Deciphering the mechanism of hafnium oxide nanoparticles perturbation in the bio-physiological microenvironment of catalase

Nanoparticles (NPs) are extensively being used in state-of-the-art nano-based therapies, modern electronics, and consumer products, so can be released into the environment with enhancement interaction with humans. Hence, the exposures to these multifunctional NPs lead to changes in protein structure and functionality, raising serious health issues. This study thoroughly investigated the interaction and adsorption of catalase (CAT) with HfO2-NPs by circular dichroism (CD), Fourier transform infrared (FTIR), absorption, and fluorescence spectroscopic techniques. The results indicate that HfO2 NPs cause fluorescence quenching in CAT by a static quenching mechanism. The negative values of Vant Hoff thermodynamic expressions (ΔH o , ΔS o , and ΔG o ) corroborate the spontaneity and exothermic nature of static quenching driven by van der Waals forces and hydrogen bonding. Also, FTIR, UV-CD, and UV–visible spectroscopy techniques confirmed that HfO2 NPs binding could induce microenvironment perturbations leading to secondary and tertiary conformation changes in CAT. Furthermore, synchronous fluorescence spectroscopy confirmed the significant changes in the microenvironment around tryptophan (Trp) residue caused by HfO2 NPs. The time depending denaturing of CAT biochemistry through HfO2-NPs was investigated by assaying catalase activity elucidates the potential toxic action of HfO2-NPs at the macromolecular level. Briefly, this provides an empathetic knowledge of the nanotoxicity and likely health effects of HfO2 NPs exposure.


Introduction
Hafnium oxide (HfO 2 ) NPs due to extraordinary physicochemical behavior possess the characteristics such as high melting point (2758°C), high dielectric constant (∼30), chemical stability, insulation potential, wide bandgap (>5.0 eV), and high neutron absorption cross-section, make them an excellent candidate to be used in a wide range of electronics and x-ray detection tools [1]. Recently, HfO 2 -NPs composites have found their way into medicine due to the demand for safety evaluation [2,3]. Europium doped hafnium dioxide NPs have developed for cellular optical imaging and as radiosensitive materials for clinical treatment [4]. In another study, inherently therapeutic polymeric silane conjugated hafnium oxide NPs (Hf-PS NPs) were proved very effective in eliminating the biofilms of Streptococcus mutans (most predominant and carious oral bacteria) by effectively killing the bacteria through 'latch and kill mechanism' in rodent models. Furthermore, these Hf-PS NPs have also enhanced targeted x-rays imaging [5]. Therefore, the protracted use of these hafnia nanocomposites would make their way into the environment, which in turn might be associated with some health issues. While the studies show biocompatibility and lower toxicity of HfO 2 -NPs toward human cells [6] and microorganisms [7], but there is no knowledge about the possible mechanism of HfO 2 NPs interaction with the biological molecules and affect their functionality. A lot of literature is available explaining the nano-material toxicity on human health [8][9][10]. There are also studies demonstrated that these NPs interact with biomolecules (proteins, lipids, and carbohydrates) and leads to alteration of native configuration and hence, the functionality of biomolecules [11,12] and accelerate the initiation and progression of diseases [13]. As soon as these biomolecules come into physical contact with NPs, biomolecules competitively adhere to the surface of nano-particles, constructing the protein corona, resulting in the mutated fortune and mechanism of nanomedicine and increased the nanotoxicology [14]. The composition of the external coating is determined by the size and surface properties of nanoparticles and equilibrium binding constants of each protein corona [15,16]. The final corona (hard corona) is permanent and able to mediate their interaction with other parts of the cell [17]. Hence, exploring the possible interactions of NPs with expected biomolecules in fluids of interest are of high significance. There is evidence that the NPs interaction induce the conformational alterations in the serum protein by distressing the secondary and tertiary architecture of albumins, and hence deviate from normal working [18,19]. Nano-carbon relentlessly distresses the transportability of serum proteins by fluctuating their architecture and anatomy [12,20]. Cobalt-doped iron oxide NPs also affects the 3D architecture and symmetry, so enhance the activity of acid phosphatases and bovine serum albumin [21,22]. Furthermore, these studies unveil the conceivable transportations of NPs into the body, demands the safety by design synthesis of NMs for biomedical applications.
Catalase is a ubiquitous oxidative stress-reducing enzyme by a disproportionation of two molecules of H 2 O 2 to one molecule of O 2 and two molecules of H 2 O [23]. As an important antioxidant enzyme, CAT plays a key role in protecting the cell from xenobiotics-induced stress tolerance [24]. Some large number of evidences provide data that the catalase is a significant player in multiple uncontrolled states such as abnormal blood sugar, aging, oxidative stress (disturbed biochemical reactions), and cancer (unwanted propagation of cells). Though, denaturing and repetition mechanism of catalase in living body lacks comprehensive understanding [25,26]. The inactivation of catalase would lead to H 2 O 2 accumulation in the cells and amplified oxidative stress leading to protein and DNA oxidation [27,28]. Furthermore, NPs exposure and inactivation of catalase links oxidative stress-mediated pathogenic pathways in tissue demolition [29]. Moreover, multiple studies ratified that the NMs attacking could significantly disturb the CAT free movement [30,31]. Though, the specific information about the interface of CAT and NPs is urgently needed.
Hence, this study aimed to analyze the conformational and purposeful transformations of CAT that began by interaction with HfO 2 -NPs. This research work revealed the likely toxicity of HfO 2 -NPs to the abundant universal antioxidant along with bonding parameters, sites, and conformational transformations in CAT with the help of bio-physical analytical techniques. Corresponding investigations would assist in understanding the toxic behavior of HfO 2 in-vitro and bring up to the minute scientific perceptions about a diverse range of nanomaterials.

Characterization of HfO 2 NPs
Morphology of HfO 2 NPs was perceived by a transmission electron microscope. XRD was done for crystalline structure analysis (PNAlytical, Netherlands) equipped with Cu Kα radiation. FTIR (Thermo Fischer Nicolet 6700, FTIR) was used to obtain the surface coatings.

Circular dichroism measurement
Secondary structure of the CAT through Circular dichroism measurements and was performed on JASCO spectropolarimeter (J-810, scan range190-260 nm, cuvette path length=100 mm) by fixing CAT at (2 μM) with graded HfO 2 NPs (50 to 250 μM). The mean residual ellipticity (MRE) was represented in deg cm 2 dem −1 according to the equation given below: Obs p Where Cp=Molarity of CAT, n=amino acid residues of CAT (240) and l=cuvette path-length (100 mm cm). The α-helical (% age) of free and conjugated CAT were mathematically determined at 208 nm from MRE considering the equation (2) Here, α i is a pre-exponential factor matching to the ith decay time constant τ i.

UV-spectrophotometry
Absorbance spectra of each suspension were graphed by scanning from 190 nm to 420 nm, through a double beam spectrophotometer (Shimadzu, Japan).

Fourier transforms infrared (FTIR) spectroscopy
The FTIR spectra of CAT were recorded with and without of HfO 2 NPs in the range from 500 cm −1 to 1800 cm −1 by fixing CAT and HfO 2 -NPs concentration at 2.0 μM in HBS buffer.

Thermogravimetric analysis
Thermogravimetric analysis (TGA) of CAT@HfO 2 -NPs conjugates were completed using a Perkin Elmer Thermal-Analyzer (Waltham, PA). The weight drops of each sample (HfO 2 NPs, CAT and CAT@HfO 2 NPs) was noted from 25°C to 700°C at a rate of 10°C, with continuous N 2 gas flow (50 ml min −1 ).   (table 1):

CAT activity assay
F o and F indicate FL strengths of CAT, with and without HfO 2 NPs, respectively. K sv is the Stern-Volmer constant and [HfO 2 ] indicates absorption of HfO 2 NPs. The mechanism of intrinsic FL quenching (static or dynamic) of Trp residue in CAT provide us very vital information about the dynamics between enzyme and quencher and examining K sv values at different temperatures could provide us with this information [35,36]. Dynamic and static quenching can be distinguished through simultaneous variation in FL and viscosity by varying temperature. High temperature endorses dynamic quenching via boosting diffusion and restricts the static quenching subsequently formation of less stable complexes [37]. The reduction in K sv (table 1) with increasing temperature, validates the supremacy of static quenching, through ground-state complex construction. Also, the mechanism of ground-state complex computed through quenching rate constant (k q ) equation (6) 298. 15 The observed kq value is 1.46×10 12 l mol −1 s −1 at 298.15 K, it's a higher quenching rate constant of the biopolymer (i-e., 2.0×10 10 l mol −1 s −1 ). The information in table 1 confirms a progressive reduction in quenching constant (k q ) with increasing temperature, indicating static quenching [11]. To extend the validation of static quenching, the number of binding sites and value of binding must decline with a temperature rise, after incubating with HfO 2 NPs, computed by the Lineweaver-Burk equation (7) [11,39].
Where K b is the binding constant (i-e., evidence of the existence of binding sites in a molecule whose occupation depends on the affinity of the ligand to the receptor-such a binding behavior is very specific and can be interpreted as a particular characteristic between the ligand and the target molecules) and n is a number of binding sites [40,41] (8) Where 'R' is the gas constant

Conformation transitions
3.3.1. Transitions in circular dichroism spectra Circular dichroism spectroscopy (CDS) was practiced for conformation transformations of proteins solutions quantitatively due to its sensitive analytical measurements [43]. The CDS spectra (figure 2) graphed two negative peaks at 210 and 225 nm, demonstrating the π→π * and n→π * electronic-transitions in CAT α-helix segment, respectively. The contents of the α-helix have shown in the inset of figure 2. Furthermore, isodichroic or isosbestic point is the wavelength where the molar absorptivity is the same for two (or more) protein spectra [44,45]. The existence of an isodichroic point for a given substance indicates a local two-state (α-helix and random coil) population computed at 203-205 nm range [46,47]. Therefore, analysis of the isodichroic point will distinguish a mutation that affects protein concentration calculation form that affecting native structure [48]. Given that, as shown in figure 2, the isodichroic point for CAT is at 203 nm demonstrates hafnium oxide NPs and catalase complex formation significantly decreases the α-helix complex formation with the increase in random coils in concentration-dependent manner. This complex formation also leads to compromising the intra protein H-bonding, hence decreasing the α-helix content with increasing random coil. Compare to the pristine CAT, α-helical number of CAT@HfO 2 NPs reduced from 81% to 53% (∼34% reduction); suggesting strong interactions among HfO 2 NPs and CAT. The reduced amount of α-helix and amplified β-sheet amount in CAT designated that HfO 2 NPs have developed attractions with aromatic amino acid residues (Trp and Tyr) of the primary polypeptide tail of CAT, destructs the hydrogen bonding systems within protein [49,50] leading to the exposing the active binding sites.

Transitions in synchronous fluorescence spectrometry (SFS)
Synchronous fluorescence spectrometry (SFS) used for fingerprinting the biological samples was introduced by Lloyd [11]. The salient features of SFS are sensitivity and simple spectra. In addition, SFS provides evidence about transformations in the molecular microenvironment of the fluorophore (e.g., residues of aromatic amino acids) functional group of CAT. In SFS, Δλ (Δλ=λ em −λ ex ) values were fixed to 20 nm for tyrosine (Tyr) and to 60 nm for tryptophan (Trp), showed the essential information of microenvironment as well as amino acid residues [51] as shown in figure 3. According to figure 3, diminishing FL intensity of Tyr residues, and the position of maximum emission wavelength temporarily shifted to a longer wavelength (redshift), by fixing Δλ to 60 nm. It corroborates transition in Trp microenvironment by the establishment of HfO 2 -CAT NPs complex. The optimum emission wavelength (λ max ) located at 330 nm to 332 nm designates that Trp residues are positioned in the hydrophobic area of CAT: λ max positioned at 345-352 nm further designates, Trp residues are less positioned in the hydrophilic region [52]. The redshift recommended poor hydrophobic microenvironment of Trp residue and ground state HfO 2 NP-CAT complex formation. Our results corroborate that HfO 2 -NPs probably bind to Trp residues of CAT, leading to subsequent conformation transition with amplified polarity around the Trp residues result in enhanced hydrophilicity [53,54]. Hereafter reasonable communication position of HfO 2 with CAT is nearby Trp amino-acid. A trivial redshift of SFS spectra for Δλ=60 nm stipulates conformation fluctuations in the micro-neighborhood of Trp residue with boosted hydrophilicity. These results are in good promise with the earlier studies [55]. Also, minor differences of SFS spectra at Δλ=15 nm clarifies  non-significant transformations in the micro-environment of Tyr residue with an increasing concentration of HfO 2 -NPs.

UV-vis Absorption spectroscopy
UV-vis absorption spectroscopy provides information related to structural transformations in a protein upon protein-NPs complex creation. The absorption band for active positioned around 192 nm is due to the π→π * transition in the polypeptide(C=O) structure [25]. The intensification of the absorbance at 192 and 278 nm indicates the HfO 2 NPs networked with CAT, shifting the CAT configurational conformation, results in a shifted micro-environment of the chromophores.
The absorption peak at about 404 nm corresponds to the Soret absorption band belonging to π→π * transitions of hematoporphyrin in catalase (inset of figure 4) [56]. The hyperchromic effect in the Soret region may be attributed to a conformational change in the vicinity of the heme moiety that leads to an alteration in the intensity of the π→π * transitions [57]. Hence, both the hyperchromic and slight red around 404 nm suggests that HfO 2 NPs are bound close to the active center of CAT and have developed relatively stronger interaction with the heme group [38,58]. Since the changes of the spectra in the Soret region correlate with the changes of CAT activity, we considered that HfO 2 NPs may result in an alteration of CAT activity. UV-vis absorption spectra have been generally commissioned to learn the mechanism of ground state protein-ligand complex formation ultimately leads to the quenching of the protein-ligand system [38]. Static quenching stimulates changes in the absorption spectra of the protein after complex formation at ground-state, on the contrary, dynamic quenching only disturbs the excited state of the fluorophores, and therefore absorption spectra indicate the slight transformations. Concisely, ground-state complex formation of HfO 2 NPs with CAT was a spontaneous process and static quenching was dominant quenching mechanism of CAT. This verified the results comprehended the time-resolved fluorescence assay.

Transitions in fourier transform infrared spectroscopy
FTIR spectroscopy is an admirable device for the scanning of protein-NPs interactions [43]. Figure 5 illustrates the comparative FTIR spectra of pristine CAT and CAT -HfO 2 NPs. The 1655.5, 1538.5, and 1393.8 cm −1 bands were linked to the α-helix primary amide, secondary amide, and tertiary amide, respectively [59]. Two bands, 1655.5 and 1538.5 cm −1 were attributed to elevated α-helix content indicated by the primary amide and secondary amide of CAT. The α-helix configuration injury was illustrated by intensity zone out at 1655.5, 1538.5, and 1393.8 cm −1 bands. The profound intensity destruction was assigned to enduring attractive forces among CAT and HfO 2 NPs [60] and initiating the transformations in secondary and tertiary structure conformation.

Energy transfer between HfO 2 NPs and CAT
Förster's non-radiative energy transfer will occur when the donor emission and absorption spectrum of the acceptor overlaps, provided the donor and acceptor distance is less than ∼8.0 nm [32,61]. The efficacy of energy transfer, E, was computed through equation (11) Here, 'r' is the mean distance linking the donor and acceptor and 'R 0 ' is critical distance with 50% transfer efficiency. The value of 'R 0 ' is computed through equation ( Here K 2 is the spatial direction linked with donor and acceptor dipoles, K 2 =2/3 for arbitrary orientation, for liquids; N is the refractive index of the medium.; and Φ is the donor quantum yield. J is the extent of spectral overlap amid donor fluorescence and acceptor absorption spectrum, and computed through equation ( Here, F(λ) is the donor fluorescence intensity at wavelength λ, and ε(λ) is the molar absorption coefficient of the acceptor at wavelength λ. Figure 6 illustrated the intersection of the absorption spectrum of HfO 2 NPs and the CAT emission spectrum. Considering equations (11)-(13), by means of K 2 =2/3, n=1.336 and Φ =0.15 for CAT, we calculated: J=92.5×10 −17 cm 3 L/mol, R 0 =2.55 nm, r=2 nm and E=68.2%. In the case of HfO 2 NPs-CAT system, observed donor-acceptor distance (r) <8 nm further confirming the static quenching was predominant. The mean tiny distance amid CAT-HfO 2 NPs advocate the stronger attractive electrostatic forces that exist between HfO 2 NPs and CAT conjugate.

Transitions in fluorescence lifetime analysis
Fluorescence lifetime is the state of the art analytical tool practiced for identification of transitions around the microenvironment of a fluorophore, and for differentiating the static and dynamic fluorescence quenching [63]. Therefore, FL lifetime analysis further confirms the dominance of static quenching in the HfO 2 -CAT system ( figure 7). CAT (Without HfO 2 NPs) illustrates the mean FL lifetime of 4.03 ns from equation (4), while a negligible decrease in FL 4.02 ns was observed with a graded concentration of HfO 2 NPs, clearly represents the agitations in the microenvironment [64]. The data obtainable in table 2 demonstrate the insignificant transformations in the FL lifetime of CAT in the presence of HfO 2 NPs. Also, the data corroborate the establishment of a non-fluorescent ground state complex between CAT and HfO 2 NPs. It is also a deep-rooted reality that the establishment of static ground state complex does not reduce the decay-time of the non-complexed fluorophore since only the life-times of unquenched fluorophores are sensed in the time-resolved spectroscopy [65]. This further corroborates the outcomes of the steady-state FL quenching. Typically, Trp lifetimes are reduced in polar environments [64].

Red-edge excitation energy shifts (REES)
For polar fluorophore with incomplete solvent reduction emission spectra shows redshifts at excitations on a longer wavelength region of the absorption spectrum, this phenomenon is known as REES [32]. This phenomenon is a signal for an abruptly dense dynamic microenvironment of Trp residue with reduced access to the solvent and stimulated through electronic coupling amid Trp indole rings and neighboring dipoles [61,66]. Consequently, REES is predominantly advantageous in tracking dynamic shifting around the Trp residues in proteins. Calculating the FL emission and red edge excitation shift (REES) of CAT after incubation with HfO 2   NPs (CAT-HfO 2 NPs complex), provide us the information about the microenvironment and mobility of Trp residue. The Aftermath of REES study for the CAT-HfO 2 conjugate (table 3) demonstrates the REES value is 20. This is significantly elevated than the REES value of CAT (without HfO 2 NPs), demonstrating HfO 2 NPs enforce the unfolding of CAT revealing the fundamental fluorophore by early complex creation by establishing the attractive forces with the hydrophobic domains of CAT limiting free motion of Trp microenvironment [61,67].

Adsorption of CAT on HfO 2 NPs
Furthermore, quantitative analysis was performed to investigate the adsorption of CAT on HfO 2 NPs through TGA. Thermograms at 700°C reveal that HfO 2 NPs (Supplementary figure 3, blue thermogram) exhibit the lowest weight loss due to solid Hf core, whereas CAT (Supplementary figure 3, black thermogram) showed optimum weight loss due to extreme protein degradation at higher temperatures. The weight loss of all CAT@HfO 2 NPs (Supplementary figure 3, red thermogram) was between those of the HfO 2 NPs and the CAT sample.
3.5. Influence of HfO 2 NPs on the functionality of CAT According to the above experimental results, attractive forces were operational between HfO 2 -NPs and CAT leads to the configurational alteration in the conformation of CAT. We are familiar that enzyme activity correlates directly to its conformation [38]. Therefore, a sensitive assay is imperative to perform with and without incubating with HfO 2 NPs to elaborate on the relationship between configurational changes and activity disparity of CAT. Every subunit possesses a single heme, an integral part of the CAT active center. The effect of HfO 2 on CAT activity was presented in figure 8; the catalase activity revealed a decline in CAT activity is associated with the incubation with HfO 2 NPs.
As the concentration of HfO 2 increased from 0 to 250 mM, the catalytic activity CAT decreased to 44%, 63%, and 69.2% compared to control with incubation times of 15, 30, and 60 min, respectively. The conformational transformation of the CAT complex in combination with the above spectral computation Table 3. REES effects of HfO 2 NPs (λ ex 280 nm and λ ex 295 nm). displayed that activity inhibition of CAT instigated by incubating with HfO 2 NPs, and results in structural transformations. However, HfO 2 NPs could also influence the activity of CAT directly through binding with the active center of CAT, (figure 8). Similar results could be found in the work of Wei et al which evidenced the graphene oxide inhibited CAT activity through attacking the active center. This hypothesis was validated by confirming the change of the microenvironment of Tyr, Phe which binds near to the intense center and has a close relationship with the activity of CAT [56].

Conclusion
The attractive forces amid CAT and HfO 2 NPs were explored by a diverse range of spectroscopic tools. We also concluded that the HfO 2 NPs attached with CAT through ground-state complex generation directing the dominance of static FL quenching and the bonding course is greatly impulsive with loss in energy. The computation of UV-visible, FTIR, and UV-CD, spectroscopy exemplify obvious transformations in secondary (2 o ) and tertiary(3 o ) configurations of CAT upon incubation with HfO 2 -NPs. Likewise, the SFS spectra at Δ60 nm authenticate the disorder simulated around the Trp residue microenvironment instigated through HfO 2 NPs. Furthermore, TGA thermograms again established a profuse coating of CAT on HfO 2 NPs surfaces. In brief, results advocated that HfO 2 NPs markedly weaken the catalytic activity and ultimately the performance of proteins. Consequently, demanded a profound consideration of NPs' impressions on biological systems before their extensive utilization in daily consumer goods.