylase macromolecules. As suggested
by the increase in peak magnitude with increasing
hydration, the addition of water may increase the
rotational mobility of side-groups responsible for the
265K peak. A complex band in the temperature range
230±260K has been reported for the protein casein, for
which the possible source of polarization is suggested to
be rotation of some side-groups affected by the bound
water molecules.10 It is noteworthy that at the same
temperature, ie around 265K, a peak is obtained in the
Figure 3. Effect of polarizing temperature on peak magnitude of 300K
peak.
Figure 5. Effect of hydration on peak magnitude of 220K peak.
Polym Int 49:699±702 (2000)
701

V Mishra, R Nath
CONCLUSIONS
The enzyme amylase has been found to manifest the
bioelectret state. The observed polarization storage
may be attributed to the rotation of polar amino acid
residues or associated side-groups, and bound water
molecules present in three different states in the
amylase macromolecule. Four relaxation polarizations
are observed, with long enough relaxation times to
sustain the FroÈhlich waves that are proposed to play a
fundamental role in the catalytic action of enzymes.
ACKNOWLEDGEMENTS
The authors wish to thank Prof R Srinivasan, the then
Director, Inter-University Consortium (IUC)-India,
for providing experimental facilities, and Dr V
Ganesan, Low Temperature Laboratory, IUC, for
his active cooperation. Financial assistance by CSIR-
India and UGC-India is also gratefully acknowledged.
Figure 6. Effect of hydration on peak magnitude of 300K peak.
TSDC spectrum of glycine,27 one of the active-site
components of amylase; even the activation energy
values are comparable in both cases, being around 1eV.
Thus, in the case of amylase also, it could be concluded
that the rotation of some amino acid residue or
associated side-group in the peptide linkage is respon-
sible for the observed peak around 265K.
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Polym Int 49:699±702 (2000)