A comparative evaluation of the activity modulation of flavo and non-flavo enzymes induced by graphene oxide

Article abstract of DOI:10.1039/c7tb00083a

Graphene, and its water soluble derivative graphene oxide, has shown great promise in various biomedical applications, such as cancer therapeutics, drug delivery, etc. and in industrial applications such as enzyme immobilization, etc. Thus, modulation of the activities of different classes of enzymes by graphene materials is an important aspect in the formulation of different biological applications. We have demonstrated here how flavin adenine dinucleotide (FAD) moieties protect the binding site from conformational change in the presence of an inhibitor, graphene oxide, and also explore differences in the mode of interactions between flavo and non-flavo enzymes. It was shown that there was a much greater loss of activity with the non-flavo enzyme, l-lactate dehydrogenase (LDH), of ～74% compared to that with the flavo-enzyme, glucose oxidase (GOX), of ～45%, in the presence of equal concentrations of GO. Furthermore, GO acts as an enzyme inhibitor and the mode of inhibition is uncompetitive for GOX and competitive for LDH. Circular dichromism measurements showed a 21% decrease in the α helix of GOX and a 31% decrease in the α helix of LDH in the presence of a given concentration of GO (0.5 mg mL^-1). There was a slight change in the average emission lifetime of tryptophan in GOX in the presence of GO from 3.2 to 2.6 ns. In contrast, there was no change in the average emission lifetime of tryptophan in LDH in the presence of GO. The extents of fluorescence quenching for GOX and LDH were 39% and 70% upon addition of a certain amount of GO. The present study provides insight into the development of sensors through the immobilization of enzymes and the possible formulation of a multifunctional protein and graphene composite system for various biomedical applications such as bio-sensing, gene and drug delivery, etc.

Full text of DOI:10.1039/c7tb00083a

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ARTICLE
A Comparative Evaluation on Activity Modulation of Flavo and
non-Flavo Enzymes Induced by Graphene Oxide
Received 00th January 20xx,
Accepted 00th January 20xx
Susmita Maiti, Somashree Kundu, Chandra Nath Roy, Debasmita Ghosh, Tushar Kanti Das and
Abhijit Saha*
DOI: 10.1039/x0xx00000x
Graphene and its water soluble derivative, graphene oxide has shown great promise in various biomedical applications,
such cancer therapeutics, drug delivery, etc. and industrial applications like enzyme immobilization, etc. Thus, modulation
of activity of different class of enzymes by graphene materials is an important aspect to formulate the different biological
applications. We have demonstrated here how flavin adenine dinucleotide (FAD) moieties protect binding site from
conformational change in the presence of inhibitor, graphene oxide and also explores differences in mode of interactions
between a flavo and non-flavo enzymes. It was shown that there was much greater loss of activity of non-flavo enzyme, L-
lactate dehydrogenase (LDH) ~74% compared to that with flavo-enzyme, glucose oxidase (GOX) that of ~45% in the
presence of equal concentration of GO. Further, GO acts as enzyme inhibitor and the mode of inhibition are uncompetitive
for GOX and competitive for LDH. The circular dichroism measurements show decrease in α helix for GOX is 21% and that
for LDH 31% in the presence of a given concentration of GO (0.5 mg/ml). There was a slight change in the average
emission lifetime of tryptophan of GOX in the presence of GO from 3.2 to 2.6 ns. In contrast, there was no change in
average emission lifetime of tryptophan in LDH in the presence of GO. Extent of fluorescence quenching for GOX and LDH
were 39% and 70% on addition of a certain amount of GO. The present study provides insight in developing sensors
through immobilization of enzymes and possible formulation of multifunctional protein and graphene composite system
for various biomedical applications like bio-sensing, gene and drug delivery, etc.
www.rsc.org/
Introduction
candidates for enzyme modulation.3,4 Further, in recent years,
Enzymes are macromolecular biological catalysts that
accelerates various biochemical processes with particular
specificity and exceedingly high turnover number.1,2 They are
valuable in the applications of biotechnology, biosensing,
biomedicine, etc. On the other case, anomalous activities of
enzymes may possibly direct to diseases and disorders like
cancers. So, modification of enzyme properties, such as
stability, activity is very important and has great importance in
the study of pharmaceutics and thrapeutics. The catalytic
functionality of most of the enzymes depends on its precise
three dimensional structure. Among various enzyme
regulators, such as proteins, peptides, synthetic organic
molecules, recently, nanomaterials are shown to be attractive
graphene and its oxidized water-soluble derivative, graphene
oxide (GO) has shown tremendous potentials in various
biological applications because of their interesting physical and
chemical properties.
Functionalized GO are promising
materials for applications in gene and drug delivery,5
biosensing,6,7 cancer therapeutics,8 cellular imaging,9 as well as
antibacterial agent.10 There are only a few reports on physico-
chemical interactions of GO with biomacromolecules (e.g.,
enzymes, DNA, etc.).11,12 GO plays, as
a matrix for
immobilization of several enzymes, such as glucose oxidase,13-
horseradish peroxidase,17-21 mostly for biosensing. De et al.
16
showed that GO can act as an artificial receptor and inhibit
activity of chymotrypsin protein.22 This has spurred our
interest to make a detailed investigation on modulation of the
kinetic and structural behavior of enzyme molecules on
interactions with water soluble graphene oxide. It is well
known that flavoenzmye is an important class of protein
molecules which are indispensable for numerous biological
processes like signal transduction, gene expression, immune
responses, metastasis, and metabolism. Folding of protein
structure and stability of those depends on the binding
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through flavin adenine dinucleotide (FAD) site. Flavoenzymes
play critical role in electron transfer processes leading to
biological oxidation and reductions both in vivo and in vitro.
Thus, it is important to compare GO-induced modulation of
catalytic functions vis-a-vis structural features of non-
flavoenzyme with flavoenzyme.
Graphite flakes (natural, 325 mesh, 99.8%) was obtained from
Alfa Aesar. Sodium nitrate (NaNO3), potassium permanganate
(KMnO4), hydrogen peroxide (H2O2) and sulfuric acid (H2SO4)
were obtained from Merck, India. Glucose oxidase (EC 1.1.3.4)
from Aspergillus niger, β-D(+) glucose and o-dianisidine
dihydrochloride were purchased from Sigma-Aldrich, USA.
Horseradish peroxidase (HRP), L-lactate dehydrogenase (EC
1.1.1.27) from rabbit muscle, pyruvic acid, β-nicotinamide
adenine dinucleotide (reduced) disodium salt (β-NADH-Na2)
were purchased from Seisco Research Laboratory, India. Tris
buffer was purchased from Merck, India. Phosphate buffer
solution (PBS, 0.1 M, pH 7.0) was made up from mixing of 61.5
mL 1 M K2HPO4 and 38.5 mL 1 M KH2PO4 and after that pH was
measured by JENWAY-3345 pH cum ion meter. The solution of
GOX in PBS (pH 7.0) was prepared just before each
experiment. The glucose stock solution was allowed to
mutarotate for at least 24 h before use. All the chemicals used
were of analytical grade or highest purity available and were
used as obtained. Milli-Q water (Millipore) was used as a
solvent.
In the present study, we have chosen two clinically
important enzymes, glucose oxidase (GOX) and L-lactate
dehydrogenase (LDH) as model enzymes representating class
of flavo- and non-flavoproteins, respectively. Glucose oxidase
is a flavoenzyme that catalyzes oxidation of β-D-glucose by
molecular oxygen to d-gluconolactone which subsequently
undergoes spontaneous hydrolysis to gluconic acid and
hydrogen peroxide.23 GOX which catalyzes oxidation of glucose
in blood, contains one tightly, non-covalently bound FAD
cofactor per monomer and is a homodimer with a molecular
mass of 160 kDa, depending on the extent of glycosylation.24
The fluorescing units with well-resolved emission profiles in
GOX, namely, tryptophan and FAD can help monitor the role of
these two key components in modulation of functions of
flavoenzyme. On the other hand, LDH considered as biomarker
in diagnosis of diseases like congestive heart disease, hepatitis
especially liver cancer, organ failure, etc.25-27 contains only
tryptophan as fluorescence unit. The fluorescence of the
tryptophan is highly sensitive and therefore, has been widely
used as an intrinsic probe of protein conformation, dynamics
and intermolecular interaction.28
Methods
UV−vis Spectroscopy
The interactions of GOX and LDH with GO were followed by
UV-Vis spectroscopy using Perkin Elmer Lambda UV/Vis/NIR
spectrometer. The concentrations of the GO, enzymes were
9.9 µg/mL and 30 µg/mL, respectively.
In this study. we have shown how GO can modulate the
catalytic functionality of the enzyme molecules in terms of
standard Michaelis−Menten (MM) model. The two Michaelis-
Menten parameters in this model are Km (Michaelis constant)
and Vmax (maximal velocity), where, Vmax represents the
maximum velocity of the enzyme substrate reaction at infinite
(saturating) substrate concentration and the Michaelis
constant, Km which is equivalent to substrate concentration at
half maximum velocity, is a means of characterizing an
enzyme's affinity for a substrate. Change in Km and Vmax values
reflects the change in activity of the enzyme and type of
enzyme inhibition. The change in activity can be quantitatively
represented by the change in Km and Vmax values. Thus, these
biochemical parameters can provide a quantitative estimate of
functionality of enzyme molecules. Michaelis-Menten values
can, therefore, furnish valuable information on the
interactions in enzyme-graphene based platform which may
have ramifications in diverse biological applications.
Photoluminescence spectroscopy
A solution of GOX (30 µg/mL) in 0.1 M sodium phosphate
buffer (pH 7.0) and LDH (30 µg/mL) in 0.2 M Tris.HCl buffer, pH
7.3 was prepared. Fluorescence measurements were
performed on
a
Perkin-Elmer LS-55 luminescence
spectrometer. For monitoring the fluorescence spectra of the
tryptophan (Trp) moiety of a GOX molecule, the excitation
wavelength of 295 nm was used, and the spectra were
recorded between 305–500 nm. The fluorescence spectra of
the FAD (flavin adenine dinucleotide) moiety were recorded in
the wavelength range of 470–700 nm upon excitation at 450
nm. A rectangular quartz cell of 1.00 cm path length was used
for these measurements. GO concentration in the range of
1.61 to 11.53 µg/mL were used in the measurements.
The present work provides a detailed description on how
GO can affect the critical enzyme functionality in terms of
kinetic parameters, like Km and Vmax, which in turn, were
correlated with concomitant changes in secondary structure of
protein molecule and in tryptophan and/or flavin moiety, key
components in enzymatic function. In addition, GO-induced
modifications have been compared between flavo and non-
flavo enzymes.
Fluorescence lifetime measurements of both GOX and LDH
in the absence and in the presence of GO were done by time-
correlated single photon counting (TCSPC) spectrophotometer
(HORIBA JOBIN YVON IBH) using Nano LED (Monochromator:
JY-IBH 5000M) with a lamp of FWHM = 45 ps. The GOX or LDH
of concentration of 0.4 mg/mL were excited at 290 nm
picosecond diode laser and emission monitored at 350 nm.
The fluorescence decays were analyzed with IBH DAS6
software considering reduced chi-square value.
Experimental
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Circular dichroism spectroscopy
the slope and intercepts of the straight line. Influence of GO,
on enzyme activity and kinetic parameters were determined in
The three dimensional conformation changes of GOX and LDH the presence of GO of different concentrations.
blank and with the interaction with GO were pursued by
circular dichroism (CD) spectroscopy. The CD spectra were
recorded on a Jasco J-815 spectropolarimeter fitted with a
xenon lamp and calibrated with (+)-D-10- camphor sulfonic
acid. Dry nitrogen was purged continuously into the
instrument before and during the experiment. All
measurements were made at 25 °C. The cuvette of an optical
path length of 0.1 cm was used. Scan was performed in
wavelength range of 190 and 250 nm, at a rate of 20 nm/min.
Concentrations of GOX and LDH were 0.4 mg/mL and 0.3
mg/mL, respectively.
ꢀ
ꢄ
ꢀ
ꢅ
ꢃ ꢆ ꢊꢋꢌ ꢍ ꢆ
(4)
ꢁ
ꢂ
ꢇꢈꢉ
ꢅꢎꢏ
Results and Discussion
Absorption spectroscopy
The optical absorption of free GO and that of GOX and LDH in
the presence of GO were shown in Fig.1. Though there was no
spectral shift in the absorption but the absorption intensity of
the mixture of both the enzymes with GO increases around
280 nm compared to that of the corresponding blank enzyme.
The absorption at 280 nm is due to the presence of aromatic
amino acids of both the enzymes. The changes in the
absorption around 280 nm may be due to the interaction
between GO and aromatic systems of the enzymes.31,32
Catalytic activities of GOX and LDH
The activity of GOX in the native state and in the presence of
GO was evaluated at 25 0C by a peroxidase-coupled assay.29
GOX
β-D-Glucose + O2 + H2O
D-Gluconic acid + H2O2 (1)
Horseradish Peroxidase
o-di-
H2O2 + o–dianisidine (reduced)
anisidine (oxidised)+ H2O
(2)
β-D-glucose, HRP, and o-dianisidine were firstly dissolved in
PBS (0.1 M, pH 7.0) at concentrations of 100 mg/mL, 2 mg/mL,
and 0.066 mg/mL, respectively. The absorbance of the mixture
at 436 nm was recorded with time using UV-Vis absorption
after addition of 0.02 mL GOX solution (0.01 mg/mL, in PBS)
into the mixture. The catalytic activity of GOX was estimated
from the slope of the plot of the absorbance against time. The
catalytic activity of GOX (7 µg/mL in PBS) in the presence of
varying the concentrations of GO (from 0.01 to 3.3 µg/mL) was
also estimated following the same assay procedure.
The activity of LDH in the native state and in the presence
of GO was evaluated at 25 0C by following the enzymatic
assay.30
Fig. 1 UV–Vis absorption spectra of blank GO, GOX and LDH and in
L-Lactate Dehydrogenase
the presence of GO of concentration of 9.9 µg/mL.
Pyruvate + β-NADH
L-lactate + β-NAD
(3)
β-NADH-Na2 and pyruvic acid were dissolved in 0.2 M Tris.HCl
buffer, pH 7.3 with the concentration of 6.6 mM and 30 mM,
respectively. The absorbance of the mixture at 340 nm was
recorded with time after 0.1 mL LDH solution (0.3 mg/mL, in
Tris.HCl) was added into the mixture. The catalytic activity of
LDH was estimated from the slope of the plot of the
absorbance against time. Further, the catalytic activity of LDH
(9.9 µg/mL in PBS) in the presence of varying concentrations of
GO (from 0.02 to 0.2 µg/mL) was estimated using the same
method.
Fluorescence spectroscopy
Aromatic amino acid residues, such as tryptophan (Trp),
tyrosine (Tyr), and phenylalanine (Phe) are significant
components of proteins and also have reasonable
fluorescence. Among them, Trp fluorescence is very important
as it carries more specific local information of proteins in its
vicinity.33 GOX and LDH both contain tryptophan residue, so
they are strongly fluorescent. The photoluminescence of the
tryptophan moiety in the presence of different concentrations
of GO was shown in Fig. 2. It is observed that GO has no
significant fluorescence when excited at 295 nm,34 so it may be
assumed that there is no interference of emission of GO in the
fluorescence spectrum of tryptophan. The observed results
Michaelis-Menten parameters (Km and Vmax
)
were
determined using Lineweaver-Burk double-reciprocal plot of
1/vo versus 1/[S]. All measurements were performed in
triplicate. From eq. 4, both Km and Vmax were obtained from
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demonstrate that quenching of tryptophan fluorescence at
350 nm is much higher in LDH (about 70%) compared to that in
GOX (about 39%) on interaction with GO molecules at a given
concentration of 9.9 µg/mL. The fluorescence intensity and
nature of spectra depend on the exposed tryptophan moieties.
For interactions with GOX, flavin part also takes part in
interactions with GO. FAD quenching of GOX is upto 43% in
presence of GO and also Stern-Volmer constant is 0.03 mL/µg
(Fig.S3).
The KSV values indicate that affinity of GO towards enzyme is
weaker than blank tryptophan. Initially the fluorescence signal
for GOX or LDH do not change their position at lower
concentration ofGO. With increase in concentration of GO, a
small consistent red shift was observed. The shifts in emission
maxima reflect change of polarity of the solvent around
tryptophan residue, which indicates conformational changes of
GOX at their tertiary structure levels. The red shifts indicate
greater exposure of tryptophan residues of the flavin domain
moving out from the core of the enzyme to the polar
environment.36
The plot of I0/I (Fig. 2: inset) against concentration of GO is
linear, which suggests that fluorescence quenching follows
Stern–Volmer relation (eq. 5).35
We have also studied KSV values for the tryptophan
emission quenching of GOX and LDH with varying solvent
polarity with dielectric constant (ϵ) of medium from 80.1 to
37.5. It was observed that KSV values was not affected. This
indicates that the interactions between GO and enzymes are
non-electrostatic which may be π-π stacking, hydrogen
bonding, etc.37
ꢐꢂ
ꢃ 1 ꢍ ꢑꢋꢆꢊꢒꢓꢌ
(5)
ꢐ
where, KSV is the Stern–Volmer quenching constant, I0 and I,
the fluorescence intensities of tryptophan moieties in the
absence and in the presence of GO, respectively. In order to
gain an insight into the quenching of tryptophan fluorescence
in GOX and LDH, similar experiments were conducted with free
tryptophan. KSV values for blank tryptophan, GOX and LDH are
0.48, 0.06 and 0.19 mL/µg, respectively. It is apparent that for
free tryptophan molecule, extent of quenching is much higher
in comparison with LDH or GOX. It may be due to the fact that
free tryptophan molecule can have maximum accessibility for
interactions with GO compared to protein trypotophan.
Further, the observed results indicate that tryptophan moiety
in GOX is relatively much less accessible for interactions with
GO compared to that in LDH. Also it may be assumed that
tryptophan residues in LDH are more exposed than that in GOX
and this facilitates greater surface binding of LDH with GO in
comparison that with GOX.
Time resolved fluorescence spectroscopy
Time resolved fluorescence spectroscopy was performedto
knowinformation
about
the
conformation
change
oftryptophan residues in the GOX and LDH molecules. Fig.3
shows the emission decay profiles of GOX and LDH in the
absence and in the presence of GO. These decay profiles are
fitted employing the eq. 6
ꢟ
ꢔ ꢖ ꢃ ꢙꢠꢀ ꢘꢙꢚꢕꢛꢜ/ꢝ ꢗ
(6)
ꢞ
ꢕ ꢗ
∑
where,τi is the lifetime of the different components and the
pre-exponential factor, Ai, is a measure of the contribution of
each component to the total decay. The average lifetime of
fluorescence decay τav is calculated using the following
equation (eq. 7).38 All the values are reported in Table 1.
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ꢣꢞꢝꢞꢤ
ꢣꢞꢝꢞ
has lifetime 0.45 ns whereas for the long lived component (τ3)
has lifetime 4.39 ns. There is about 46% decrease in lifetime
(0.45 - 0.24) for the fast component in the presence of GO (0-
110 µg/mL). In contrast, the longer lifetime component
decreases relatively to a smaller extent in comparison with the
fast component. The relative contribution of longer lifetime
component increases with increasing concentration of GO. It
has been reported that buried tryptophan residues show
shorter lifetime (τ3) and the exposed moieties display longer
lifetime (τ2), because the buried part remains more contact
with the peptide bonds than the exposed part. The most of the
tryptophan residues buried in the flavin domain get exposed
with the increasing concentration of GO through hydrophobic
or π-π stacking interaction and this is in agreement with red
shift in PL spectra. Blank LDH shows two lifetime components
but on addition of GO there are three lifetime components,
due to more exposed tryptophan moieties. There is almost no
change in average lifetime of LDH after addition of GO solution
(8-63.6 µg/mL), which implies that GO-induced LDH quenching
is static in nature.
ꢟ
∑
ꢡꢢꢁ
ꢃ
(7)
ꢙꢠꢀ
The value of τav for native GOX (0.4 mg/mL) is found to
beabout 3.18 ns, which is in good agreement with those
reported previously (2.28–4.1 ns).38,39 With the increasing
concentration of GO, the average lifetime values decreases
due to the change of conformation of protein molecules (Table
1). In the absence of GO, the fast component (τ2) of emission
decay
Table
1. Luminescence lifetime analysis of tryptophan
fluorescence of GOX in the presence of different
concentrations of GO.
Conc.
of
GO
τ1
τ2
τ3
A1
A2
A3
τav
(ns)
χ2
We have reported that fluorescence intensity is quenched
upon addition of GO molecules. The decrease in fluorescence
intensity may be due to π-π stacking interaction and/or charge
transfer reaction. Consideration of redox potential values of
tryptophan ( 1.0 V),40 at pH 7 and that of graphene ( 1.8 V)41
suggests a possible electron transfer from tryptophan to
graphene oxide.
(ns) (ns) (ns)
(µg/mL)
0.0
4.0
1.7
1.7
1.3
1.2
0.5
0.5
0.3
0.2
4.4 54.1 16.4 29.5 3.2
4.4 54.2 16.9 28.9 3.2
3.3 46.6 11.5 41.9 2.7
3.3 46.2 11.2 42.6 2.6
1.0
1.1
1.2
1.0
77.5
110.0
Circular Dichroism Spectroscopy
To investigate the conformational aspects in GOX and LDH in
the native state and in the absence of GO, circular dichrosim
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spectroscopy was carried out. The amide chromophore of the
peptide bond in proteins dominates the CD spectrum below
250 nm. The far UV-CD spectra in the wavelength range 190-
250 nm of GOX and LDH were shown in Fig. 4 and near UV-CD
in Fig. S4. The CD spectra of blank GOX in PBS buffer (pH7.0)
and LDH in tris HCl buffer (pH 7.3) are characterized by two
main negative peaks at far UV region at about 209 and 220 nm
in GOX and LDH,respectively, which are characteristics of an
α-helical structure.42,43 From these results, it is assumed that
ꢱꢲ
ꢬꢭꢮꢯꢪꢰ
ꢃ
(9)
ꢀꢪꢳꢴꢵꢶ
where, θ is the ellipticity value of the samples; M, the
molecular weight in Dalton, C; the sample concentrations
(mg/mL); L, sample path length in cm and Nr, number of amino
acid residues present in the samples. From these values, we
can calculate MRE at 208 nm wavelength which reflects α helix
content of proteins (Table 2). From these results it is shown
that native GOX and LDH contain 32 and 42% α-helix,
respectively. With increasing concentration (0-0.5 mg/mL) of
the structure of blank GOX predominates in
α -helical
structure. On addition of GO, there was decrease in intensities
values of two negative bands indicating the loss of α -helix
structure and the occurrence of protein unfolding induced by
GO.40 So, the result shows that there is a dependence of
changes in the secondary structure on the concentration of
GO. In addition with the increasing concentration of GO
besides the decrease in intensities of two negative
bandsfurther also they move along together in the region
between 209 and 220 nm indicating loss of α-helix and enrich
of β- sheet structure since β- sheet has negative band at 215
nm.42 The extent of decrease of α-helix and β- sheet is small
for GOX with respect to the LDH. In the near-UV CD spectra of
GOX, a strong positive peak appears at 274 nm due to coupling
between FAD and tryptophan in the active site similar to other
flavo proteins. On addition of GO there is a decrease in
intensity about ca. 15% and also a blue shift occurs in the
resultant CD spectra. This suggests definite change in the
tertiary structure of GOX in the FAD binding centre.44,45
Table 2. Changes in α-helical content of glucose oxidase and
LDH in the presence of different concentrations of graphene
oxide.
Glucose Oxidase
Lactate Dehydrogenase
Concentration
α helix %
Concentration
of GO
(mg/mL)
0.00
α helix %
of GO
(mg/mL)
0.00
32
29
28
27
25
42.0
39.3
35.4
32.8
29.0
0.02
0.05
0.05
0.06
0.25
0.13
0.50
0.51
α helix content of both the enzymes GOX and LDH was
calculated using the following relations (eq. 8 and 9).46
α helix (%) = ꢊꢛꢥꢦꢧ ꢛꢩꢪꢪꢪꢌ ꢫ 100
(8)
ꢤꢂꢨ
ꢊꢟꢟꢪꢪꢪꢛꢩꢪꢪꢪꢌ
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In order to understand the effect GO on thekinetics of two
enzymes, GOX or LDH, we have measured the Vmax and Km
values of two enzymes in the presence of GO. The kinetic
parameters obtained from the enzyme kinetic assay in the
presence of different concentrations of GO are summarized in
Table 3 and 4. For GOX we obtain Michalis-Menten constant as
99.6 mM and that for LDH as 0.11 mM which are agreed well
to the reported values.47,48
GO, those decreased to 21.9% in GOX and 31% in LDH. Since,
catalytic activity significantly depends on secondary structure
of enzyme, these results indicate significant effect on catalytic
functions of these enzymes on interactions with GO. Further,
secondary structure in GOX is relatively less affected
compared to that in LDH on interactions with GO in far-UV
region of CD spectra. There is also chnges in the tertiary
structures in the near-UV CD region. This result also indicates
binding of GO with GOX occurs through FAD binding centre.
Table 3. Kinetic parameters of GOX in the presence of varying
concentrations of GO when GO was mixed with the enzyme
before substrate addition.
Effect of GO on catalytic activity and kinetic parameters
of enzymes
It is well known that the activity of an enzyme is closely
correlated with its conformation.42 From the above spectral
studies, it is clear that enzyme produced conformational
changes in the interactions with GO. To determine
corresponding changes in terms of activity, the enzyme assay
of GOX or LDH was carried out under different concentrations
of GO. The changes of percentage of activities of GOX and LDH
was shown in (Fig. 5). It was observed that activity of enzyme
decreased with increasing concentration of GO. The nature of
the plot for GOX or LDH is exponential which indicates
activities of enzymes effected strongly in the presence of GO.
The calculated activity of GOX was 0.87 (U/mL) which
decreased to 0.44 (U/mL) on addition of GO ( 2.0 µg/mL).
Similarly,the calculated activity of LDH was 0.91 (U/mL) which
Concentration of
% of activity
Km (mM)
Vmax (min-1)
GO in µg/mL
0.00
remaining
100
97
87
55
53
50
99.60
36.74
26.43
21.57
18.34
16.85
24.88
21.65
20.40
11.57
8.39
0.01
0.30
0.60
1.20
2.00
5.95
Table 4. Kinetic parameters of LDH in the presence of varying
concentrations of GO when GO was mixed with the enzyme
before substrate addition.
decreased to 0.24 (U/mL) in the presence of
a given
Concentration of
% of activity
remaining
100.0
Km (mM)
Vmax (min-1)
concentration of GO (0.6 µg/mL). From these results, we
observed that on addition of GO (0.3 µg/mL), activity of GOX
decreased only to 13.4%, whereas activity of LDH decreased to
56% on addition of GO concentration only 0.2 µg/mL. The loss
in GO-induced activity of lesser magnitude in GOX in
comparison with that in LDH may be due to the protection
imparted by FAD moieties towards conformational changes.
GO in µg/mL
0
0.11
0.29
0.54
1.41
3.45
4.50
10.1
10.6
9.7
0.02
88.7
0.03
73.5
0.20
44.0
9.9
0.40
32.0
10.9
9.3
0.60
26.0
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Journal Name
From these kinetic data, it was shown that both of Km and Vmax of enzyme from GO-induced perturbations which was also
of GOX decrease on interactions with GO which occurs in case observed from CD spectral measurement. The present study
of uncompetitive inhibition. But in case of LDH, Michaelis- gives valuable information how GO can modulate enzyme
Menten constant (Km) increases while Vmax remains constant activity as well as changes of secondary structures of different
and this is exactly seems to be competitive inhibition. When class of enzymes which have great implications in fabrication
affinity of enzyme towards substrate increases, Km decreases. of protein-graphene composite platforms for various
Thus, Km has inverse relation to the enzyme affinity towards biomedical and industrial applications involving enzyme
substrate and the decrease in Vmax values indicates a decrease immobilization, etc.
in the rate of reaction between enzyme and substrate which
implies that enzyme converts lesser amount substrate to
Acknowledgements
product per unit time. In the presence of GO molecules,
activity of GOX towards glucose oxidation decreases which
could be due to structural changes in the active site of enzyme.
However, in case of LDH, affinity of enzyme towards substrate
decreases in the presence of GO and the amount of active
enzyme remains constant under the influence of various
concentrations of GO. This presumably suggests that the
presence of GO has not caused significant modification of the
active site. To understand the mode of inhibition, we plotted
the velocity of enzyme substrate reaction in the presence of
inhibitor with regard to the function of substrate
concentration following the eq.10.22,49
The authors are grateful to Prof. S. K. Pal of S N Bose National
Centre for Basic Sciences for providing Circular dichroism
facility. One of the authors (S. Maiti) is grateful to DST, Govt of
India, for the award of INSPIRE Fellowship. The authors S.
Kundu and D. Ghosh are thankful to the University Grants
Commission for NET fellowship and D. S. Kothari post doctoral
fellowship respectively. One of the authors T. K. Das is grateful
to CSIR for NET fellowship.
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Graphical Abstract
A Comparative Evaluation on Activity Modulation of Flavo and non-Flavo
Enzymes Induced by Graphene Oxide