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Ecofriendly gold nanoparticles – Lysozyme interaction: Thermodynamical perspectives
Accepted Manuscript Ecofriendly gold nanoparticles Thermodynamical perspectives
–
Lysozyme
interaction:
Swarup Roy, Shailendra K. Saxena, Suryakant Mishra, Priyanka Yogi, P.R. Sagdeo, Rajesh Kumar PII: DOI: Reference:
S1011-1344(17)30063-5 doi: 10.1016/j.jphotobiol.2017.08.009 JPB 10944
To appear in:
Journal of Photochemistry & Photobiology, B: Biology
Received date: Revised date: Accepted date:
12 January 2017 6 June 2017 5 August 2017
Please cite this article as: Swarup Roy, Shailendra K. Saxena, Suryakant Mishra, Priyanka Yogi, P.R. Sagdeo, Rajesh Kumar , Ecofriendly gold nanoparticles – Lysozyme interaction: Thermodynamical perspectives, Journal of Photochemistry & Photobiology, B: Biology (2017), doi: 10.1016/j.jphotobiol.2017.08.009
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ACCEPTED MANUSCRIPT Ecofriendly gold nanoparticles – lysozyme interaction: thermodynamical perspectives
Swarup Roy1, Shailendra K Saxena1, Suryakant Mishra1, Priyanka Yogi1, P.R. Sagdeo1 and
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Rajesh Kumar1, 2, *
Discipline of Physics and MEMS, Indian Institute of Technology Indore, Simrol, India 453552
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Centre of Bioscience and Biomedical Engineering, Indian Institute of Technology Indore,
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*
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Simrol, India 453552
Email: [email protected]
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ACCEPTED MANUSCRIPT Abstract In the featured work interaction between biosynthesized gold nanoparticles (GNP) and lysozyme (Lys) has been studied using multi-spectroscopic approach. A moderate association constant (Kapp) of 2.66×104 L/mol has been observed indicative of interactive nature. The binding constant (Kb) was 1.99, 6.30 and 31.6 ×104 L/mol at 291, 298 and 305 K respectively and the number of binding sites (n) was found to be approximately one. Estimated values of thermodynamic parameters (Enthalpy change, ΔH = 141.99
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kJ/mol, entropy change, ΔS = 570 J/mol/K, Gibbs free energy change, ΔG = -27.86 kJ/mol at 298K)
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suggest hydrophobic force as the main responsible factor for the Lys-GNP interaction and also the process of interaction is spontaneous. The average binding distance (r = 3.06 nm) and the critical energy
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transfer distance (Ro = 1.84 nm) between GNP and Lys was also evaluated using Förster’s non-radiative energy transfer (FRET) theory and results clearly indicate that nonradiative type energy transfer is
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possible. Moreover, the addition of GNP does not show any significant change in the secondary structure of Lys as confirmed from circular dichroism (CD) spectra. Furthermore, NMR spectroscopy also
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indicates interaction between Lys and GNP. The resulting insight is important for the better understanding
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of structural nature and thermodynamic aspects of binding between the Lys and GNP.
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Keywords: Lysozyme; Green Synthesis; GNP; Spectroscopy; Hydrophobic force
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ACCEPTED MANUSCRIPT Introduction Nanoscience and nanotechnology are growing very rapidly in all the discipline of science and engineering. There is a rapid development in nanoscience in the last couple of decades and the research work continues for further development in this field. Day-to-day more novel nanomaterials are getting synthesized and research work is going on to apply these various nanomaterials for the development of consumer products as well as in the advancement of medical science. There has been already lots of
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development in nanomaterials which have various applications but the toxicity of these nanomaterials has
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been the concerning issue for the further use of nanomaterials.
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Among the metallic nanomaterials, Gold nanoparticle (GNP) is a key material of interest as it has been found to be less toxic, inert and has enormous application capabilities in different fields. Synthesis of
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GNP is possible using chemical [1,2] and biological methods[3–9] but the shortcomings of chemical methods include use of toxic chemicals, being expensive and hazardous to the environment [10]. Among all available methods for synthesis of nanomaterials, most preferable one is green synthesis. The specialty
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of green synthesis is in this process there is no requirement of reducing agent as well as capping/stabilizing agent. Green synthesized nanoparticles can quickly form conjugates with proteins
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through either covalent bonds or physical interactions, and these conjugates have been extensively used in
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biomedical fields [11–13].
Understanding in interaction (interacting forces, binding sites, binding affinity, and conformational changes) of nontoxic nanoparticles with bio-macromolecules are very important for effective use of
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nanoparticles in biomedical application [14,15]. After entry of nanoparticles in blood stream they may quickly interact with plasma proteins leading to an alteration in the native conformation or function that
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may further result in astonishing biological reactions to reduce toxicity [16]. The interactions between protein and green synthesized nanoparticles have not been completely explored. We have selected
proteins.
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lysozyme for the interaction study due to the natural abundance and it is one of the most widely used
Lysozyme (Lys) is a 14.6 kDa small monomeric globular protein which consists of 129 amino acid residues containing 6 tryptophan (Trp) and 3 tyrosine residues & 4 disulfide bonds [17–19]. In Lys, six Trp residues are located at the substrate binding sites, of which two are in the hydrophobic matrix box, while the lone Trp residue is separated from the others [20–23]. Trp-62 and Trp-108 have considered the most dominant fluorophores in Lys among the Trp residues [20,24]. From the crystal structure, it has been observed that Trp-62 and Trp-108 residues are placed close to the active substrate binding site [25]. Furthermore, Lys has also many physiological and pharmaceutical functions such as, antibacterial,
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ACCEPTED MANUSCRIPT antivirus as well as detumescence and as a results extensively used in the pharmaceutical and food fields [19]. Another very significant role of Lys is its ability to carry drugs [26]. It is already known that Lys is capable of reversible binding to various endogenous and exogenous compounds [27].Understanding the binding characteristics of medically important GNP with Lys may expand the development of possible delivery modes for facilitating availability at required sites, leading to therapeutic usage. Recently, reports are available on the interaction of proteins with various
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nanoparticles in pure form or functionalized with a suitable group [18,21,28–37]. Lys is very useful to
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carry drugs, such as antibiotics to treat inflammation, abscess, stomatitis and rheum [38]. Accordingly,
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the interaction of Lys with biologically important green synthesized GNP is of great interests. Here in this featured work, we have used spectroscopic techniques like UV–visible, intrinsic fluorescence, and circular dichroism to describe the interaction between Lys and GNP. Systematic study of the binding
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mechanism between GNP and Lys with respect to the binding sites, the binding distance between the two and the effect on the secondary structure of Lys has been highlighted here.
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2. Experimental Details
Hen egg white lysozyme was used as received commercially from Sigma-Aldrich, USA. Tris–base was
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purchased from Merck, Germany. All the other chemicals were of analytical reagent grade and double distilled water was used throughout. Emission spectra were taken using a Fluoromax-4p
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Spectrofluorimeter from Horiba Jobin Yvon (Model: FM-100) well equipped with attached temperature controller. The absorption spectra were obtained from a Cary 60 UV-Vis Spectrophotometer Agilent
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Technologies. For the time resolved studies, a picosecond time correlated single photon counting (TCSPC) system from Horiba Yovin (Model: Fluorocube-01-NL) was used. Circular dichroic spectra
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were analyzed by a Jasco J-815 CD spectrometer. NMR spectra were examined by an Ascend Bruker BioSpin International AG spectrometer. All the details of the experimental and computational procedures
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are elaborated in the supporting information (SI). 3. Results and Discussion 3.1 Effect of GNP concentration on the Lys stability As the intensity and position of the surface plasmon band of colloidal gold are related to the size, shape, and dispersity of the GNP, UV-Vis absorption spectroscopy can be used for characterizing the optical properties of GNP. Here an aqueous solution of pure green synthesized GNPs shows a narrow and symmetric absorption peak at approximately 530 nm (Fig. 1), which agrees with an earlier report [39] and indicates that the GNPs are monodispersed and spherical. The UV–Vis absorption spectra of Lys in presence of increasing concentration of GNP are measured under stimulative physiological condition. In 4
ACCEPTED MANUSCRIPT the presence of increasing concentration of GNP (0, 2, 4, 6, 8, 10, 12, 14 and 16 µM) the overall absorbance of pure Lys gradually increases (Fig. 2) after the conjugation with GNP with a little variation in the shape of the band. This increase in intensity is likely due to the creation of ground state complex between Lys and GNP.
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1.2
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0.8
0.0 300
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0.4
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Absorbance
535 nm
400
500
600
700
800
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Wavelenght (nm)
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1.5
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Fig. 1 UV-Vis absorption spectrum of green synthesized GNP
16M
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Absorbance
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1.2
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0.9
0.6
0.3
0.0 250
275
300
325
Wavelength (nm)
Fig. 2 Spectrophotometric interaction of Lys in presence of GNP (0, 2, 4, 6, 8, 10, 12, 14 and 16 µM)
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ACCEPTED MANUSCRIPT To know the extent of interaction between Lys and GNP apparent association constant (Kapp) has to be calculated. The ground state complex forms as follows. ,
(1)
The Kapp value has been calculated by following Benesi and Hildebrand [40] equation. ,
(2)
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Here, Aobs is the absorbance of the Lys solution containing different concentrations of GNP at 280 nm, α
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is the degree of association between Lys and GNP, εLys and εC are the molar extinction coefficients at the
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defined wavelengths for Lys and the complex formed respectively, C0 is the initial concentration of Lys and la is the optical path length. Eq. 2 can be now expressed as Eq. 3
(3)
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,
Here, A0 and AC are the absorbance of Lys and the complex at 280 nm respectively with the concentration
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of C0. At relatively higher GNP concentration, α can be equated to (Kapp [GNP])/(1+ Kapp [GNP]) where
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1/(Aobs-Ao)
(4)
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25
20
,
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[GNP] is the concentration of GNP in mol/L. Thus Eq. 3 now converts to Eq.4 as follows:
10
5
0 0.00
1.80x105
3.60x105
5.40x105
1/[GNP]
Fig. 3 Benesi-Hildebrand plots for Lys in presence of GNP, Kapp = 2.66×104 L/mol
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ACCEPTED MANUSCRIPT A plot of 1/(Aobs−A0) vs 1/[GNP] yield a linear plot (Fig. 3) and from this plot (R value = 0.9999, R2 value = 0.9998) the value of Kapp is found to be 2.66×104 L/mol which indicates the formation of a moderate complex between Lys and GNP. This means that interaction studies must be carried out in detail to get the comprehensive information of Lys-GNP interaction. 3.2. Fluorescence quenching studies of Lys in presence of GNP Many techniques are available to study the interaction between nanomaterials and Lys, but the utmost
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suitable one is fluorescence quenching. The fluorescence spectra (Fig. 4) have been recorded for Lys
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alone and in presence of different GNP concentrations of (0, 2, 4, 6, 8, 10, and 12 µM. Figure 4 clearly
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shows that the Lys has a strong emission band at 350 nm when excited with 280 nm wavelength. The intensity of this fluorescence band decreases steadily with increasing concentration of GNP. This alteration in the fluorescence characteristic of the Lys suggests the presence of binding between GNP and
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Lys to form a certain complex.
12M
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Intensity (a.u)
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320
360
400
440
Wavelength (nm)
Fig. 4 Fluorescence spectra of Lys in the presence of GNP (0, 2, 4, 6, 8, 10, and 12 µM) 3.3. Binding mechanisms between Lys and GNP The fluorescence quenching generally occurs through either dynamic or static mode and classified depending on the way of interaction between quencher and biomolecule [41,42]. The fluorescence quenching data at variable temperatures can be analyzed using very well-known Stern–Volmer equation as follows: ,
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ACCEPTED MANUSCRIPT Here, F0 and F denote the steady-state fluorescence intensities in the absence and presence of GNP respectively, KSV is the Stern–Volmer quenching constant, [Q] is the concentration of GNP, Kq is the quenching rate constant of the biological macromolecule, and τ0 is the average lifetime of the molecule without any quencher. The Stern–Volmer plot of F0/F vs [Q] is presented in Fig. 5 and slope, after linear regression of this plot, yields the Stern–Volmer constant (Table 1).
coefficient, R2= Coefficient of determination) R2
R
KSV (L/mol) ×104
291
0.9994
0.9997
4.29
298
0.9982
0.9992
3.97
305
0.9878
0.9951
4.07
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3.54 3.28 3.36
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1.6
291K 298K
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Fo/F
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1.4
1.2
Kq (L/mol/s)×1013
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T(K)
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Table 1: Quenching rate constants and Stern Volmer constant for Lys binding to GNP (R= Correlation
305K
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1.0
4.0x10-6
8.0x10-6
1.2x10-5
[Q]
Fig. 5 Stern–Volmer curves at 293, 300, 303 and 310 K of GNP–Lys It can be overserved that Kq increases with increase in temperature indicating that fluorescence quenching occurs due to specific binding between GNP and Lys by forming complexes which are stabilized at higher temperatures. The value of Kq can be calculated by considering conventional fluorescence lifetime of the biopolymer of 10−8 s. The lifetime value of Lys as obtained using time resolved experiment was used to determine Kq value. In the present case (Table 1) the quenching constant Kq value suggests that the 8
ACCEPTED MANUSCRIPT interaction between the Lys and GNP is taking place through static quenching. Du & Xia [43] previously reported the interaction between chemically synthesized GNP and Lys which was through static quenching mode and the observed results corroborates our findings. It has been observed that KSV, Kq as obtained here in case of green synthesized GNP are 104 times and 103 times lower respectively as compared to chemically synthesized GNP. So, it can be concluded that chemically synthesized GNP has more quenching ability than biologically synthesized GNP towards Lys. KSV, Kq obtained in case of Lys-
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silver nanoparticle interaction was 5.66 ×103L/mol and 9.94×1011 L/mol/s respectively[44], which are
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also comparable to our findings. This result is also suggests that quenching takes place only due to proper interaction rather than the collision effect. For confirmation of the quenching phenomena as a result of
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Lys-GNP interaction further studies has been carried out in the following section. 3.4. Time resolved fluorescence measurement of Lys with GNP
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The fluorescence lifetime measurement is the most definitive method for discriminating static and dynamic quenching processes [45]. Time resolved fluorescence lifetime measurement has been carried
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out to validate static quenching mechanism for Lys-GNP interaction. The time resolved fluorescence decay curve of Lys in presence of GNP is presented in Fig. 6. As can be seen from Fig. 6, the
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fluorescence decay curve of Lys is single exponential with a lifetime value of τ = 1.21 ns. Details of
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results are given in Table S1 in the SI.
1000
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6000
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Counts
8000
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10000
Lys+8M GNP 600
400 1.02x10-8
4000
2000
Lys
800
1.10x10-8
1.17x10-8
Lys Prompt
0 7.50x10-9
1.50x10-8
2.25x10-8
Time (s)
Fig. 6 Time-resolved fluorescence decays of Lys (0.1 µM) in the presence of varying concentrations of GNP. The line profile in black represents the instrument response function (λex 280 nm, λem 345 nm).
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ACCEPTED MANUSCRIPT After addition of GNP, the excited state lifetime of Lys showed no characteristic change (τ = 1.31 ns). It is well known that the static quenching mechanism is generally associated with stable fluorescence lifetime values, whereas, in case of excited-state quenching the fluorescence lifetime values are altered significantly. From the above analysis, it is apparent that the unaltered fluorescence lifetime of Lys in the presence of GNP is mainly initiated by static quenching process [46–48]. 3.5. Number of binding sites and binding locality
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In case of quenching interaction, relationship between the fluorescence intensity and the quencher
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,
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concentration can be derived from the following equation [49].
(6)
where, L is the biomolecule with a fluorophore, Q is the quencher; Qn..L is the quenched biomolecule and
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the resultant constant K is given by the following equation.
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,
(7)
If the overall amount of biomolecules (bound and unbound with the quencher) is L0, then, (8)
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where, [L] is the concentration of unbound biomolecules, the relationship between fluorescence intensity
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and the unbound biomolecules is as following.
(9)
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,
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Now from using above equation, one can write the following equation. ,
(10)
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where, Kb is binding constant of GNP with Lys and n is the number of binding site. The values of Kb and n can be determined from the linear plot between log [(F0 –F)/F] vs log [Q] as shown in Fig. 7. The values of Kb and n at 291, 298, and 305 K temperatures for Lys-GNP are shown in Table 2. Table 2 Binding constants and binding sites for Lys binding to GNP T(K)
R2
R
Kb (L/mol)×104
n
291
0.9999
0.9998
1.99
0.95
298
0.9994
0.9997
6.30
1.02
305
0.9978
0.9991
31.6
1.19 10
ACCEPTED MANUSCRIPT The results demonstrated that there is a strong binding force between GNP and Lys, and single binding site would be formed. The Kb as obtained here in case of interaction between Lys and green synthesized GNP are 105 times lower as compare to chemically synthesized GNP[43]. These results clearly indicate that chemically synthesized GNPs have greater ability to form strong binding with Lys compared with green synthesized GNPs. In case of Lys-silver nanoparticles interaction, Kb was found to be 1.26 ×104 L/mol and the value of n was approximately equal to 1, these results are also analogues to our
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experimental findings [44]. To understand the details of thermodynamic properties of Lys and GNP
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interaction the following study has been carried out.
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291K
-0.8
298K
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log[(Fo-F)/F]
-0.4
305K
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-5.8
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-1.2
-5.4
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-5.0
-4.8
log[Q]
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Fig. 7 Fluorescence quenching [(F0-F)/F] vs log [Q] plots for Lys in the presence of the GNP 3.6. Thermodynamic parameters and nature of the binding forces
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Four types of forces namely hydrophobic forces, electrostatic interaction, hydrogen bond, and van der Waals forces are found in case of interactions between small molecules and biological macromolecules. The interaction model is as following [50,51], 1) ΔH > 0 and ΔS > 0, hydrophobic forces; 2) ΔH < 0 and ΔS < 0, van der Waals interactions and hydrogen bonds; 3) ΔH < 0 and ΔS > 0, electrostatic interaction. If the enthalpy change has not changed significantly within a temperature range, then ΔH and ΔS can be calculated using following Van’t Hoff equation. ,
(11)
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ACCEPTED MANUSCRIPT where Kb is analogous to the binding constant at the corresponding temperature, R is gas constant. The values of ΔH and ΔS have been obtained from the ln Kb vs 1/T plot (Fig. 8). The free energy change (ΔG) can be obtained from the following equation. ,
(12)
Using Eq. 11 and Eq. 12 the values of ΔG, ΔH and ΔS were obtained as shown in Table 3. From the negative value of ΔG it appears that the process of interaction is spontaneous. Positive values of ΔH and
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ΔS indicate that the major contribution in complexation is hydrophobic force [52]. Similar results of
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thermodynamic parameters have been found in case of interaction between chemically synthesized GNPs
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and Lys [43]. From the resulting insight, it is clear that the interaction between GNPs (Chemical & biological) and Lys takes place by means of hydrophobic effect. According to the thermodynamic data,
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the formation of the Lys–GNP complex is in favor of formation of the aforesaid complex.
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11
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lnK
12
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3.28x10-3
3.34x10-3
3.40x10-3
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1/T(K-1)
Fig. 8 Van’t Hoff plot for the interaction between Lys and GNP. Table 3 Thermodynamic parameters for the binding of GNP to Lys ΔS (J/mol/K)
ΔG (kJ/mol)
141.99
570
-23.87
298
141.99
570
-27.86
305
141.99
570
-31.88
T(K)
R2
R
291
0.9974
0.9993
ΔH (kJ/mol)
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ACCEPTED MANUSCRIPT Thermodynamical parameters have been studied in various reports dealing with interaction to get an insight about the binding force. Existence of van der Waals interaction & hydrogen bonding and their significant contribution in the binding process have been observed in case of Lys-rhodamine b and Lysdrospirenone [48,53]. Electrostatic forces played a major role in the interaction between Lys and cefepime hydrochloride [54], electrostatic and hydrophobic force was the most important force to stabilize the Lysheparine complex [55]. In case of interactions between cyclophosphamide and aspirin with Lys results
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found using DFT calculations suggested that Trp 62 has the most affinity to cyclophosphamide and
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aspirin drugs as the ligands [56]. The binding of chelerythrine to Lys also suggested Trp 62 in the βdomain of the protein was closer to the binding site[57]. Accordingly, it is expected that in the present
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case of GNP-Lys interaction Trp62 may have an important role. To understand structural effect of Lys in presence of GNP the following study has been carried out.
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3.7. Circular dichroism spectra
To get an idea of structural change of Lys, CD spectral measurements at room temperature have been
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carried out in absence and in presence of GNP (Fig. 9). There are two strong negative bands in the UV region at 208 and 222 nm and this contribution are due to n→Π* transfer for the peptide bond of α-helical
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form.
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-40
-80
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(mdegcm2mol-1)
0
Lys+20M GNP
-120
Lys+10M GNP Lys
200
220
240
260
Wavelength (nm)
Fig. 9 CD spectra of Lys in the presence of different concentration of GNP (0, 10, and 20 µM)
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ACCEPTED MANUSCRIPT The CD results are expressed in terms of mean residue ellipticity (MRE) in degree-cm2/mol according to the following equation [58,59]. ,
(13)
where, Cp is the concentration of Lys in µM, Ɵ is observed rotation in degrees, l is the path length in cm and na is the number of amino acid residues of protein (129 for Lys). The α-helical contents of Lys have
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been estimated from MRE values at 222 nm using Eq. 14 and then cross checked by K2D software
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[60,61].
(14)
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It is observed that the percentage of helicity of Lys is 40% and in presence of GNP helicity is almost
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unchanged and this is due to the fact that the α-helical structure of Lys is unaffected and may induce very small or no conformational changes. This observation strongly indicates that the binding of GNP to Lys
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induces little change in conformation of Lys and the secondary structure of Lys retains mainly its α-helix character.
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3.8. NMR studies
NMR results show that the interaction of GNP with Lys take place as indicated through the line widths
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and chemical shifts of the 1H-NMR signal (Fig. S1 in SI). This result also shows that new signals were formed by means of the significant interaction between the Lys and GNP [62]. The comparative 1H-NMR
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spectra of Lys in the presence and absence of GNP were given in Fig. S1 (a–b). After addition of GNP to Lys, there has been no significant shift in signal rather new signals are formed which indicates interaction
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between Lys and GNP. Resulting insights are clearly indicative of interaction between Lys and green GNP. To understand the energy transfer process of Lys-GNP complex following study has been carried
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out.
3.9. Energy transfer between Lys and GNP In process of Forster’s resonance energy transfer, donor fluorophore (Lys) in its excited state transfers energy to an acceptor (GNP) molecule through nonradiative dipole–dipole coupling [16]. The efficacy of this energy transfer has been used for estimation of distance (r) between GNP and fluorophore Lys [63]. Energy transfer process mainly depends on the magnitude of overlap of emission spectrum of the donor with absorption spectrum of the acceptor. The emission spectrum of Lys is well overlapped with absorption spectrum of GNP which indicates that there is a possibility of energy transfer from excited state of Lys to GNP (Fig.10).
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Intensity (a.u)
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Emission of Lys
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Absorption of GNP
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600
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Fig. 10 Overlap plots of fluorescence spectra of Lys with absorption spectra of GNP. The efficacy (E) of FRET is inversely proportional to the sixth power of distance between donor and
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acceptor [64].
(15)
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The magnitude of R0 is dependent on the spectral characteristics of the donor emission and acceptor
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absorption of the molecules. R0 is the critical distance when the transfer efficiency is 50 % and given by the following equation.
(16)
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,
where φ is the fluorescence quantum yield of the donor, N is the refractive index of the medium, K2 is the spatial orientation factor of the dipole, J is the overlap integral of the fluorescence emission spectrum of the donor and the absorption spectrum of the acceptor as given by following equation. ,
(17)
where F(λ) is the fluorescence intensity of the donor and ε(λ) is the molar absorptivity of the acceptor when the wavelength is λ.
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ACCEPTED MANUSCRIPT Using Eqs. 15-17 along with the values for φ = 0.15, K2 = 2/3 and N = 1.36 [65,66], the values of R0 and r were calculated to be 1.84 nm and 3.06 nm, respectively and J = 1.40×10-15 cm3 L/mol, E = 0.045. The result shows that GNP is strong quencher and they are situated in close proximity to the Lys. The average distances between a donor fluorophore and acceptor fluorophore are in the range of 2–7 nm [65] which denotes that the energy transfer occurs between Lys and GNP. This also indicates that the fluorescence quenching of Lys was a non-radiative transfer process.
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4. Conclusion
Spectroscopic studies reveal the quenching of Lys fluorescence through static quenching processes
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induced by gold nanoparticles (GNPs). A moderately strong binding affinity between GNP and Lys has also been observed along with the existence of a single binding site in Lys for GNP. A thorough analysis
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of thermodynamic data clearly indicates that the Lys-GNP interaction is dominated by hydrophobic forces. The binding process is thermodynamically favorable and spontaneous. A non-radiative transfer of energy between GNP and Lys, 3.06 nm apart, is also revealed. GNP addition to Lys does not make any
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significant changes in Lys secondary structure in terms of decrease in α-helix. The interactive nature of Lys-GNP complex is not strong enough to induce significant structural modifications to make the
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complex to be appropriately used in certain applications. The binding phenomenon of green synthesized nontoxic GNP with Lys is of great importance in pharmacy and biochemistry. Furthermore, it is expected
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that fluorescence technique is a promising tool for designing GNP based biomaterials and nano drugs that will be noteworthy for present day’s medical research.
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Acknowledgement
Authors are thankful to Department of Science and Technology (DST) project (No. SB/S2/CMP-012-
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2014) for the financial support and also SIC facility of Indian Institute of Technology Indore for the instrumental facility. Authors also thank MHRD, Govt. of India for fellowship. Valuable discussion with
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Dr. Hem C. Jha (IIT Indore) is also greatly acknowledged. Supporting Information Experimental and computational details, TCSPC results in Table S1, and Fig.S1. Notes The authors declare no competing financial interest.
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Highlights
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Graphical abstract
1. Lys fluorescence is quenched by green GNP through static quenching. 2. Hydrophobic forces stabilize the Lys-GNP complex. 3. GNP does not induce conformational changes in Lys.
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Lysozyme
interaction:
Swarup Roy, Shailendra K. Saxena, Suryakant Mishra, Priyanka Yogi, P.R. Sagdeo, Rajesh Kumar PII: DOI: Reference:
S1011-1344(17)30063-5 doi: 10.1016/j.jphotobiol.2017.08.009 JPB 10944
To appear in:
Journal of Photochemistry & Photobiology, B: Biology
Received date: Revised date: Accepted date:
12 January 2017 6 June 2017 5 August 2017
Please cite this article as: Swarup Roy, Shailendra K. Saxena, Suryakant Mishra, Priyanka Yogi, P.R. Sagdeo, Rajesh Kumar , Ecofriendly gold nanoparticles – Lysozyme interaction: Thermodynamical perspectives, Journal of Photochemistry & Photobiology, B: Biology (2017), doi: 10.1016/j.jphotobiol.2017.08.009
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ACCEPTED MANUSCRIPT Ecofriendly gold nanoparticles – lysozyme interaction: thermodynamical perspectives
Swarup Roy1, Shailendra K Saxena1, Suryakant Mishra1, Priyanka Yogi1, P.R. Sagdeo1 and
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Rajesh Kumar1, 2, *
Discipline of Physics and MEMS, Indian Institute of Technology Indore, Simrol, India 453552
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Centre of Bioscience and Biomedical Engineering, Indian Institute of Technology Indore,
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Simrol, India 453552
Email: [email protected]
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ACCEPTED MANUSCRIPT Abstract In the featured work interaction between biosynthesized gold nanoparticles (GNP) and lysozyme (Lys) has been studied using multi-spectroscopic approach. A moderate association constant (Kapp) of 2.66×104 L/mol has been observed indicative of interactive nature. The binding constant (Kb) was 1.99, 6.30 and 31.6 ×104 L/mol at 291, 298 and 305 K respectively and the number of binding sites (n) was found to be approximately one. Estimated values of thermodynamic parameters (Enthalpy change, ΔH = 141.99
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kJ/mol, entropy change, ΔS = 570 J/mol/K, Gibbs free energy change, ΔG = -27.86 kJ/mol at 298K)
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suggest hydrophobic force as the main responsible factor for the Lys-GNP interaction and also the process of interaction is spontaneous. The average binding distance (r = 3.06 nm) and the critical energy
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transfer distance (Ro = 1.84 nm) between GNP and Lys was also evaluated using Förster’s non-radiative energy transfer (FRET) theory and results clearly indicate that nonradiative type energy transfer is
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possible. Moreover, the addition of GNP does not show any significant change in the secondary structure of Lys as confirmed from circular dichroism (CD) spectra. Furthermore, NMR spectroscopy also
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indicates interaction between Lys and GNP. The resulting insight is important for the better understanding
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of structural nature and thermodynamic aspects of binding between the Lys and GNP.
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Keywords: Lysozyme; Green Synthesis; GNP; Spectroscopy; Hydrophobic force
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ACCEPTED MANUSCRIPT Introduction Nanoscience and nanotechnology are growing very rapidly in all the discipline of science and engineering. There is a rapid development in nanoscience in the last couple of decades and the research work continues for further development in this field. Day-to-day more novel nanomaterials are getting synthesized and research work is going on to apply these various nanomaterials for the development of consumer products as well as in the advancement of medical science. There has been already lots of
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development in nanomaterials which have various applications but the toxicity of these nanomaterials has
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been the concerning issue for the further use of nanomaterials.
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Among the metallic nanomaterials, Gold nanoparticle (GNP) is a key material of interest as it has been found to be less toxic, inert and has enormous application capabilities in different fields. Synthesis of
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GNP is possible using chemical [1,2] and biological methods[3–9] but the shortcomings of chemical methods include use of toxic chemicals, being expensive and hazardous to the environment [10]. Among all available methods for synthesis of nanomaterials, most preferable one is green synthesis. The specialty
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of green synthesis is in this process there is no requirement of reducing agent as well as capping/stabilizing agent. Green synthesized nanoparticles can quickly form conjugates with proteins
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through either covalent bonds or physical interactions, and these conjugates have been extensively used in
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biomedical fields [11–13].
Understanding in interaction (interacting forces, binding sites, binding affinity, and conformational changes) of nontoxic nanoparticles with bio-macromolecules are very important for effective use of
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nanoparticles in biomedical application [14,15]. After entry of nanoparticles in blood stream they may quickly interact with plasma proteins leading to an alteration in the native conformation or function that
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may further result in astonishing biological reactions to reduce toxicity [16]. The interactions between protein and green synthesized nanoparticles have not been completely explored. We have selected
proteins.
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lysozyme for the interaction study due to the natural abundance and it is one of the most widely used
Lysozyme (Lys) is a 14.6 kDa small monomeric globular protein which consists of 129 amino acid residues containing 6 tryptophan (Trp) and 3 tyrosine residues & 4 disulfide bonds [17–19]. In Lys, six Trp residues are located at the substrate binding sites, of which two are in the hydrophobic matrix box, while the lone Trp residue is separated from the others [20–23]. Trp-62 and Trp-108 have considered the most dominant fluorophores in Lys among the Trp residues [20,24]. From the crystal structure, it has been observed that Trp-62 and Trp-108 residues are placed close to the active substrate binding site [25]. Furthermore, Lys has also many physiological and pharmaceutical functions such as, antibacterial,
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ACCEPTED MANUSCRIPT antivirus as well as detumescence and as a results extensively used in the pharmaceutical and food fields [19]. Another very significant role of Lys is its ability to carry drugs [26]. It is already known that Lys is capable of reversible binding to various endogenous and exogenous compounds [27].Understanding the binding characteristics of medically important GNP with Lys may expand the development of possible delivery modes for facilitating availability at required sites, leading to therapeutic usage. Recently, reports are available on the interaction of proteins with various
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nanoparticles in pure form or functionalized with a suitable group [18,21,28–37]. Lys is very useful to
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carry drugs, such as antibiotics to treat inflammation, abscess, stomatitis and rheum [38]. Accordingly,
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the interaction of Lys with biologically important green synthesized GNP is of great interests. Here in this featured work, we have used spectroscopic techniques like UV–visible, intrinsic fluorescence, and circular dichroism to describe the interaction between Lys and GNP. Systematic study of the binding
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mechanism between GNP and Lys with respect to the binding sites, the binding distance between the two and the effect on the secondary structure of Lys has been highlighted here.
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2. Experimental Details
Hen egg white lysozyme was used as received commercially from Sigma-Aldrich, USA. Tris–base was
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purchased from Merck, Germany. All the other chemicals were of analytical reagent grade and double distilled water was used throughout. Emission spectra were taken using a Fluoromax-4p
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Spectrofluorimeter from Horiba Jobin Yvon (Model: FM-100) well equipped with attached temperature controller. The absorption spectra were obtained from a Cary 60 UV-Vis Spectrophotometer Agilent
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Technologies. For the time resolved studies, a picosecond time correlated single photon counting (TCSPC) system from Horiba Yovin (Model: Fluorocube-01-NL) was used. Circular dichroic spectra
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were analyzed by a Jasco J-815 CD spectrometer. NMR spectra were examined by an Ascend Bruker BioSpin International AG spectrometer. All the details of the experimental and computational procedures
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are elaborated in the supporting information (SI). 3. Results and Discussion 3.1 Effect of GNP concentration on the Lys stability As the intensity and position of the surface plasmon band of colloidal gold are related to the size, shape, and dispersity of the GNP, UV-Vis absorption spectroscopy can be used for characterizing the optical properties of GNP. Here an aqueous solution of pure green synthesized GNPs shows a narrow and symmetric absorption peak at approximately 530 nm (Fig. 1), which agrees with an earlier report [39] and indicates that the GNPs are monodispersed and spherical. The UV–Vis absorption spectra of Lys in presence of increasing concentration of GNP are measured under stimulative physiological condition. In 4
ACCEPTED MANUSCRIPT the presence of increasing concentration of GNP (0, 2, 4, 6, 8, 10, 12, 14 and 16 µM) the overall absorbance of pure Lys gradually increases (Fig. 2) after the conjugation with GNP with a little variation in the shape of the band. This increase in intensity is likely due to the creation of ground state complex between Lys and GNP.
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Fig. 1 UV-Vis absorption spectrum of green synthesized GNP
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Fig. 2 Spectrophotometric interaction of Lys in presence of GNP (0, 2, 4, 6, 8, 10, 12, 14 and 16 µM)
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ACCEPTED MANUSCRIPT To know the extent of interaction between Lys and GNP apparent association constant (Kapp) has to be calculated. The ground state complex forms as follows. ,
(1)
The Kapp value has been calculated by following Benesi and Hildebrand [40] equation. ,
(2)
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Here, Aobs is the absorbance of the Lys solution containing different concentrations of GNP at 280 nm, α
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is the degree of association between Lys and GNP, εLys and εC are the molar extinction coefficients at the
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defined wavelengths for Lys and the complex formed respectively, C0 is the initial concentration of Lys and la is the optical path length. Eq. 2 can be now expressed as Eq. 3
(3)
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Here, A0 and AC are the absorbance of Lys and the complex at 280 nm respectively with the concentration
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of C0. At relatively higher GNP concentration, α can be equated to (Kapp [GNP])/(1+ Kapp [GNP]) where
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1/(Aobs-Ao)
(4)
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[GNP] is the concentration of GNP in mol/L. Thus Eq. 3 now converts to Eq.4 as follows:
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Fig. 3 Benesi-Hildebrand plots for Lys in presence of GNP, Kapp = 2.66×104 L/mol
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ACCEPTED MANUSCRIPT A plot of 1/(Aobs−A0) vs 1/[GNP] yield a linear plot (Fig. 3) and from this plot (R value = 0.9999, R2 value = 0.9998) the value of Kapp is found to be 2.66×104 L/mol which indicates the formation of a moderate complex between Lys and GNP. This means that interaction studies must be carried out in detail to get the comprehensive information of Lys-GNP interaction. 3.2. Fluorescence quenching studies of Lys in presence of GNP Many techniques are available to study the interaction between nanomaterials and Lys, but the utmost
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suitable one is fluorescence quenching. The fluorescence spectra (Fig. 4) have been recorded for Lys
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alone and in presence of different GNP concentrations of (0, 2, 4, 6, 8, 10, and 12 µM. Figure 4 clearly
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shows that the Lys has a strong emission band at 350 nm when excited with 280 nm wavelength. The intensity of this fluorescence band decreases steadily with increasing concentration of GNP. This alteration in the fluorescence characteristic of the Lys suggests the presence of binding between GNP and
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Lys to form a certain complex.
12M
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CE
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Intensity (a.u)
M
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0M
320
360
400
440
Wavelength (nm)
Fig. 4 Fluorescence spectra of Lys in the presence of GNP (0, 2, 4, 6, 8, 10, and 12 µM) 3.3. Binding mechanisms between Lys and GNP The fluorescence quenching generally occurs through either dynamic or static mode and classified depending on the way of interaction between quencher and biomolecule [41,42]. The fluorescence quenching data at variable temperatures can be analyzed using very well-known Stern–Volmer equation as follows: ,
(5) 7
ACCEPTED MANUSCRIPT Here, F0 and F denote the steady-state fluorescence intensities in the absence and presence of GNP respectively, KSV is the Stern–Volmer quenching constant, [Q] is the concentration of GNP, Kq is the quenching rate constant of the biological macromolecule, and τ0 is the average lifetime of the molecule without any quencher. The Stern–Volmer plot of F0/F vs [Q] is presented in Fig. 5 and slope, after linear regression of this plot, yields the Stern–Volmer constant (Table 1).
coefficient, R2= Coefficient of determination) R2
R
KSV (L/mol) ×104
291
0.9994
0.9997
4.29
298
0.9982
0.9992
3.97
305
0.9878
0.9951
4.07
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3.54 3.28 3.36
M
AN
1.6
291K 298K
CE
PT
Fo/F
ED
1.4
1.2
Kq (L/mol/s)×1013
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T(K)
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Table 1: Quenching rate constants and Stern Volmer constant for Lys binding to GNP (R= Correlation
305K
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1.0
4.0x10-6
8.0x10-6
1.2x10-5
[Q]
Fig. 5 Stern–Volmer curves at 293, 300, 303 and 310 K of GNP–Lys It can be overserved that Kq increases with increase in temperature indicating that fluorescence quenching occurs due to specific binding between GNP and Lys by forming complexes which are stabilized at higher temperatures. The value of Kq can be calculated by considering conventional fluorescence lifetime of the biopolymer of 10−8 s. The lifetime value of Lys as obtained using time resolved experiment was used to determine Kq value. In the present case (Table 1) the quenching constant Kq value suggests that the 8
ACCEPTED MANUSCRIPT interaction between the Lys and GNP is taking place through static quenching. Du & Xia [43] previously reported the interaction between chemically synthesized GNP and Lys which was through static quenching mode and the observed results corroborates our findings. It has been observed that KSV, Kq as obtained here in case of green synthesized GNP are 104 times and 103 times lower respectively as compared to chemically synthesized GNP. So, it can be concluded that chemically synthesized GNP has more quenching ability than biologically synthesized GNP towards Lys. KSV, Kq obtained in case of Lys-
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silver nanoparticle interaction was 5.66 ×103L/mol and 9.94×1011 L/mol/s respectively[44], which are
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also comparable to our findings. This result is also suggests that quenching takes place only due to proper interaction rather than the collision effect. For confirmation of the quenching phenomena as a result of
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Lys-GNP interaction further studies has been carried out in the following section. 3.4. Time resolved fluorescence measurement of Lys with GNP
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The fluorescence lifetime measurement is the most definitive method for discriminating static and dynamic quenching processes [45]. Time resolved fluorescence lifetime measurement has been carried
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out to validate static quenching mechanism for Lys-GNP interaction. The time resolved fluorescence decay curve of Lys in presence of GNP is presented in Fig. 6. As can be seen from Fig. 6, the
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fluorescence decay curve of Lys is single exponential with a lifetime value of τ = 1.21 ns. Details of
ED
results are given in Table S1 in the SI.
1000
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6000
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Counts
8000
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10000
Lys+8M GNP 600
400 1.02x10-8
4000
2000
Lys
800
1.10x10-8
1.17x10-8
Lys Prompt
0 7.50x10-9
1.50x10-8
2.25x10-8
Time (s)
Fig. 6 Time-resolved fluorescence decays of Lys (0.1 µM) in the presence of varying concentrations of GNP. The line profile in black represents the instrument response function (λex 280 nm, λem 345 nm).
9
ACCEPTED MANUSCRIPT After addition of GNP, the excited state lifetime of Lys showed no characteristic change (τ = 1.31 ns). It is well known that the static quenching mechanism is generally associated with stable fluorescence lifetime values, whereas, in case of excited-state quenching the fluorescence lifetime values are altered significantly. From the above analysis, it is apparent that the unaltered fluorescence lifetime of Lys in the presence of GNP is mainly initiated by static quenching process [46–48]. 3.5. Number of binding sites and binding locality
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In case of quenching interaction, relationship between the fluorescence intensity and the quencher
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,
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concentration can be derived from the following equation [49].
(6)
where, L is the biomolecule with a fluorophore, Q is the quencher; Qn..L is the quenched biomolecule and
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the resultant constant K is given by the following equation.
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,
(7)
If the overall amount of biomolecules (bound and unbound with the quencher) is L0, then, (8)
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,
where, [L] is the concentration of unbound biomolecules, the relationship between fluorescence intensity
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and the unbound biomolecules is as following.
(9)
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,
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Now from using above equation, one can write the following equation. ,
(10)
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where, Kb is binding constant of GNP with Lys and n is the number of binding site. The values of Kb and n can be determined from the linear plot between log [(F0 –F)/F] vs log [Q] as shown in Fig. 7. The values of Kb and n at 291, 298, and 305 K temperatures for Lys-GNP are shown in Table 2. Table 2 Binding constants and binding sites for Lys binding to GNP T(K)
R2
R
Kb (L/mol)×104
n
291
0.9999
0.9998
1.99
0.95
298
0.9994
0.9997
6.30
1.02
305
0.9978
0.9991
31.6
1.19 10
ACCEPTED MANUSCRIPT The results demonstrated that there is a strong binding force between GNP and Lys, and single binding site would be formed. The Kb as obtained here in case of interaction between Lys and green synthesized GNP are 105 times lower as compare to chemically synthesized GNP[43]. These results clearly indicate that chemically synthesized GNPs have greater ability to form strong binding with Lys compared with green synthesized GNPs. In case of Lys-silver nanoparticles interaction, Kb was found to be 1.26 ×104 L/mol and the value of n was approximately equal to 1, these results are also analogues to our
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experimental findings [44]. To understand the details of thermodynamic properties of Lys and GNP
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interaction the following study has been carried out.
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291K
-0.8
298K
M
log[(Fo-F)/F]
-0.4
305K
-5.6
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-5.8
ED
-1.2
-5.4
-5.2
-5.0
-4.8
log[Q]
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Fig. 7 Fluorescence quenching [(F0-F)/F] vs log [Q] plots for Lys in the presence of the GNP 3.6. Thermodynamic parameters and nature of the binding forces
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Four types of forces namely hydrophobic forces, electrostatic interaction, hydrogen bond, and van der Waals forces are found in case of interactions between small molecules and biological macromolecules. The interaction model is as following [50,51], 1) ΔH > 0 and ΔS > 0, hydrophobic forces; 2) ΔH < 0 and ΔS < 0, van der Waals interactions and hydrogen bonds; 3) ΔH < 0 and ΔS > 0, electrostatic interaction. If the enthalpy change has not changed significantly within a temperature range, then ΔH and ΔS can be calculated using following Van’t Hoff equation. ,
(11)
11
ACCEPTED MANUSCRIPT where Kb is analogous to the binding constant at the corresponding temperature, R is gas constant. The values of ΔH and ΔS have been obtained from the ln Kb vs 1/T plot (Fig. 8). The free energy change (ΔG) can be obtained from the following equation. ,
(12)
Using Eq. 11 and Eq. 12 the values of ΔG, ΔH and ΔS were obtained as shown in Table 3. From the negative value of ΔG it appears that the process of interaction is spontaneous. Positive values of ΔH and
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ΔS indicate that the major contribution in complexation is hydrophobic force [52]. Similar results of
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thermodynamic parameters have been found in case of interaction between chemically synthesized GNPs
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and Lys [43]. From the resulting insight, it is clear that the interaction between GNPs (Chemical & biological) and Lys takes place by means of hydrophobic effect. According to the thermodynamic data,
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the formation of the Lys–GNP complex is in favor of formation of the aforesaid complex.
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13
M 10
ED
11
PT
lnK
12
CE
3.28x10-3
3.34x10-3
3.40x10-3
AC
1/T(K-1)
Fig. 8 Van’t Hoff plot for the interaction between Lys and GNP. Table 3 Thermodynamic parameters for the binding of GNP to Lys ΔS (J/mol/K)
ΔG (kJ/mol)
141.99
570
-23.87
298
141.99
570
-27.86
305
141.99
570
-31.88
T(K)
R2
R
291
0.9974
0.9993
ΔH (kJ/mol)
12
ACCEPTED MANUSCRIPT Thermodynamical parameters have been studied in various reports dealing with interaction to get an insight about the binding force. Existence of van der Waals interaction & hydrogen bonding and their significant contribution in the binding process have been observed in case of Lys-rhodamine b and Lysdrospirenone [48,53]. Electrostatic forces played a major role in the interaction between Lys and cefepime hydrochloride [54], electrostatic and hydrophobic force was the most important force to stabilize the Lysheparine complex [55]. In case of interactions between cyclophosphamide and aspirin with Lys results
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found using DFT calculations suggested that Trp 62 has the most affinity to cyclophosphamide and
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aspirin drugs as the ligands [56]. The binding of chelerythrine to Lys also suggested Trp 62 in the βdomain of the protein was closer to the binding site[57]. Accordingly, it is expected that in the present
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case of GNP-Lys interaction Trp62 may have an important role. To understand structural effect of Lys in presence of GNP the following study has been carried out.
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3.7. Circular dichroism spectra
To get an idea of structural change of Lys, CD spectral measurements at room temperature have been
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carried out in absence and in presence of GNP (Fig. 9). There are two strong negative bands in the UV region at 208 and 222 nm and this contribution are due to n→Π* transfer for the peptide bond of α-helical
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form.
ED
CE
PT
-40
-80
AC
(mdegcm2mol-1)
0
Lys+20M GNP
-120
Lys+10M GNP Lys
200
220
240
260
Wavelength (nm)
Fig. 9 CD spectra of Lys in the presence of different concentration of GNP (0, 10, and 20 µM)
13
ACCEPTED MANUSCRIPT The CD results are expressed in terms of mean residue ellipticity (MRE) in degree-cm2/mol according to the following equation [58,59]. ,
(13)
where, Cp is the concentration of Lys in µM, Ɵ is observed rotation in degrees, l is the path length in cm and na is the number of amino acid residues of protein (129 for Lys). The α-helical contents of Lys have
T
been estimated from MRE values at 222 nm using Eq. 14 and then cross checked by K2D software
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[60,61].
(14)
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,
It is observed that the percentage of helicity of Lys is 40% and in presence of GNP helicity is almost
US
unchanged and this is due to the fact that the α-helical structure of Lys is unaffected and may induce very small or no conformational changes. This observation strongly indicates that the binding of GNP to Lys
AN
induces little change in conformation of Lys and the secondary structure of Lys retains mainly its α-helix character.
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3.8. NMR studies
NMR results show that the interaction of GNP with Lys take place as indicated through the line widths
ED
and chemical shifts of the 1H-NMR signal (Fig. S1 in SI). This result also shows that new signals were formed by means of the significant interaction between the Lys and GNP [62]. The comparative 1H-NMR
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spectra of Lys in the presence and absence of GNP were given in Fig. S1 (a–b). After addition of GNP to Lys, there has been no significant shift in signal rather new signals are formed which indicates interaction
CE
between Lys and GNP. Resulting insights are clearly indicative of interaction between Lys and green GNP. To understand the energy transfer process of Lys-GNP complex following study has been carried
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out.
3.9. Energy transfer between Lys and GNP In process of Forster’s resonance energy transfer, donor fluorophore (Lys) in its excited state transfers energy to an acceptor (GNP) molecule through nonradiative dipole–dipole coupling [16]. The efficacy of this energy transfer has been used for estimation of distance (r) between GNP and fluorophore Lys [63]. Energy transfer process mainly depends on the magnitude of overlap of emission spectrum of the donor with absorption spectrum of the acceptor. The emission spectrum of Lys is well overlapped with absorption spectrum of GNP which indicates that there is a possibility of energy transfer from excited state of Lys to GNP (Fig.10).
14
ACCEPTED MANUSCRIPT
4
Intensity (a.u)
T
Emission of Lys
2
Absorption of GNP
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Absorbance
3
0 300
500
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400
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1
600
AN
Wavelength (nm)
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Fig. 10 Overlap plots of fluorescence spectra of Lys with absorption spectra of GNP. The efficacy (E) of FRET is inversely proportional to the sixth power of distance between donor and
ED
acceptor [64].
(15)
PT
,
The magnitude of R0 is dependent on the spectral characteristics of the donor emission and acceptor
CE
absorption of the molecules. R0 is the critical distance when the transfer efficiency is 50 % and given by the following equation.
(16)
AC
,
where φ is the fluorescence quantum yield of the donor, N is the refractive index of the medium, K2 is the spatial orientation factor of the dipole, J is the overlap integral of the fluorescence emission spectrum of the donor and the absorption spectrum of the acceptor as given by following equation. ,
(17)
where F(λ) is the fluorescence intensity of the donor and ε(λ) is the molar absorptivity of the acceptor when the wavelength is λ.
15
ACCEPTED MANUSCRIPT Using Eqs. 15-17 along with the values for φ = 0.15, K2 = 2/3 and N = 1.36 [65,66], the values of R0 and r were calculated to be 1.84 nm and 3.06 nm, respectively and J = 1.40×10-15 cm3 L/mol, E = 0.045. The result shows that GNP is strong quencher and they are situated in close proximity to the Lys. The average distances between a donor fluorophore and acceptor fluorophore are in the range of 2–7 nm [65] which denotes that the energy transfer occurs between Lys and GNP. This also indicates that the fluorescence quenching of Lys was a non-radiative transfer process.
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4. Conclusion
Spectroscopic studies reveal the quenching of Lys fluorescence through static quenching processes
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induced by gold nanoparticles (GNPs). A moderately strong binding affinity between GNP and Lys has also been observed along with the existence of a single binding site in Lys for GNP. A thorough analysis
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of thermodynamic data clearly indicates that the Lys-GNP interaction is dominated by hydrophobic forces. The binding process is thermodynamically favorable and spontaneous. A non-radiative transfer of energy between GNP and Lys, 3.06 nm apart, is also revealed. GNP addition to Lys does not make any
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significant changes in Lys secondary structure in terms of decrease in α-helix. The interactive nature of Lys-GNP complex is not strong enough to induce significant structural modifications to make the
M
complex to be appropriately used in certain applications. The binding phenomenon of green synthesized nontoxic GNP with Lys is of great importance in pharmacy and biochemistry. Furthermore, it is expected
ED
that fluorescence technique is a promising tool for designing GNP based biomaterials and nano drugs that will be noteworthy for present day’s medical research.
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Acknowledgement
Authors are thankful to Department of Science and Technology (DST) project (No. SB/S2/CMP-012-
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2014) for the financial support and also SIC facility of Indian Institute of Technology Indore for the instrumental facility. Authors also thank MHRD, Govt. of India for fellowship. Valuable discussion with
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Dr. Hem C. Jha (IIT Indore) is also greatly acknowledged. Supporting Information Experimental and computational details, TCSPC results in Table S1, and Fig.S1. Notes The authors declare no competing financial interest.
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Highlights
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Graphical abstract
1. Lys fluorescence is quenched by green GNP through static quenching. 2. Hydrophobic forces stabilize the Lys-GNP complex. 3. GNP does not induce conformational changes in Lys.
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