Modulation of catalytic functionality of alkaline phosphatase induced by semiconductor quantum dots: Evidence of substrate-mediated protection

Article abstract of DOI:10.1039/c5ra22591d

Enzymes provide the critical means by which almost all biological reactions are catalyzed in a controlled manner. Methods to harness and exploit their properties are of strong current interest to the growing field of biotechnology. In view of many unique

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PAPER
Modulation of Catalytic Functionality of Alkaline Phosphatase
Induced by Semiconductor Quantum Dots: An Evidence of
Substrate Mediated Protection
Received 00th January 20xx,
Accepted 00th January 20xx
Debasmita Ghosha, Chandra Nath Roya, Somrita Mondala, Somashree Kundua, Susmita Maitia,
Prasanta Kumar Bagb and Abhijit Sahaa*
DOI: 10.1039/x0xx00000x
www.rsc.org/
Enzymes provide the critical means by which it catalyzes almost all biological reactions in a controlled manner. Methods to
harness and exploit their properties are of strong current interest to the growing field of biotechnology. In view of many
unique physical and optical characteristics that are advantageous for studying enzyme activity at quantum dot-
bioconjugate interfaces, we endeavored to explore the possible influence on conformation and catalytic functionality of
alkaline phosphatase (ALP), a clinical marker enzyme, by conducting in-vitro enzymatic activity assay and using molecular
spectroscopic techniques such as UV–visible absorption, fluorescence, circular dichorism (CD) and fourier transform
infrared (FT-IR) spectroscopy. The resulting experimental data were then analyzed in the context of the classical
Michaelis−Menten model. The results show almost 60% and 30% decrease in enzyme acꢀvity on interaction with 10 nM
Cys-CdTe or Cys-CdS QDs, respectively, suggesting inhibition dependence on particle nature. CD spectra measurements
indicated that QD induced the decrease of α-helical content and the increase of β-sheet structure in ALP, resulting in the
loosening and unfolding of the protein skeleton. Interestingly, when QDs and substrate were added simultaneously to
enzyme molecules, catalytic inhibition was significantly reduced indicating protection of enzyme structure by substrate
molecules. Further, we demonstrate that protein encapsulated quantum dots do not affect the catalytic functionality of
enzyme molecules. The present study provides valuable information which may have significant ramifications in various
biomedical applications, such as biosensing, drug delivery, cellular imaging, etc.
applications that include their use as a new type of fluorescent
probes as well as active components in nanostructure−biomolecule
complexes.12,13 Further, there are some reports where
Introduction
Enzymes known as nature’s catalysts are highly efficient at
accelerating biochemical processes with precise specificity.1,2 This
has made enzymes of considerable interest for utilization in a
myriad of biotechnological applications, such as artificial
biosynthesis, biosensing, nanomedicine, industrial product
preparation processes, etc.3−5 In recent years, the rapid
advancement in fabrication of a variety of nanostructured materials
has opened up possible avenues of utilizing nanoparticles as
carriers of enzymes for specific applications.4,5 The conjugation of
proteins to nanoscale colloidal particles has led to the development
of immunoprobes.6 Among available nanomaterials, semiconductor
quantum dots (QDs) has gained considerable attention because its
unique physical and optical properties can be employed in
understanding enzyme activity at NP-bioconjugate interfaces.7-11
Semiconductor quantum dots (QDs) have a broad variety of
nanoparticles have been shown to promote both the retention of
the native configuration and subsequently the activity of
immobilized enzymes as compared to micro or macro surfaces due,
in part, to their high surfacecurvature.5,14 Againfor understanding
the potential toxic effects of nanoparticles in biological systems, a
detailed description of the binding of proteins to nanoparticles is
needed. Each of the binding parameters is likely to be strongly
dependent on both the particle and protein identity.
As enzymes are very important biologically active protein, we
endeavoured to examine the influence of semiconductor QDs on
enzymatic activity, kinetics and associated structural changes. For
this purpose, we have chosen calf intestinal alkaline phosphatase as
a model enzyme, since it occurs widely in nature, and is found in
many organisms from bacteria to human.15 Alkaline Phosphatase
(ALP) is an important enzyme in recycling phosphate within living
cells.
Alkaline
phosphatase
(ALP)
is
a
nonspecific
phosphomonoesterase that hydrolyzes phosphomonoesters as well
as the phosphate termini of DNA. It exists in organisms of all kinds
and plays a vital role in survival under phosphate starvation.16 In
mammals, alkaline phosphatase is a particularly ubiquitous enzyme
that is very important in physiological functions and medical
a. UGC-DAE Consortium for Scientific Research, Kolkata Centre, III/LB-8
Bidhannagar, Kolkata 700 098, India. Fax: +91-33-2335 7008 ; E-mail:
abhijit@alpha.iuc.res.in
b.Department of Biochemistry, University of Calcutta, 35 Ballygunge Circular Road
Kolkata-700 019, India
Electronic
DOI: 10.1039/x0xx00000x
Supplementary
Information
(ESI)
available:
See
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Assay procedure
diagnosis. ALP, which concentrates in the liver and bones, and
monitoring its activity is an indicator for liver or bone diseases.17
Therefore, it is interesting to obtain information on how the
nature of particles vis-à-vis its surface capping groups can modulate
the catalytic functionality of the enzyme molecules in terms of
classic Michaelis−Menten (MM) model. The two important catalyꢀc
parameters in this model are Km (Michaelis constant) and Vmax
(maximal velocity), where Vmax represents the maximum rate
achieved by the system, at maximum (saturating) substrate
concentrations and the Michaelis constant, Km 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. If for any reason the enzyme
conformation is changed, the active site becomes pretty messed up
and 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-nanoparticle
based platform which has spurred interest in recent years in diverse
biological applications.
Alkaline phosphatase hydrolyzes the colourless, synthetic substrate
p-nitrophenyl phosphate to produce a yellow-colour product, p-
nitrophenol and inorganic phosphate.
The catalytic activity was assayed in DEA buffer of pH 9 by
monitoring the increase in absorbance at 405 nm from p-
nitrophenol (pNP) (€M= 18.5×103 M-1cm-1) generated in an
enzyme catalyzed hydrolysis of pNPP at 37 oC using UV-Vis
absorption (Perkin Elmer Lambda UV/Vis/NIR spectrometer)
based on the protocol described in the literature.18,19 The
concentration of ALP was taken 1 μg/ml.
Kinetic measurements of alkaline phosphatase cleavage
were calculated from the enzyme’s initial velocity (vo) as a
function of the six different substrate concentrations. The
initial velocity (vo) of the reaction, in micromoles of product
produced/minute, was calculated from the increase in
absorbance at 405 nm (ΔA405) measured at timed intervals
during the reaction. Michaelis-Menten parameters (Km and
The present work provides a detailed description on how the
variation of chemical nature of QDs, like CdS and CdTe, and its
surface groups can affect the critical enzyme functionality in terms
of kinetic parameters like Km and Vmax of ALP, which in turn, were
correlated with concomitant changes in secondary structure of
protein molecule and in tryptophan moiety, a key component in
enzymatic function. In the present study we have shown, for the
first time, substrate can protect the enzyme damage from
nanoparticle-induced modifications.
Vmax) were determined using Lineweaver-Burk double-
reciprocal plot of 1/v0 versus 1/[S] (eq. 1).
1
Km
1
ꢀ
ꢃ
(1)
V0
Vmax ꢁSꢂ
Vmax
All measurements were performed in triplicate. Both Km and
Vmax were obtained from the slope and intercepts of the
straight line. Influences of the nanoparticles, in question, on
enzyme activity and kinetic parameters were determined in
the presence of the particles of different concentrations.
Experimental
Materials
Photoluminescence spectroscopy
L-Cysteine hydrochloride and cadmium chloride were
purchased from Merck, India. Calf intestinal alkaline
phosphatase, telluric acid (H2TeO4, 2H2O), sodium borohydrate
(NaBH4), and bovine serum albumin (BSA) were purchased
from Sigma-Aldrich. Na2S and diethanolamine buffer (DEA)
were purchased from Seisco Research Laboratory, India. All
the chemicals were of analytical grade or highest purity
available and were used as obtained. Milli-Q water (Millipore)
was used as a solvent.
A solution of ALP (0.01 M) was prepared in 10 mM sodium
phosphate buffer (pH 7.4).The as-prepared Cys-CdTe and CYs-
CdS QD-solutions were further diluted 10 times with
phosphate buffer of pH 7.4 and used for studying the
interactions with ALP. To the solution of ALP, a small amount
of the different QD solutions were added gradually and PL
spectra were recorded in each step. The fluorometric study
was done with
a
Perkin-Elmer LS-55 luminescence
spectrometer. To monitor fluorescence of ALP, the excitation
wavelength was selected at 295 nm, and emission was
recorded at 350 nm. For following luminescence of QDs, the
excitation wavelength chosen was 390 nm, and the PL
intensities of CdS and CdTe were recorded at 500 and 540 nm,
respectively.
Cysteine capped CdS, CdTe and BSA capped CdS
nanoparticles were synthesized following the methods
reported earlier (See supplementary information).
Characterization of the particles by UV-vis spectroscopy (Fig.
S1-S3), transmission electron microscopy (TEM) (Fig. S4) and
Dynamic light scattering (DLS) (Fig. S5) has been mentioned in
the supplementary information. Concentrations of the
synthesized nanoparticles have been determined based on the
procedure proposed by Peng et al. (details are presented in
Supplementary information).
Fluorescence lifetimes were determined from time-
resolved intensity decays at 350 nm by using a time-
correlated-single photon-counting (TCSPC) spectrophotometer
(Edinburgh) with a lamp of FWHM = 1.2 ns and a repetition
rate of 25 kHz. The details of the set-up were described
elsewhere.20 Quality of the fits is judged by the reduced χ2
criterion and the randomness of the fitted function to the raw
data. Mean (average) fluorescence lifetimes (τav) for the bi-
2 | J. Name., 2012, 00, 1-3
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Figure 1. The relationship between the increase in absorbance
at 405 nm (ΔA405) and time in the hydrolysis of p-nitrophenyl
phosphate by alkaline phosphatase
exponential fittings were calculated from the decay times (τ1
and τ2) and the relative amplitudes (a1 and a2) using the
relation
ꢄꢆꢇꢈꢆꢉ ꢄꢈꢇꢈꢈ
τꢄꢅ
ꢀ
(2)
ꢊꢄꢆꢇꢆꢉ ꢄꢈꢇꢈꢋ
Fluorescence lifetimes of ALP in phosphate buffer (pH 7.4),
both in the absence and in the presence of QDs, were
measured.
UV−vis Spectroscopy
Figure 2. ALP activity at different concentrations of (A) CdTe
and (B) CdS; Inset: Plot of decrease in activity of ALP with
increasing concentration of (A) CdTe and (B) CdS where A0 and
A are the activity of ALP in absence and presence of QDs
respectively.
The interactions of ALP with Cysteine capped CdTe and CdS
quantum dots were also followed by UV-Vis spectroscopy
using Perkin Elmer Perkin Elmer Lambda UV/Vis/NIR
spectrometer. The concentrations of the QDs and ALP were
taken as mentioned in photoluminescence study.
the linear dependence of change in absorbance (ΔA405) with
time of catalysis followed. An important assumption in the
derivation of the Michaelis-Menten equation is that the
concentration of the enzyme-substrate complex ([ES]) instantly
reaches a steady state and remains unchanged during initial
velocity measurements. In this assay, this presents no problem
as the linear relationship between the increase in absorbance
and time holds for at least 10 min, twice the duration of the
assay. This prolonged linear phase is particularly useful if
reaction rates are measured in the presence of high enzyme
inhibitor concentrations.
FTIR Spectroscopy
For FTIR spectroscopic measurements, a film of ALP solution in
the absence and in the presence of different QDs of 10 nM
concentration was prepared on ZnSe window. The spectra
were recorded on Perkin Elmer spectrometer (Spectrum GX)
with a resolution of 2 cm−1 and scan range of 400–4000 cm−1.
Circular dichroism spectroscopy
The changes in 3D conformation of ALP due to interaction with
QDs were followed by circular dichroism spectroscopy. The CD
spectra were recorded on a Jasco J-600 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 the
measurements were made at 25 °C. The cuvette of an optical
path length of 2 mm was used. Scan was performed at
wavelengths between 190 and 250 nm, at a rate of 20 nm/min.
The enzyme concentration was 0.1 mg/mL.
The UV-vis spectra of the semiconductror QDs were also
studied for the same time period of 10 min. No change in
spectral distribution or absorbance indicates the stability of
the QDs for the time period studied.
When same concentration QDs of Cys-CdTe or Cys-CdS was
mixed with ALP solution before addition of substrate, the
measured catalytic activity declined to different extent with
varying type of QDs. To illustrate, we have observed that there
is almost 60% and 30% decrease in activity of ALP in the
presence of 10 nM Cys-CdTe and Cys-CdS, respectively. The
results show that CdS QDs can affect the enzymatic function to
relatively lesser extent when compared with CdTe. In addition,
to look into the influence of protein encapsulation of QDs, the
enzyme activity was measured in the presence of BSA-CdS. It
was observed that even in the presence of very high
Results and Discussion
The influence of QDs on the kinetics of ALP catalysed pNPP
hydrolysis reaction as
a function of concentration and
sequence of addition of QDs was investigated. Fig. 1 illustrates
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concentration of BSA-CdS QDs, such as 100 nM, the decrease to the mixture, the effect on catalytic function of enzyme is
in activity was about 16% only (Table S1). very less in comparison to that observed in the previous case
To investigate the concentration dependence of QDs on (Table 2). Thus, the observed results suggest that simultaneous
enzyme activity, we carried out enzyme assay in the presence addition of substrate and QDs to enzyme does not have much
of different concentrations of these nanoparticles. It was impact on catalytic function. It may be mentioned here, the
observed that with the increasing concentration of QDs, interaction of substrate, pNPP with Cys-CdTe or Cys-CdS QDs
enzyme activity decreases. A typical bar diagram of change in was investigated by UV-Vis spectroscopy. No change in the
activity of ALP in the presence of different concentration of spectral distribution or in absorbance suggests that there is no
cysteine capped CdTe and CdS QDs is shown in Fig. 2 and plot apparent interaction between the substrate and the QDs. It is
of change in enzyme activity (A/A0) vs concentration of these apparent that substrate can protect the possible QD-induced
QDs is shown in Fig. 2:inset. An apparent exponential decay of enzyme inhibition. In this context, it may be mentioned that
activity with increasing concentration of QDs suggests that substrate binding can protect enzyme from gastrointestinal
catalytic function is strongly affected with initial small amount deactivation as reported earlier.21,22
of QDs and these nanoparticles can interact with both active
and inactive molecules to almost equal extent.
Table 2. Variation in catalytic activity of ALP in the presence
In order to understand the effect of different QDs on the of QDs
kinetics of ALP, we have measured the Vmax and Km values of
ALP in the presence of QDs, in question. The kinetic
parameters obtained from the enzyme kinetic assay in the
presence of different concentrations of QDs are summarized in
Table 1.
Type of
QDs
Concentration
of QDs in nM
% of Activity remaining
QDs
incubated
with
QDs
incubated
with
Enzyme
100.0
Substrate
100.0
Table 1: Kinetic parameters of ALP in the presence of QDs
Cys-CdTe
0
when QDs were mixed with the enzyme before substrate
addition
10
50
40.0
13.7
10.5
100.0
70.4
56.4
50.0
90.0
70.2
50.4
100.0
98.7
90.5
80.7
Types Concentration
Km
Vmax
(µ moles min-1)
100
0
of
of QDs (nM)
(M)
Cys-CdS
QDs
Cys-
CdTe
10
0
5.6×10-4
3.1×10-4
1.6×10-4
5.6×10-4
4.2×10-4
3.0×10-4
44.70
22.97
10.73
42.40
33.75
28.27
50
10
50
0
100
Cys-
CdS
The protection of QD-induced enzyme activity by the substrate
is studied at different concentrations of pNPP in the presence
of 10 nM cysteine capped CdS or CdTe QDs. From Table 3 it
appears that at a particular concentration of QDs, the
protection of enzyme activity decreases with decreasing
concentration of the substrate.
10
50
Inspection of these kinetic data reveals that the value of Km
and Vmax of ALP decreases on interaction with the QDs. With
the increase in the concentration of the nanoparticles there is
a decrease in both Km and Vmax values. A low Km value indicates
a strong affinity between enzyme and substrate; however a
low Vmax value reflects that the enzyme does not convert much
substrate to product per unit of time when the enzyme is
saturated with substrate. From the observed results it may be
indicated that in the presence of QDs, although the substrate
affinity increases, the rate of hydrolysis decreases, which may
be a reflection of possible subtle conformational changes of
the enzyme molecules. It is observed that Km and Vmax of ALP
are maximally affected in the presence of Cys-CdTe
nanoparticles. However, the effect of BSA encapsulated CdS on
catalytic function of ALP is not significant.
Table 3: Protection of catalytic activity of ALP when 10 nM
QDs incubated with substrate
Type
of
Concentration
Activity remaining (%)
Protection
by
of pNPP (mM)
QDs
substrate
(%)
QDs
QDs
incubated incubated
with
Enzyme
45.4
with
Substrate
87.0
Cys-
11.2
2.24
38.6
20.5
16.5
22.9
13.3
6.1
CdTe
68.7
68.9
71.6
85.4
81.0
89.2
85.4
95.5
98.7
87.1
0.224
11.2
We have noticed when QDs were added to enzyme and the
mixture was subsequently added to the substrate, catalytic
activity of the enzyme is severely affected (Table 2). In
contrast, when the same concentration of QDs were first
added to substrate pNPP and the enzyme ALP was then added
Cys-
CdS
2.24
0.224
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Figure 3. Photoluminescence spectra of ALP in presence of
different concentrations (from a = 0 nM to h = 80 nM) Cys-
CdTe (A) and Cys-CdS (B) NPs, Inset: Stern-Volmer plot of PL Figure 4. (A) PL enhancement of Cys-CdS QDs and (B) PL
quenching of ALP (Here, I0 and I are the PL intensities of ALP in quenching of Cys-CdTe QDs in the presence of varying
the absence and in the presence of QDs, respectively).
concentrations of ALP
Further, when QDs were incubated with the substrate against concentration of QDs is linear in the range of 10-220
instead of the enzyme, even for high concentration of cys- nM (Fig. 3: inset), where it follows Stern-Volmer relation (eq.
CdTe QDs, Km and Vmax values were determined as 5.1×10-4
M
3),
and 41.2 µmoles min-1, which are close to the values of ALP in
the absence of QDs. The observed protection by the substrate
when added to enzyme simultaneously with QDs may be
attributed to the facts that diffusion rate of substrate (being
much smaller in size compared to QDs) is relatively high and
the substrate binds preferentially the specific catalytic sites of
enzymes.
I0
I
ꢀ 1 ꢃ KSVꢁNanoparticleꢂ
(3)
where, KSV is the Stern-Volmer constant.23 The Stern-Volmer
quenching constant (KSV) was determined to be 6.03×10-4 M−1
and 4.2×10-4 M−1 in case of CdTe and CdS, respectively. This
indicates weak binding of NP to ALP. The fluorescence
quenching of ALP by nanoparticles indicates modification of
tryptophan residues of ALP by interaction with the QDs. To
determine the nature of quenching, time-resolved
fluorescence spectroscopy was carried out. Figure S6 (see
supporting information) illustrates luminescence decay profile
of ALP, which follows bi-exponential decay kinetics. The
fluorescence lifetime data were analysed by considering
reduced chi-square value. The average lifetimes (τav) were
estimated to be 2.06±0.15 ns for ALP. The addition of CdTe
NPs to the ALP solution decreases the luminescence intensity,
but does not affect average lifetime suggesting the quenching
to be static one.
In order to understand the observed influence on the
catalytic functions of the enzyme molecules, the possible
interactions between these functionalized QDs and alkaline
phosphatase have been investigated by different spectroscopic
techniques. The optical absorption and emission spectra of the
synthesized cysteine capped CdS, and CdTe QDs and BSA
capped CdS QDs are shown in Supporting information Fig.S1,
S2 and S3, respectively.
The presence of nanoparticles in ALP solution was signalled
by intrinsic fluorescence quenching (FQ) of tryptophan residue
of ALP at 350 nm, when excited at 295 nm.
The FQ has been determined from the ratio of the
fluorescence intensities ALP in the absence of QDs (I0) and in
the presence of QDs (I). Figure 3 displays quenching of
luminescence of tryptophan residue of ALP with increasing
concentration of Cys-CdS or Cys-CdTe QDs. The plot of I0/I
We have also checked if luminescence of QDs is
affected by the presence of ALP. The presence of ALP in Cys-
CdS QDs solution caused significant enhancement in
luminescence (Fig. 4A). In contrast, the presence of ALP in Cys-
CdTe QDs solution caused luminescence quenching (Fig. 4B).
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Figure 6. FTIR spectra of ALP in the presence of different QDs
electronic defects by different groups can result in
enhancement of luminescence of QDs.27,28 Thus, it may
reasonably be assumed that surface defects in CdS QDs are
more dominant than that in CdTe QDs and consequently, the
luminescence increase due to surface passivation of CdS QDs
by attached ALP molecules overshadows the quenching of PL
through electron transfer. In addition, we observed no change
Figure 5. Different UV-Vis absorption spectra of ALP in the in luminescence of BSA-CdS QDs on addition of ALP, which is
presence of (a) 0, (b) 20 and (c) 80 nM cysteine capped CdTe consistent with the observed results on enzyme inhibition as
and CdS QDs
described earlier.
To look into the possible interactions at the ground state,
On having a closer look at the electron transfer involved in absorption behaviour of ALP in the presence of Cys-CdTe or
the quenching process, it appears that photo-excited CdTe QDs Cys-CdS NPs was investigated. It is evident from Fig.5, although
might give up the electron to ALP. The absorption of sufficient there was no change in spectral distribution of ALP; however,
light energy causes excitation of electrons from valence band addition of QDs decreased the absorbance at 280 nm
(highest occupied molecular orbital, HOMO) to the conduction corresponding to the absorption peak of tryptophan residue of
band (lowest unoccupied molecular orbital, LUMO).
ALP. The observed changes in absorption spectra suggest a
possible ground state binding of QDs with ALP resulting in
modification of tryptophan residue of ALP. Binding of protein
molecules with QDs in the ground state is reflected in FTIR
CdTe + hν → CdTe (e- + h+)
Such charge separation leads to formation of reactive electron- spectral data (figure 6). It can be noticed that on interaction
hole pair, which may migrate to the particle surface and get with Cys-CdTe or Cys-CdS QDs, band appearing at 1652 cm-1 in
de-excited, transferring the energy to the quencher molecules. ALP splits into two bands and is shifted to 1657 and 1636 cm-1
In quantitative terms, for the electron transfer to be feasible, in case of Cys-CdTe and 1656 and 1639 cm-1 in Cys-CdS. In case
the redox potential of the conduction band edge should be of BSA-CdS, this band is shifted to 1656 cm-1. This particular
more negative than that of the redox couple of the quencher band can be assigned to C=O, amide I (α-helix)29 stretching
species. The redox potential of tryptophan at pH7 is ~1.0 V24 mode of ALP and the observed shift could be due to
whereas the redox potential of the conduction band edge in coordination C=O group of ALP with the QDs. Again, band
CdTe nanoparticles lies in the range of −1.9 to −2.7 V.25 appearing at 1455 cm-1 of ALP gets shifted to 1467 cm-1 in case
Therefore, thermodynamic data favour the reduction of of Cysteine capped CdTe or CdS. This particular band is
tryptophan by photo-excited CdTe QDs. These new-formed assigned to amide II stretching mode of ALP and the observed
non-radiative channels for electron annihilation effectively shift could be due to coordination N-H amide II group of ALP
compete with the radiative electron-hole recombination with QDs. It is well known that tryptophan residue is essential
within the QDs giving rise to fluorescence quenching. The for catalytic activity of the enzyme. Thus, it may be inferred
redox potential of conduction band edge of CdS NPs is -0.85 from observed results that change in tryptophan residue is
V26 which is also more negative than redox potential of likely to contribute to loss in enzyme activity.
tryptophan. It is well known that passivation of surface
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Figure 7. CD spectra of ALP in presence of (A) Cys-CdTe, (B) Cys-CdS and (C) BSA-CdS QDs at 0, 10 and 50 nM
In addition, to explore the change in conformation of ALP by Cys-CdTe and the least affected by BSA-CdS. Again between
in the presence of QDs which may contribute to the QDs- Cys-CdS and BSA-CdS, enzyme catalysis is maximally affected in
induced inactivation of enzyme, the secondary structures of the presence of Cys-CdS. So, it is evident from these results
ALP in their action system were determined by CD that type of the nanoparticle as well as capping group play a
spectroscopy (Fig. 7) which is
a valuable spectroscopic role in modulating the enzyme catalytic activity. The
technique for studying protein and its complex. Circular conformational studies suggest that QDs can significantly
dichroism is observed when molecules absorb left and right change the secondary structure of ALP, which can lead to
circularly polarized light to different extent. The amide enzyme inactivation. It is demonstrated that substrate can
chromophore of the peptide bond in protein dominates their protect the possible QD-induced enzyme modifications. In
CD spectra below 250 nm. In α helix structure of protein, a addition, it has been shown that protein-encapsulated QDs can
negative band near 220 nm is observed due to the strong hardly affect catalytic functions of enzyme molecules. The
hydrogen-bonding environment of this conformation, a second observed results will help in developing the strategy for
transition at 190 nm splits into a negative band near 208 nm different applications of QDs in biological systems, such as
and a positive peak near 192 nm. The CD spectra of β-sheet drug delivery, cellular imaging, etc.
display a negative band near 216 nm, a positive band between
195 and 200 nm, and a negative band near 175 nm.30,31 CD Acknowledgements
spectra of pure ALP and ALP in the presence of QDs of The authors are grateful to the Prof. S. Basu of Saha Institute
different concentrations are presented in Fig. 7. From the of Nuclear Physics for providing Circular dichroism and lifetime
figure, it can be seen that, the CD curve of ALP is different measurement facilities. One of the authors (D. Ghosh) is
from that obtained in the presence of QDs. In the presence of grateful to CSIR, Govt of India, for the award of senior
10 nM concentration of the QDs, the CD spectra of the enzyme Research Fellowship. The authors, C. N. Roy, S. Mondal and S.
changed noticeably and the average molecular ellipticity at Kundu are thankful to the University Grants Commission for
222 nm decreased. The circular dichroism signal measured at NET fellowship. One of the authors, Susmita Maiti, is grateful
222 nm is mainly due to the helical structure of protein to DST for inspire fellowship.
backbone. It was observed that the average molecular
ellipticity at 222 nm decreased with increase in nanoparticle
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Graphical Abstract
Modulation of Catalytic Functionality of Alkaline Phosphatase Induced by Semiconductor
Quantum Dots: An Evidence of Substrate Mediated Protection