A systematic investigation on the interaction of L-cysteine functionalised Mn₃O₄ nanoparticles with lysozyme

Abstract

Biological interactions of magnetic nanoparticles have attracted enormous interest because of their wide applications in medical diagnostics, imaging, and biosensors. Mn₃O₄ nanoparticles are potentially significant among magnetic nanoparticles and have not yet been investigated for their interactions with proteins. Here we report a systematic investigation of the interactions between hen egg white lysozyme and L-cysteine functionalised Mn₃O₄ nanoparticles using calorimetric and spectrophotometric techniques. The L-cysteine functionalised nanoparticles were synthesized by a simple chemical precipitation method and characterized by X-ray diffraction, FTIR, HRTEM, UV-Vis absorption and fluorescence spectroscopy. Mn₃O₄ nanoparticles are in the tetragonal phase with an average size of 10 nm. Thermodynamic parameters, the binding constant and the number of binding sites associated with the interactions were studied using Isothermal Titration Calorimetry (ITC). Quenching of lysozyme fluorescence is observed upon complex formation with Mn₃O₄ nanoparticle and the time resolved fluorescence measurements confirm static quenching. The Circular Dichroism (CD) spectra reveal minor alterations in the secondary structure of lysozyme in the complex indicating that Mn₃O₄ nanoparticles are potential candidates for therapeutic applications.

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1. Introduction

Magnetic nanoparticles have attracted pronounced interest because of their large number of applications including catalytic, magnetic, electronic, dry cell batteries, pigments etc.^1–3 In biomedical applications magnetic nanoparticles are used for targeted drug delivery, cell labelling and targeting, tissue repair and magnetic resonance imaging (MRI).^4–7

Among the various magnetic nanoparticles, Mn₃O₄ (trimanganese tetraoxide, hausmannite) is one of the most stable manganese oxides having stimulated interest because of its special electronic configuration and distorted spinel structure.⁸ Due to the different oxidation states of manganese (Mn²⁺, Mn³⁺, and Mn⁴⁺), manganese oxide crystals mainly exist as MnO, Mn₃O₄, Mn₂O₃ and MnO₂ forms.⁹ The special attraction of Mn₃O₄ nanoparticles is their applications in diverse fields such as magnetism, solar energy transformation, varistors, and electrochemistry. They are used as catalysts in air purification and hollow Mn₃O₄ nanoparticles as positive MRI contrast agents.^10–15 In order to avoid the agglomeration and to stay longer time in blood circulation, the surface of these nanoparticles should be modified with suitable organic or inorganic materials.¹⁶ The surface modification also improves the biocompatibility, prevents protein absorption, enhance their targeting and reduce the toxicity of the nano materials.^17–20

The effective use of nanoparticles in biomedical applications requires the complete understanding of the molecular level interactions of nanoparticles with bio macromolecules,²¹ providing the information regarding the interacting forces, binding sites, binding affinity, conformational changes, and stability of the interacting molecules.^22–24 When nanoparticles enter into biological environment, such as blood stream they quickly interact with blood plasma proteins and the interaction may alter the native conformation or perturb the normal protein function leading to unexpected biological reactions and toxicity.^25,26 The interactions between protein and nanoparticles functionalized with suitable groups have not been completely explored. We selected lysozyme as a model protein for the interaction study because of its high natural abundance and is one among the proteins investigated thoroughly for its structural and functional characterization. Hen egg white lysozymes (N-acetylmuramide glycanhydrolase), is a small monomeric globular protein having formula weight of about 14.6 kDa and it contains structural elements such as α-helix, β-sheet and turns which are commonly found in proteins. Lysozyme has 129 tactic amino acid residues including 6 tryptophanes (Trp-28, Trp-62, Trp-63, Trp-108, Trp-111 and Trp-123), 3 tyrosines and 4 disulfide bonds.^27,28 Among six tryptophan residues, Trp-62 and Trp-108 contribute to tryptophan emission, Trp-62 is located on the active site of lysozyme while Trp-108 is not in the active site (Scheme 1). Lysozyme is used in pharmaceutical zones, and has many functions such as antibacterial, antiviral detumescence and ability to carry drugs. Lysozyme can bind with antibiotics in order to treat inflammation, abscess, stomatitis and rheum.²⁹

Scheme 1 Schematic representation of hen egg white lysozyme (PDB no. 3ZEK) (the tryptophan residues are marked).

Here, we have studied the systematic interactions between L-cysteine capped Mn₃O₄ nanoparticles (Cy-Mn₃O₄ NPs) and hen egg white lysozyme (HEWL) using spectroscopic techniques such as UV-vis spectrophotometry, fluorescence, time resolved measurements and circular dichroism. Spectroscopic techniques are most powerful techniques to investigate the interactions between nanostructured materials and biological molecules. The thermodynamic changes during the interaction of Mn₃O₄ nanoparticles with lysozyme were studied using isothermal titration calorimetry (ITC). Langmuir absorption isotherm and van't Hoff equations gave the thermodynamic parameters associated with the binding. The thermodynamic and spectroscopic data on the interaction between proteins and nanoparticles is essential to understand how cellular systems will respond to the presence of nanoparticles.³⁰

2. Experimental

2.1. Reagents

L-Cysteine (C₃H₇NO₂S), potassium permanganate (KMnO₄) and hen egg white lysozyme were purchased from Sigma-Aldrich. Lysozyme was dissolved in phosphate buffer solution of pH 7. All of the reagents were of analytical grade and used without further purification.

2.2. Instrumentation

The high-resolution transmission electron microscopy (HRTEM) images were taken using JEOL JEM 2100 with LaB6 filament with an operating voltage of 200 kV. X-ray diffraction spectrum was recorded by PANalytical X-ray diffractometer with CuKα radiation (λ = 1.5406 Å) in the range of 10–90° (2θ) at a scanning rate of 0.01° min⁻¹. UV-Vis absorption studies were performed by Schimadzu 2401 UV-Vis spectrophotometer. The FT-IR spectrum was measured on a Shimadzu Model: IR Prestige 21, with ZnSe ATR crystal (Pike technologies) spectrometer. Fluorescence measurement was carried out using Fluoromax-4 Spectrophotometer with an excitation wavelength of 280 nm. Fluorescence lifetime measurements were carried out at an excitation wavelength of 278 nm using a picosecond diode (Spectra-NanoLED source S-278). Circular dichroism (CD) data was collected from JASCO 810 circular dichroism spectropolarimeter using quartz cuvette of 1 cm path length. ITC experiments were carried out in a VP-ITC isothermal titration calorimeter from Microcal (Northampton, MA, USA). Thermodynamic parameters associated with the binding experiments were carried out using isothermal titration calorimetry (ITC). ITC directly provides the enthalpy change (ΔH), entropy change (ΔS) number of binding sites (n), binding constant (K), binding free energy (ΔG) and the entropic contribution (TΔS). Approximately 14 μM HEWL was prepared in phosphate buffer at pH 7. The Cy-Mn₃O₄ nanoparticles were prepared in the same buffer. The prepared solutions were degassed. The experiments were carried out at 25 °C, a reference power of 15 μcal s⁻¹ and with a syringe stirring speed of 394 rpm. A typical titration involved 30 injections of nanoparticles into the sample cell containing HEWL. Data analysis was conducted using ORIGIN 7.0 software. From the fitted data we will get binding constant (K), enthalpy change (ΔH), entropy change (ΔS), and number of binding sites (n). ΔG and TΔS can be calculated using the following equations.³¹

ΔG = −RT ln K

ΔG = ΔH − TΔS

2.3. Procedure

2.3.1. Synthesis of L-cysteine stabilized Mn₃O₄ nanoparticles. For the synthesis Mn₃O₄ nanoparticles we selected L-cysteine and potassium permanganate (KMnO₄). Here we employed a facile one-step method to reduce potassium permanganate (KMnO₄) to Mn₃O₄ nanoparticle with L-cysteine. In a typical synthesis procedure, 0.5 gm of KMnO₄ was dissolved in 60 mL of distilled water under constant stirring and 0.2 gm of L-cysteine was introduced into the above solution. After stirring at 60 °C for 1 hour and a brown precipitate was formed. This was collected and washed several times with distilled water in order to remove excess of capping agents and impurities. Finally dried at 60 °C for 12 hours in a hot air oven.

Lysozyme stock solution was prepared in PBS of pH 7 by dissolving 0.01 g of lysozyme in 50 mL buffer, and used for further studies. Cy-Mn₃O₄ NPs at various concentrations (0.01–0.07 mM) were allowed to interact with a constant concentration of lysozyme at room temperature.

3. Results and discussions

3.1. Characterization of L-cysteine capped Mn₃O₄ nanoparticles

The X-ray diffraction pattern of as-prepared L-cysteine capped Mn₃O₄ nanoparticles is shown in Fig. 1a. The dominant peaks in the diffraction patterns were indexed to (101), (112), (103), (211), (004), (220) (105) and (224) planes corresponding to the tetragonal structure (JCPDS 076088). The average particle size of Mn₃O₄ nanoparticles was estimated as 9 nm according to the Scherer formula.

Fig. 1 (a) XRD spectrum of Cy-Mn₃O₄ NPs (b) FTIR spectrum of Cy-Mn₃O₄ nanoparticles (c) TEM images, inset figure shows the HRTEM image (d) SAED pattern of L-Cy Mn₃O₄ nanoparticles.

The microstructure and morphology of the Cy-Mn₃O₄ NPs were studied from the HRTEM (Fig. 1c) showing that most of the particles are spherical in shape with an average size less than 10 nm. The well-defined diffraction rings in the SAED pattern (Fig. 1d) indicates the crystalline nature of the sample. The most prominent rings in the SAED pattern of Mn₃O₄ nanoparticles can be indexed to the tetragonal structure, consistent with the XRD data.

Fig. 1b is the FTIR spectrum of Cy-Mn₃O₄ NPs. The NH₂ or OH vibration of cysteine is observed at 3313 cm⁻¹ (ref. 32) and the –COOH group vibrations are at 1597, 1404 cm⁻¹ (ref. 33) confirming the presence of L-cysteine on the surface of nanoparticles.

3.2. Characterization of lysozyme–Cy-Mn₃O₄ system

3.2.1. Absorption studies of HEWL in the presence of Cy-Mn₃O₄ NPs. Fig. 2a shows the absorption spectra of HEWL and HEWL complexed with different concentrations (0.01–0.07 mM) of Cy-Mn₃O₄ NPs. The absorbance of HEWL at 278 nm is gradually increasing with increase in concentration of nanoparticles. The interactions of Cy-Mn₃O₄ NPs with HEWL lead to the formation of a ground state complex,³⁴ as a consequence the absorbance of HEWL increases progressively on increasing concentration of nanoparticles.

Fig. 2 (a) Absorption spectra of HEWL and its complex with Cy-Mn₃O₄ NPs showing a gradual increase in the absorbance with increase in concentrations of Cy-Mn₃O₄ NPs. The absorption spectrum of Cy-Mn₃O₄ NPs is shown in the inset. (b) The plot showing the dependence of 1/A_obs − A₀ on the reciprocal concentration of Cy-Mn₃O₄ NPs.

The binding constant can be calculated using Benesi–Hildebrand equation.^35–38

HEWL + Cy-Mn₃O₄ ↔ HEWL………Cy-Mn₃O₄ (1)

K_p represents the apparent association constant and the value of K_p is calculated from the equation.

A_obs = (1 − α)C₀ε_Lyzl + αε_cl (3)

where A_obs is the observed absorbance of HEWL in the presence of different concentrations of Cy-Mn₃O₄ NPs, α is the degree of association between HEWL and Mn₃O₄ nanoparticles, ε and ε_c are the molar extinction coefficients at the defined wavelength (λ = 278 nm) for HEWL alone and HEWL–Cy-Mn₃O₄ complex respectively, C₀ is the initial concentration of HEWL and ‘l’ is the optical path length. Eqn (3) can be expressed as

A_obs = (1 − α)A₀ + αA_c (4)

where A₀ and A_c are the absorbance of HEWL in the absence and presence of Mn₃O₄ at 278 nm respectively.

At relatively high Cy-Mn₃O₄ NPs concentration α can be equated to (K_p [capped Mn₃O₄]/(1 + K_p [capped Mn₃O₄]) and eqn (4) can be expressed as

The enhancement in the absorbance at 278 nm is due to the absorption of HEWL–Cy-Mn₃O₄ complex. The linear relationship between 1/A_obs − A₀ and reciprocal concentration of [Mn₃O₄] were obtained (Fig. 2b) with a slope of 1/K_p (A_c − A₀) and an intercept at 1/A_c − A₀. The value of apparent association constant (K_p) calculated from the graph is 5.29 × 10³ M⁻¹.

3.2.2. Fluorescence quenching study. The binding affinity between Cy-Mn₃O₄ NPs and HEWL was studied using fluorescence quenching measurements. HEWL has a strong emission band at 348 nm when excited at 278 nm. By adding Cy-Mn₃O₄ NPs having different concentrations (0.01–0.07 mM) into HEWL the fluorescence intensity of HEWL decreases progressively (Fig. 3a) indicating the complex formation between HEWL and Cy-Mn₃O₄ NPs.

Fig. 3 (a) Fluorescence spectra of HEWL and HEWL with various concentrations of Cy-Mn₃O₄ NPs. Quenching of fluorescence intensity on increasing the concentration of Cy-Mn₃O₄ NPs is observed. Inset shows the fluorescence spectrum of Cy-Mn₃O₄ NPs. (b) Stern–Volmer plot between I₀/I and [Q].

The quenching of HEWL emission intensity by Cy-Mn₃O₄ NPs can be analysed by Stern–Volmer equation^39–41

I₀/I = 1 + K_sv[Q]

where I₀ is the fluorescence of HEWL and I denote the fluorescence of HEWL in the presence of Cy-Mn₃O₄ NPs. K_sv is the Stern–Volmer quenching constant and [Q] is the concentration of the Mn₃O₄ nanoparticles. By plotting I₀/I against [Q], (Fig. 3b) K_sv can be calculated from the slope and is found to be 5.2728 × 10³ M⁻¹. Stern–Volmer constant (K_sv) and quenching rate constant are related by

K_q = K_sv/τ

where τ is the lifetime of HEWL and the value of the quenching rate constant is 2.775 × 10¹² M⁻¹ s⁻¹.

Fluorescence quenching data can be used for calculating the binding constant (K) and the number of binding sites (n) in HEWL–Cy-Mn₃O₄ complex and the values can be evaluated from the equation.^41,42

Fig. 4 shows the plot of versus log[Q]. The values of n and K are obtained from the slope and Y intercept respectively and the corresponding values are 7.126 × 10³ M⁻¹ and 1.003.

Fig. 4 The plot of versus log[Q] for HEWL with Cy-Mn₃O₄ NPs.

HEWL has six tryptophan residues, Trp-28, 62, 63, 108, 111 and 123 (Scheme 1). Among the six tryptophan residues, Trp-62 and Trp-108 contribute significantly to the fluorescence of HEWL. The tryptophan residues at 28, 108, 111, and 123 are located in the α domain, and tryptophan at 62, 63, and 108 are at the substrate binding cleft. The Trp-62 and Trp-63 are not in the hydrophobic core located near to the active-site and are most exposed to the solvent, whereas Trp-108 is in the hydrophobic environment and is almost completely buried. Trp-62 has an important role in the catalysis of HEWL, particularly in substrate binding. Thus the chemical modifications will affect the two Trp residues (Trp-62 and Trp-63) and the exposed moieties could be the main residues affected and quenched by binding. During emission, inter-tryptophanyl energy transfer among Trp residues 108, 63, and 62 has also been proposed. Therefore, the exposed Trp-62 and 63 moieties could be mainly affected and hence the fluorescence can be quenched by ligand binding. The tryptophan fluorescence is highly sensitive to the local environment and any change in their micro environment can alter the fluorescence. Therefore it is reasonable to conclude that the nanoparticle will bind in the micro environment of Trp-62 and Trp-63 (ref. 43–47) (Scheme 2).

Scheme 2 The possible interaction site of hen egg white lysozyme, also shown the buried and exposed tryptophan residues.

3.2.3. Time resolved fluorescence decay. The decays of HEWL in the absence and presence of Cy-Mn₃O₄ NPs are shown in Fig. 5 and the relevant parameters are listed in Table 1. Time resolved measurement is a reliable technique to distinguish the quenching mechanism of HEWL by Cy-Mn₃O₄ NPs. The observation is that there is no considerable change in the lifetime value upon the addition of Cy-Mn₃O₄ NPs indicating static fluorescence quenching. HEWL exhibits multi-exponential decay and the mean life time value can be calculated using the equation

τ_m = τ₁a₁ + τ₂a₂ + τ₃a₃

where a₁, a₂, a₃, τ₁, τ₂, and τ₃ are the amplitudes and decay times of the multi exponential components of the fluorescence decay. The lifetime value for bare HEWL is 1.90 ns and after the addition of various concentrations of Cy-Mn₃O₄ NPs, the lifetime value changes to 1.79 ns. This marginal change indicates that the quenching is static in nature and a ground state complex is formed between HEWL and Cy-Mn₃O₄ NPs.^48–50

Fig. 5 Fluorescence decay of HEWL in the absence and presence of Cy-Mn₃O₄ NPs.

Table 1 Fluorescence lifetime parameters of HEWL in the absence and presence of Cy-Mn₃O₄ NPs

Mn3O4 (10^−5 M)	τ1 (ns)	τ2 (ns)	τ3 (ns)	a1	a2	a3	τm (ns)	χ^2
0	0.65	20.35	1.86	65.92	3.98	13.73	1.90	1.19
1	0.81	30.05	2.15	63.4	4.94	6.55	1.93	1.17
2	0.73	24.51	1.91	62.04	3.93	13.44	1.89	1.10
3	0.67	23.59	1.92	66.02	4.26	10.4	1.87	1.17
4	0.69	23.24	1.84	64.12	3.96	12.64	1.84	1.16
5	0.54	18.11	1.72	65.62	3.65	16.27	1.82	1.21
6	0.71	25.75	1.89	64.03	4.15	10.22	1.81	1.15
7	0.65	24.06	1.86	65.58	4.10	10.36	1.79	1.17

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3.2.4. Energy transfer to the nanoparticles and distance measurements. The Forster theory of dipole–dipole resonance energy transfer (FRET) is used to determine the distance between Cy-Mn₃O₄ NPs and HEWL protein. In FRET the energy transfer process is highly dependent on the distance between the donor and the acceptor molecules and the energy transfer from donor to acceptor is through dipole–dipole interaction.^51,52 FRET can happen only when, there is spectral overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor and the distance between the donor and the acceptor is within 2–8 nm. The energy transfer efficiency E is inversely related to the sixth power of the critical energy transfer radius (R₀) and the distance between the donor and acceptor (r),⁵³ E can be expressed as
I, I₀ are the fluorescence intensities of the donor in the absence and presence of the acceptor. R₀ is the critical distance when the transfer efficiency is 50%, and the value of R₀ can be calculated using the equation,

R₀⁶ = 8.8 × 10⁻²⁵k²N⁻⁴ϕJ

where k² is the spatial orientation factor, N is the refractive index of the medium, ϕ is the quantum yield of the donor and J is the overlap integral and is given by
where F(λ) and ε(λ) are the fluorescence intensity of the donor and molar absorptivity of the acceptor at λ, respectively.

The spectral overlap of the absorption spectrum of Cy-Mn₃O₄ NPs with the fluorescence emission spectrum of HEWL is shown in Fig. 6. Using the values of J = 1.522 × 10⁻¹⁶ cm³ L mol⁻¹, k² = 2/3, N = 1.36 and ϕ = 0.14 (ref. 54) the values of R₀, E and r can be determined. The calculated values are R₀ = 1.393 nm, E = 0.0578 and r = 2.218 nm. Therefore, the binding distance between Cy-Mn₃O₄ NPs and the Trp residue in the HEWL is 2.218 nm which is lower than the proposed 8 nm indicating a large probability of energy transfer between the Trp residue and the nanoparticles.

Fig. 6 Spectral overlaps of the absorption spectrum of Cy-Mn₃O₄ NPs and emission spectrum of HEWL.

3.2.5. Isothermal titration calorimetry (ITC). In order to understand the heat effects taking place during the interaction of HEWL with Mn₃O₄ nanoparticles ITC experiments were carried out. ITC results also provide the information about the number of binding sites (n) and the binding constant (K). ITC curve of the binding of HEWL with Mn₃O₄ nanoparticles is shown in Fig. 7. The upper portion of the figure represents the raw ITC curves obtained as a result of the injection of Cy-Mn₃O₄ NPs in to HEWL. A number of injections were done until the desired concentration range was attained and each curve in the figure represents the single injection of nanoparticles into HEWL. After each injection, the heat flow was recorded and plotted against time t. The thermodynamic parameters for binding were obtained by integrating the areas under the peak and subtracting the heat of dilution. Solid rectangular points represent the experimental injection heats and the solid line is the calculated fit of the data. From the data, it is clear that the interaction of Cy-Mn₃O₄ nanoparticles with HEWL is an exothermic process (ΔH < 0). The values of binding constant (K), the number of binding sites (n), enthalpy change (ΔH), ΔG and TΔS are listed in Table 2. The data were best fitted to a single site of binding model.^55–59

Fig. 7 ITC data from the titration of HEWL with Cy-Mn₃O₄ (PBS buffer, pH 7.0) at 25 °C.

Table 2 Binding parameters of Cy-Mn₃O₄ nanoparticles with HEWL

Parameter	Cy-Mn3O4–HEWL
K	1.49 × 10^4 M^−1
ΔH	−54.2 kcal mol^−1
ΔG	−8.303 kcal mol^−1
−TΔS	54.046

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Depending on the sign and magnitude of the thermodynamic parameters the interactions between HEWL and Cy-Mn₃O₄ NPs take place by three different mechanisms, namely hydrophobic interactions (ΔG −ve, ΔH +ve, and ΔS +ve), electrostatic interactions (ΔG −ve, ΔH is +ve or −ve, and ΔS +ve) and hydrogen bonding interactions (ΔG −ve, ΔH −ve, and ΔS −ve). The thermodynamic parameters (Table 1) show that the interaction is exothermic and HEWL interacts with Cy-Mn₃O₄ nanoparticles via hydrogen bonding.⁶⁰

3.2.6. Circular dichroism spectroscopy. To investigate the conformation of proteins in aqueous solution CD spectroscopy is extensively used.⁶¹ The changes in the secondary structure of HEWL upon binding with Cy-Mn₃O₄ nanoparticles at various concentrations were monitored using CD spectrum (Fig. 8). The spectrum of HEWL exhibits two characteristic negative bands at 209 nm and 222 nm.^62,63 The band at 209 nm originates from π–π* transfer of peptide bond and the band at 222 nm is from n–π* transfer of peptide bond in the α-helix.⁶⁴ The ellipticity of HEWL decreases after binding with the nanoparticles indicating some kind of partial unfolding upon binding with Cy-Mn₃O₄ NPs. The α-helical content of HEWL and HEWL with different concentrations of nanoparticles can be calculated from the following equation⁶⁵

Fig. 8 Circular dichroism spectra of HEWL in the absence and presence of Cy-Mn₃O₄ NPs and the ellipticity of HEWL decreases with increasing concentration of Cy-Mn₃O₄ NPs.

MRE (mean residue ellipticity) can be determined from the following equation

C is the concentration of HEWL, n and l are the number of the amino acid residues (129 for HEWL) and path length (1 mm) respectively. The addition of Cy-Mn₃O₄ NPs reduces the α-helical content by ∼6% (Table 3) indicating a conformational change in the secondary structure of HEWL although, this is not appropriate to alter the overall conformation of HEWL.^66,67

Table 3 The variations of α-helix% of HEWL in the presence of Cy-Mn₃O₄ nanoparticle

Concentration of Cy-Mn3O4 NPs (10^−4 M)	% of α-helix content (at 209 nm) HEWL
0	38.34
1	35.79
3	33.43
5	32.16

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3.2.7. FT-IR spectroscopy. The structural alterations of HEWL resulting from the interactions of Cy-Mn₃O₄ NPs are also studied using FT-IR spectroscopy. The FT-IR spectra of HEWL in the absence and presence of Cy-Mn₃O₄ NPs are shown in the Fig. 9. In proteins, amide I and amide II bands are observed in the regions 1600–1700 cm⁻¹ and 1500–1600 cm⁻¹ (ref. 31, 68) respectively. When Cy-Mn₃O₄ NPs are added to HEWL the amide I peak of HEWL is shifted from 1639 to 1643 cm⁻¹ and the amide II peak from 1544 to 1551 cm⁻¹ signifying minor conformational changes in the HEWL. This result supports the minor conformational changes in HEWL observed from the CD spectra.^36,38

Fig. 9 FT-IR spectrum of HEWL in the absence and presence of Cy-Mn₃O₄ NPs.

Fluorescence quenching is the decrease in the fluorescence intensity of a fluorophore due to various reasons such as excited state reactions, energy transfer, ground state complex formation, collisional quenching etc.^69–71 Two types of mechanisms, static and dynamic are proposed for fluorescence quenching. In static quenching a non-fluorescent ground state complex is formed between the quencher and the fluorophore while in dynamic quenching the collisional encounters between them reduces the fluorescence intensity.⁷² Fluorescence lifetime measurement is a technique to distinguish between the two types of quenching.

The UV-Vis spectroscopy (Fig. 2a) shows that the absorbance of HEWL at 278 nm increases progressively with the increase in concentration of Cy-Mn₃O₄ NPs. This is due to the formation of a ground state complex between the HEWL and Cy-Mn₃O₄ NPs. The addition of nanoparticles increases the micro environmental hydrophobicity of the amino acid residues. The ground state complexation is usually associated with the non-covalent interactions such as hydrogen bonding, electrostatic, hydrophobic, and π–π stacking.^73,74 ITC experiment revels the complexation between Cy-Mn₃O₄ NPs and HEWL is associated with hydrogen bonding along with other interactions. On increasing the concentration of Cy-Mn₃O₄ NPs the fluorescence intensity of HEWL decreases progressively (Fig. 3a). When Cy-Mn₃O₄ NPs were added in to HEWL a non-fluorescent ground state complex is formed and the fluorescence is observed from the uncomplexed fluorophore. The measured lifetime of pure HEWL and HEWL with various concentrations of Cy-Mn₃O₄ NPs shows only marginal differences (1.90–1.79 ns) (Table 1) and therefore, we can conclude that, the quenching mechanism is static in nature. Upon increasing the concentration of Cy-Mn₃O₄ NPs the fluorescence intensity decreases, without any change in the excited state life time. Therefore, it is reasonable to say that the addition of Cy-Mn₃O₄ NPs a non-fluorescent ground state complex is formed between HEWL and Cy-Mn₃O₄ NPs. The CD spectra revealed the conformational changes in HEWL after the addition of Cy-Mn₃O₄ NPs indicating minor variations in the local environment of HEWL.

4. Conclusions

In conclusion, we have explored the interactions of hen egg white lysozyme with cysteine capped Mn₃O₄ nanoparticles using spectroscopic techniques. The thermodynamic parameters associated with the binding were determined from ITC and it shows an exothermic binding. HEWL fluorescence quenching is observed due to the complex formation by Cy-Mn₃O₄ NPs and the static nature of quenching is revealed from fluorescence decay measurements. From the fluorescence spectroscopy, it is inferred that the Cy-Mn₃O₄ NPs bind near Trp-62 and Trp-63 residues in lysozyme. The binding constant and the number of binding sites determined from fluorescence and ITC are consistent with each other. FRET studies reveal that there is enough possibility of energy transfer between tryptophan residues and Cy-Mn₃O₄ NPs. The secondary structure of lysozyme complexed with Cy-Mn₃O₄ NPs was determined from circular dichroism and it shows partial unfolding of α-helical peptide strands (∼6%). This conformational change is not appropriate to induce any relevant conformational change in the overall conformation of HEWL and hence the function of HEWL is unaltered upon complexation with Cy-Mn₃O₄ NPs. The current investigations highlight the behaviour of Mn₃O₄ nanoparticles in the biological medium, which will certainly enhance the applications of nanoparticles in therapeutics and nano biotechnology.

Acknowledgements

The authors SP, DRR and RKT thank UGC for BSR-RFSMS fellowship and Dr M. Haridas Inter University Centre for Bioscience, Kannur University, Thalassery Campus, Palayad, Kerala, India for providing access to ITC.

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