Supplementary material
ZnO Nanoparticle -Protein Interaction: Corona Formation with
Associated Unfolding
A. K. Bhunia1, P. K. Samanta2, S. Saha1, T. Kamilya3,a
1
Department of Physics & Technophysics, Vidyasagar University, Paschim Medinipur, -721102,
India
2
Department of Physics, Ghatal R.S. Mahavidyalaya, Paschim Medinipur-721212, India
3
Department of Physics, Narajole Raj College, Paschim Medinipur-721211, India
Hill equation
We have characterized the strength and association cooperativity of the adsorption of the BSA
onto the ZnO NPs by the Hill coefficient (n). To study the Hill coefficient we have fitted the
fluorescence data into Hill equation:
Q = (I0-I)/I0
(1)
Q/Qmax = [NP] n/(knD + [NP] n)
(2)
Here I and I0 are fluorescence intensities in absence and in presence of nanoparticles,
respectively. Qmax is the saturation value of Q, relative intensity. kD is the protein-nanoparticle
equilibrium constant (dissociation constant), and n, the Hill coefficient. In favor of positive
cooperative reaction, n›1, reveals that once one protein molecule is bound to the NPs, its affinity
for the nanoparticle gradually increases in a superlinear fashion. However, in case of negative
cooperative reaction, n‹1, the binding strength of the protein with the NPs becomes weaker as
further proteins adsorb. As well as, for a noncooperative association, n=1, the affinity of the
proteins to nanoparticle does not depend on whether other protein molecules are already bound
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and Q follows the Langmuir adsorption equation. The “binding constant” K represent the
reciprocal of kD.
TABLE-1
Table-1 Fitting parameters of Fig. 3 and Fig. S4 fitted by equation 2, 3
BSA
TRY
Qmax
0.38
0.22
n
1.18
1.95
kd
0.03
0.02
K
33.33
50.00
R2
0.999
0.999
CD Analysis
The value of α and β component of BSA is obtained by K2D2 fitting. The fitting is done online
by (http://www.ogic.ca/projects/k2d2/).
FTIR Spectroscopy
FTIR spectroscopy is a valuable tool to study the unfolding protein. The unfolding, intra and
intermolecular associations of protein were studied by monitoring the peak positions and width
Supplementary material
of amide bands within a fixed range by FTIR analysis. The FTIR absorption spectra of amide –I
band (1600-1700 cm-1) due to C=O stretching modes of peptide linkages of BSA. The vibrational
energies of the carboxyl group depend in reality on the different conformations of the protein,
such as -helix, -sheet, -turns and intra and intermolecular aggregates. The determination and
the assignment of the spectral components of the amide- I band can then give the information on
the protein secondary structure. A Gaussian multiple-peak-fitting procedure has been employed
to the amide I band of FTIR spectra by using Microcal Origin 7.5 software after baseline
correction. The quality of the fitting was evaluated based on the χ2 values (on the order of 10-6)
and the square of the correlation coefficient (R2) values 0.999. The multiple peaks resulted from
the deconvolution will provide us the conformations of BSA in different condition and to
identify its component and, in particular, to determine the corresponding peak frequencies. The
percentage area of the deconvoluted peaks gives the relative area of the components. It is worth
noting that, in all the spectra considered in the present work, the maximum number N of the
components which can be safely identified in the deconvoluted amide I band does not exceed
N=5 to have a meaningful fitting.
Table-2 Fitting parameters of amide-I band of (A) pure BSA and (B) BSA-ZnO NPs complex.
Area (%) = 100 represents the total area under curve.
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TABLE-2
Position (cm-1)
Area (%)
Conformers
A
B
A
B
A1
17.34
17.68
1605.2
1623.0

12.50
07.69
1626.3
1638.9

62.82
27.20
1650.7
1649.1
T
04.68
03.13
1674.2
1669.8
A2
02.66
44.30
1688.1
1680.2
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Fig. S1: (Color online) Absorption spectra
(a) Pure BSA, (b) pure ZnO, (c) BSA-ZnO NPs complex with CBSA=0.01 mg/mL and CZnO= 1
mg/mL.
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Fig. S2: (Color online) Absorption spectra of BSA-ZnO NPs complex with CBSA=0.01 mg/mL
and CZnO= 0.01 (a) and 0.03 mg/mL (b), respectively. Baseline is done w. r. t. ZnO in these
cases. (c) Absorption of pure BSA with CBSA=0.01 mg/mL for comparison.
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Fig. S3: (Color online) Graphical representation of interaction and conjugate formation of ZnO
NPs and BSA.
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Fig. S4: (Color online) Fluorescence spectra of BSA and BSA-ZnO complex (a) Pure TRY with
concentration of TRY (CTRY) =0.01 mg/mL, (b-d) TRY-ZnO NPs complex with CTRY=0.01
mg/mL and CZnO= 0.01, 0.03, 0.06, 0. 1 mg/mL. Inset shows the fluorescence quenching plot of
TRY with CBSA=0.01 mg/mL and CZnO= 0.01, 0.03, 0.06, 0. 1 mg/mL. Red lines represent the
curves fitted by Eq. 2.
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Fig. S5: (Color online) CD Spectra
(a) Pure BSA with CBSA=0.01 mg/mL (b): BSA-ZnO NPs complex with CBSA=0.01 mg/mL and
CZnO=0.01 mg/mL.
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Fig. S6: (Color online) FTIR Spectra
(a) Amide-I band of BSA-ZnO NPs complex. Green lines represent the curves fitted by multiple
peaks fitting by Microcal Origin. (b): Bar diagram of fitting result.