Investigation of the effects of nanoplastic polyethylene terephthalate on environmental toxicology using model Drosophila melanogaster.

Plastic pollution has become a major environmental concern, and various plastic polymers are used daily. A study was conducted to examine the toxic effects of polyethylene terephthalate (PET) nanoplastics (NPLs) on Drosophila melanogaster. We have successfully synthesized PET NPLs and characterized using DLS, Zeta potential, TEM, HRTEM, SAED, XRD, FTIR, and Raman spectroscopy to gain crucial insights into the structure and properties. We fed PET NPLs to Drosophila to assess toxicity. ROS was quantified using DCFH-DA and NBT, and the nuclear degradation was checked by DAPI staining. Quantification of protein and activity of antioxidant enzymes like SOD, catalase depicted the adverse consequences of PET NPLs exposure. The dorsal side of the abdomens, eyes, and wings were also defective when phenotypically analyzed. These results substantiate the genotoxic and cytotoxic impact of nanoplastics. Notably, behavioral observations encompassing larval crawling and climbing of adults exhibit normal patterns, excluding the presence of neurotoxicity. Adult Drosophila showed decreased survivability, and fat accumulation enhanced body weight. These findings contribute to unraveling the intricate mechanisms underlying nanoplastic toxicity and emphasize its potential repercussions for organismal health and ecological equilibrium.

Antibacterial and cytotoxicity studies of pyrrolo-based organic scaffolds and their binding interaction with bovine serum albumin.

Two pyrrolo-based compounds, 1H-pyrrolo[3,2-b]pyridine-3-carboxylic acid (L1) and 1H-pyrrolo[3,2-c]pyridine-4-carboxylic acid (L2), were employed for the detection of bovine serum albumin (BSA) by UV-Vis and fluorescence spectroscopic methods in phosphate buffer solution (pH = 7). In the presence of L1 and L2, the fluorescence emission of BSA at 340 nm was quenched and concomitantly a red-shifted emission band appeared at 420 nm (L1)/450 nm (L2). The fluorescence spectral changes indicate the protein-ligand complex formation between BSA and L1/L2. An isothermal titration calorimetry (ITC) experiment was conducted to determine the binding ability between BSA and L1/L2. The binding constants are found to be 4.45 ± 0.22 × 104 M-1 for L1 and 2.29 ± 0.11 × 104 M-1 for L2, respectively. The thermodynamic parameters were calculated from ITC measurements (i.e. ∆rH = -40 ± 2 kcal/mol, ∆rG = -4.57 ± 0.22 kcal/mol and -T∆rS = 35.4 ± 1.77 kcal/mol), which indicated that the protein-ligand complex formation between L1/L2 with BSA is mainly due to the electrostatic interactions. The protein-ligand interactions were studied by performing molecular docking. Further, the antibacterial assay of L1 and L2 was conducted against gram-positive and gram-negative bacterial strains in an effort to address the difficulties caused by the co-occurrence of antimicrobial and multidrug-resistant bacteria. E. coli and S. aureus were significantly inhibited by L1 and L2. The L1 exhibits 13, 12 and 15 mm, whereas L2 exhibits a 2, 3 and 5 mm zone of inhibition against S. aureus, S. pyogenes and E. coli, respectively. In silico molecular docking of L1 and L2 was performed with bacterial DNA gyrase to establish the intermolecular interactions. Finally, the in vitro cytotoxicity activities of the ligands L1 and L2 have been carried out using drosophila.

Sulfation of hyaluronic acid reconfigures the mechanistic pathway of bone morphogenetic protein-2 aggregation.

Bone morphogenetic protein-2 (BMP-2) is a critical growth factor of bone extracellular matrix (ECM), pivotal for osteogenesis. Glycosaminoglycans (GAGs), another vital ECM biomolecules, interact with growth factors, affecting signal transduction. Our study primarily focused on hyaluronic acid (HA), a prevalent GAG, and its sulfated derivative (SHA). We explored their impact on BMP-2's conformation, aggregation, and mechanistic pathways of aggregation using diverse optical and rheological methods. In the presence of HA and SHA, the secondary structure of BMP-2 underwent a structured transformation, characterized by a substantial increase in beta sheet content, and a detrimental alteration, manifesting as a shift towards unstructured content, respectively. Although both HA and SHA induced BMP-2 aggregation, their mechanisms differed. SHA led to rapid amorphous aggregates, while HA promoted amyloid fibrils with a lag phase and sigmoidal kinetics. Aggregate size and shape varied; HA produced larger structures, SHA smaller. Each aggregation type followed distinct pathways influenced by viscosity and excluded volume. Higher viscosity, low diffusivity of protein and higher excluded volume In the presence of HA promotes fibrillation having size in micrometer range. Low viscosity, high diffusivity of protein and lesser excluded volume leads to amorphous aggregate of size in nanometer range.

Coumarin-Based Noncytotoxicity Fluorescent Dye for Tracking Actin Protein in In-Vivo Imaging.

Drosophila shares maximum homology with the human disease-causing genes and thus has been employed to evaluate the toxicity of numerous compounds. Further, its distinguishable developmental stages, easy rearing, and short lifespan make it a perfect model organism to study toxicological properties of any new compound. The current study evaluates the toxic effect of a coumarin-based organic fluorescent dye, 7-hydroxy-4-methyl-8-((4-(2-oxo-2H-chromen-3-yl)thiazol-2-ylimino)methyl)-2H-chromen-2-one (CTC), using Drosophila melanogaster as a model organism by studying different behavioral, screening, and staining techniques using Oregon-R flies. For toxicity assessment, one control fly group was compared with various flies that had been subjected to fed CTC dye orally of different concentrations (0.5, 1, 2.5, and 5 µg/mL). The 3rd instar larvae were checked for the larvae crawling assay. The crawling assay demonstrates that the speed and path of the treated larvae are almost equal to the control ones, which signifies the non-neurotoxic property of CTC. Trypan blue assay further suggested that the dye does not cause any major damage to the gut. Phalloidin staining revealed that the actin composition remains unaltered even after the CTC treatment, while the DAPI staining experiment indicates that CTC does not cause any nuclear damage to fly gut cells. However, at a concentration of 5 µg/mL, CTC causes developmental delay. The flies hatched after larval treatment of CTC do not show any structural defects, suggesting clearly that CTC is also nongenotoxic to Drosophila. The current studies propose CTC as a noncytotoxic and nongenotoxic dye to track actin protein in the model organism D. melanogaster.

Probing the interaction between niobium pentoxide nanoparticles and serum albumin proteins by Spectroscopic approaches.

Nanoparticles (NPs) can directly or indirectly enter into the body because of their small size; then they tend to alter the conformation and function of proteins upon interaction with them. Thus, it is crucial to understand the impact of NPs in a biological medium. Recently, niobium pentoxide nanoparticles (Nb2O5 NPs) are finding increasing applications in the biological system, for example, bone tissue and dental material, matrix for biosensing of proteins, etc. In all such applications, the Nb2O5 NP interacts with proteins and other biomolecules. Hence, the study of such interactions is of considerable importance. Here in this work, we present the impact of Nb2O5 NP on the structure, stability and activity of blood proteins, bovine serum albumin (BSA) and human serum albumin (HSA) by means of various spectroscopic approaches. Steady-state fluorescence studies indicated that intrinsic fluorescence intensities of both serum albumin proteins got quenched upon their interaction with NP. The nature of the quenching was elucidated by time-resolved fluorescence and absorption measurements. Using circular dichroism (CD) and synchronous fluorescence spectroscopy (SFS), the structural perturbations of the protein molecules after interaction with NP were investigated. Moreover, the role of temperature on protein stability upon complexation with NP was also explored. In addition, the effect of NP on protein functionality was probed by esterase-like activity assays.Communicated by Ramaswamy H. Sarma.

Synthesis of First Coumarin Fluorescent Dye for Actin Visualization.

Selective fluorescence imaging of actin protein hugely depends on the fluorescently labeled actin-binding domain (ABD). Thus, it is always a challenging task to image the actin protein (in vivo or in vitro) directly with an ABD-free system. To overcome the limitations of actin imaging without an ABD, we have designed a facile and cost-effective red fluorescent coumarin dye 7-hydroxy-4-methyl-8-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-ylimino)methyl-2H-chromen-2-one (CTC) for actin binding. The selective binding of the dye was investigated using the gut and eye of the model organism Drosophila melanogaster and C2C12 and SCC-9 cell lines. Our result suggests two major advantages of CTC over the dyes presently used for imaging actin proteins. First, the dye can bind to actin efficiently without any secondary intermediate. Second, it is much more stable at room temperature and exhibits excellent photostability. To the best of our knowledge, this is the first fluorescent dye that can bind to the actin protein without employing any secondary intermediate/actin-binding domain. These findings could pave the way for many biologists and physicists to successfully employ the CTC dye for imaging and tracking actin proteins by fluorescence microscopy in various in vivo and in vitro systems.

Impact of chromate and dichromate on lysozyme stability: A spectroscopic and molecular docking investigation.

A comparative study of interaction between chicken egg white lysozyme (Lyz) with two hexavalent chromate ions; chromate and dichromate; which are prevalently known for their toxicity, was investigated using different spectroscopic techniques along with a molecular docking study. Both steady-state and time-resolved studies revealed that the addition of chromate/dichromate is responsible for strong quenching of intrinsic fluorescence in Lyz and the quenching is caused by both static and dynamic quenching mechanisms. Different binding and thermodynamic parameters were also calculated at different temperatures from the intrinsic fluorescence of Lyz. The conformational change in Lyz and thermodynamic parameters obtained during the course of interaction with chromate/dichromate were well-supported by the molecular docking results.

Conformational dynamics of myoglobin in the presence of vitamin B12: A spectroscopic and in silico investigation.

Myoglobin is an essential transport protein of heart and muscle tissues that acts as a local oxygen reservoir and a marker in different diseased conditions. On the other hand, Vitamin B12 is a vital nutrient that helps synthesize red blood cells, DNA, and proteins. To understand the ability of vitamin B12 to bind to the excess of myoglobin produced in the body under certain conditions (muscle injuries, severe trauma, etc.), it is essential to dig into the interaction between them. Therefore, the present study reports the binding interaction of vitamin B12 and myoglobin employing different spectroscopic and computational methods. The myoglobin's intrinsic fluorescence is quenched by vitamin B12 via static nature as observed from steady-state as well as time-resolved fluorescence measurements. The microenvironment of myoglobin's tryptophan residue gets affected, but there is no change observed in its α-helical content by vitamin B12 as seen from synchronous fluorescence and circular dichroism measurements. The probable binding of vitamin B12 on myoglobin was elucidated through molecular docking, and the interaction stability was studied by molecular dynamics simulation. The determination of vitamin B12's affinity to myoglobin and its effect on the conformational transitions of myoglobin might afford valuable insight for clinical pharmacology.

Spectroscopic and computational insight into the conformational dynamics of hemoglobin in the presence of vitamin B12.

Protein-ligand interactions play a significant role in all living organisms, thereby affecting the design and application of drugs and other biomaterials. The current study reports the binding of vitamin B12 to hemoglobin, employing optical spectroscopy and computational methods. It is observed that vitamin B12 quenched the intrinsic fluorescence of hemoglobin. The nature of quenching appears to be static according to the steady-state and time-resolved fluorescence measurements. The conformational changes of hemoglobin caused by vitamin B12 interactions were studied by synchronous fluorescence spectroscopy and protein secondary structure analyses. The synchronous fluorescence spectra indicate the tryptophan residue microenvironment change while no secondary structural change is observed from circular dichroism spectra and molecular dynamics (MD) simulation study. The computational molecular docking elucidated the probable binding of vitamin B12 at the active site of hemoglobin, whereas the stability of the hemoglobin-vitamin B12 complex was studied by MD simulation. The study might be helpful for the treatment of pernicious anemia, hereditary transcobalamin deficiency, and performance enhancement of elite athletes.

Conformational changes of GDNF-derived peptide induced by heparin, heparan sulfate, and sulfated hyaluronic acid - Analysis by circular dichroism spectroscopy and molecular dynamics simulation.

Glial-cell-line-derived neurotrophic factor (GDNF) is a protein that has therapeutic potential in the treatment of Parkinson's disease and other neurodegenerative diseases. The activity of GDNF is highly dependent on the interaction with sulfated glycans which bind at the N-terminus consisting of 19 residues. Herein, we studied the influence of different glycosaminoglycan (i.e., glycan; GAG) molecules on the conformation of a GDNF-derived peptide (GAG binding motif, sixteen amino acid residues at the N-terminus) using both experimental and theoretical studies. The GAG molecules employed in this study are heparin, heparan sulfate, hyaluronic acid, and sulfated hyaluronic acid. Circular dichroism spectroscopy was employed to detect conformational changes induced by the GAG molecules; molecular dynamics simulation studies were performed to support the experimental results. Our results revealed that the sulfated GAG molecules bind strongly with GDNF peptide and induce alpha-helical structure in the peptide to some extent.

Polyethylene glycol perturbs the unfolding of CRABP I: A correlation between experimental and theoretical approach.

The importance of macromolecules paves the way towards a detailed molecular level investigation as all most all cellular processes occurring at the interior of cells in the form of proteins, enzymes, and other biological molecules are significantly affected because of their crowding. Thus, exploring the role of crowding environment on the denaturation and renaturation kinetics of protein molecules is of great importance. Here, CRABP I (cellular retinoic acid binding protein I) is employed as a model protein along with different molecular weights of Polyethylene glycol (PEG) as molecular crowders. The experimental evaluations are done by accessing the protein secondary structure analysis using circular dichroism (CD) spectroscopy and unfolding kinetics using intrinsic fluorescence of CRABP I at 37 °C to mimic the in vivo crowding environment. The unfolding kinetics results indicated that both PEG 2000 and PEG 4000 act as stabilizers by retarding the unfolding kinetic rates. Both kinetic and stability outcomes presented the importance of crowding environment on stability and kinetics of CRABP I. The molecular dynamics (MD) studies revealed that thirteen PEG 2000 molecules assembled during the 500 ns simulation, which increases the stability and percentage of ß-sheet. The experimental findings are well supported by the molecular dynamics simulation results.

Fe3O4 coated guargum nanoparticles as non-genotoxic materials for biological application.

The current study aims to check various behavioural, developmental, cytotoxic, and genotoxic effects of Fe3O4-GG nanocomposite (GGNCs) on Drosophila melanogaster. Fe3O4 nanoparticles were prepared by the chemical co-precipitation method and cross-linked with guargum nanoparticles to prepare the nanocomposites. The nanocomposites were characterized by using transmission electron microscopy (TEM), X-ray diffraction (XRD), and FTIR techniques. To investigate the biomolecular interaction, GGNCs was further tagged with Fluorescein isothiocyanate. Various concentrations of nanocomposites were mixed with the food and flies were allowed to complete the life cycle. The life cycle of the flies was studied as a function of various concentrations of GGNCs. The 1st instar larvae after hatching from the egg start eating the food mixed with GGNCs. The 3rd instar larvae were investigated for various behavioural and morphological abnormalities within the gut. The 3rd instar larva has defective crawling speed, crawling path, and more number of micronuclei within the gut. Similarly, in adult flies thermal sensitivity, climbing behaviour was found to be altered. In adult flies, a significant reduction in body weight was found which is further correlated with variation of protein, carbohydrate, triglyceride, and antioxidant enzymes. Altogether, the current study suggests GGNCs as a non-genotoxic nanoparticle for various biological applications.

Exploring the chemistry behind protein-glycosaminoglycan conjugate: A steady-state and kinetic spectroscopy based approach.

The impact of glycosaminoglycan (chondroitin sulphate, CS) on bone morphogenetic protein - 2 (BMP - 2) structure, stability (thermal and chemical), association kinetics and conformation was monitored by multiple spectroscopic techniques (UV-Visible, fluorescence and circular dichroism). The absorbance in peptide region and fluorescence intensity of BMP - 2 was quenched in presence of CS; thus, confirming the formation of a ground-state complex. As there was an increase in Stern-Volmer constant observed as a function of temperature, idea of dynamic quenching was established. However, the negligible changes in lifetime indicated static quenching; thus, making the process a combination of static-dynamic quenching. Basically, the protein - glycan interaction was driven by entropy of the system and mediated by hydrophobic interactions. Secondary structure (CD spectroscopy) of native protein was significantly affected (intensity became more negative) in presence of CS, thus, introducing more compactness in the protein. CS infused thermal and chemical stability into BMP - 2 via alteration in its conformation. The rate of association was inversely proportional to concentration of quencher (CS), which confirmed the correlation between large size (~ 5 times the size of protein) and structural complexity of CS with fewer binding sites present in BMP - 2. The rate of association in presence of urea, suggested a decrease in association rate as a function of urea concentration for 15 µM CS. Experimental evidences suggested an interaction between protein and glycan mediated by hydrophobic interactions, which deciphers structural, thermal and chemical stability into protein.

Tyrosine mediated conformational change in bone morphogenetic protein - 2: Biophysical implications of protein - phytoestrogen interaction.

The biophysical aspects of the binding interaction between a phytoestrogen (quercetin, QT) and bone morphogenetic protein - 2 (BMP - 2) was analyzed by various spectroscopic, calorimetric and molecular docking techniques. Interaction studies represented a loss in the absorbance of protein (only the amide region) along with a prominent red shift indicating ground-state complexation which was further confirmed by quenching with significant blue shift observed from steady-state fluorescence measurements. To narrow down the involvement of aromatic residues (Tyr & Trp), synchronous fluorescence spectroscopy was employed. Both Tyr and Trp fluorescence intensity was quenched, however, shifting was noticed only in case of Tyr residues; thus, confirming the alteration in confirmation was mediated upon reduction in polarity around tyrosine residues. It was further validated by quenching studies which highlighted the existence of a buried fraction of fluorophore upon interaction. The nature of fluorescence quenching was static and the binding efficiency was low (binding constant K ~ 10-2 M). Mechanistically, the involvement of van der Waals and hydrogen bonding interaction was confirmed from both van't Hoff plot and molecular docking studies. Secondary structure and thermal stability of the protein was not significantly affected by quercetin. All these investigations confirmed a significant effect on the structure and conformation of BMP - 2 in presence of quercetin which might serve as a potential therapeutic for the treatment of osteoporosis in postmenopausal women.

Biophysical study on complex formation between ß-Lactoglobulin and vitamin B12.

Biophysical insight into the binding interaction between the major whey protein, ß-Lactoglobulin (ßLG) and vitamin B12, was studied using different spectroscopic tools such as steady-state & time-resolved fluorescence spectroscopy, Circular Dichroism (CD) and Fluorescence Correlation Spectroscopy (FCS). The intrinsic fluorescence of ßLG was quenched by vitamin B12. From the time-resolved fluorescence experiment, the nature of quenching was found to be static suggesting ground-state complex formation between ßLG and vitamin B12, which was also supported by the excitation spectra. Synchronous fluorescence spectra revealed that the tryptophan residue microenvironment of ßLG was affected by the vitamin B12. The CD spectra suggested that the secondary structure of the ßLG remains unaffected by vitamin B12. From the FCS experiment, the tertiary structure of ßLG was observed to be stable in the presence of vitamin B12 at the single-molecule level. The outcome of this study might have potential applications in the food and pharmaceutical industry.

Molecular interactions of MnO2@RGO (manganese dioxide-reduced graphene oxide) nanocomposites with bovine serum albumin.

Graphene based materials have attracted global attention due to their excellent properties. GO-metal oxide nanocomposites have been conjugated with biomolecules for the development of novel materials and potentially used as biomarkers. Herein, a detailed study on the interaction of Bovine serum albumin (BSA) with MnO2@RGO (manganese dioxide-reduced graphene oxide) nanocomposites (NC) has been carried out. MnO2@RGO nanocomposites were prepared through a template/surfactant free hydrothermal route at 180 °C for 12 h by varying the graphene oxide (GO) concentration. Different biophysical experiments have been carried out to evaluate molecular interactions between BSA and NCs. Intrinsic fluorescence has been used to quantify the quenching efficiency of NCs and the binding association of BSA-NC complexes. NCs effectively quenched the intrinsic fluorescence of BSA via static and dynamic mechanism. Further, the results indicate that the molecular interactions of NC with BSA are dependent on the GO percentage in NC. Circular dichroism results demonstrate nominal changes in the secondary structure of BSA in presence of NCs. Also, the esterase-like activity of BSA was marginally affected after adsorption upon NCs. In addition, the FESEM micrographs reveal that the protein-NC complexes consist of nanorod and sheet-like morphologies are forming aggregates of different sizes. We hope that this study will provide a basis for the design of novel graphene based and other related nanomaterials for several biological applications.Communicated by Ramaswamy H. Sarma.

Impact of bone extracellular matrix mineral based nanoparticles on structure and stability of purified bone morphogenetic protein 2 (BMP-2).

Bone Morphogenetic Protein 2 (BMP-2) is an osteoinductive protein which has been overexpressed, refolded (Refolding is often the bottle-neck step in producing recombinant proteins from inclusion bodies of Escherichia coli, especially for dimer proteins) and purified by using Heparin affinity chromatography. Refolding of BMP-2 was based on gradient dialysis in presence of lower urea concentration. The main objective of the present work is to unravel the impact of the extracellular matrix components on BMP-2 conformation and stability. We tried to elucidate the interaction of the bone matrix minerals in the form of nanoparticles with the bone protein. We chose hydroxyapatite nanoparticles (HAp NPs) which is the most abundant bone mineral and the other being a trace mineral in our bones i.e., zinc oxide nanoparticles (ZnO NPs) as the potential nanoparticles for this study. The isolated protein is found to be a ß- sheet type with melting temperature being approximately 70.66 °C. Upon interaction with HAp NPs and ZnO NPs, the absorbance and the fluorescence intensity indicates the interaction with the protein as there was an upsurge in both the cases. Circular Dichroism (CD) spectroscopy revealed that ZnO NPs are having more dominant secondary structure and thermal stabilizing effect as compared to HAp NPs.

Phosphate and sulphate-mediated structure and stability of bone morphogenetic protein - 2 (BMP - 2): A spectroscopy enabled investigation.

Impact of different monovalent and divalent cationic salts of sulphates and phosphates on conformation and stability of BMP - 2 was unraveled by absorbance, fluorescence and circular dichroism (CD) spectroscopy. Increase in absorbance of protein confirms the ground-state complexation between salt and BMP - 2. Phosphate salts, with the exception of sodium phosphate quenched the fluorescence intensity. The nature of quenching was static, as revealed by temperature-dependent fluorescence studies (Stern-Volmer constant (KSV) decreased with rise in temperature). Moreover, kq (bimolecular quenching constant) was in the range of 1012 M-1 s-1, confirming binding of phosphate salts with the protein. Contrary to this, sulphate salts increased the fluorescence intensity and excited-state lifetime of BMP - 2 (2.668 ns), with the maximum calculated for 300 mM sodium sulphate (3.216 ns). Phosphates reduced the lifetime of protein, with the least observed in presence of 300 mM magnesium phosphate (1.480 ns). Thermal stability of the protein (Tm = 70.66 °C) was altered significantly upon interaction with phosphate salts; however, it did not vary significantly in case of sulphates (exception - magnesium sulphate). Experimental evidences confirm the role played by anionic group on protein conformation and stability and identifies monovalent and divalent cations as insignificant contributor.

The contribution of nanostructures towards the wing patterning of yellow Catopsilia pomona. How it differs from the lime?

Polyphenism, an adaptation to survive throughout the year, is shown by many butterflies including Catopsilia pomona. With the variation of seasons, different morphs were found. Among all the morphs, lime exists throughout the year whereas the yellow one is available only in the winter season. The current study deciphers the colouration mechanism of yellow morph using various microscopic and spectroscopic techniques. The scanning electron microscopy analysis reveals various types of scales on the dorsal as well as the ventral side. The shape of the cover scale varies from region to region. The fine structural arrangement of the scale like window, ridge, microrib, crossrib and pigments vary throughout the wing. The pigment present in the wing is pterin as evidenced from the shape and its isolation technique. Absorption spectroscopy further confirms the presence of various types of pterin within the wing. Scanning electron microscopy discloses the dense amount of pigments within the wing. The fine structural arrangement of the wing of yellow C. pomona is compared with the yellow region of the lime C. pomona. All together, the current study describes the fine structural arrangement of the wing of yellow C. pomona and the various types of pterin which contribute towards the wing colouration. The advantage of yellow morph over lime is also discussed in this paper.

Insights into the binding interaction between copper ferrite nanoparticles and bovine serum albumin: An effect on protein conformation and activity.

The binding affinity between bovine serum albumin (BSA) and copper ferrite (CuFe2 O4 ) nanoparticles in terms of conformation, stability and activity of protein was studied using various spectroscopic methods. The quenching involved in BSA-CuFe2 O4 NP interaction was static quenching as analysed by different techniques (steady-state and time-resolved fluorescence along with temperature-dependent fluorescence measurements). Among all types of possible interactions, it was revealed that the major binding forces were van der Waals interaction and hydrogen bonding, which were explored from negative values of enthalpy change (∆H = -193.85 kJ mol-1 ) and entropy change (∆S = -588.88 J mol-1  K-1 ). Additionally, synchronous, circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy measurements confirmed the conformational changes in BSA upon the addition of CuFe2 O4 NP. Furthermore, thermal denaturation observations were consistent with the circular dichroism results. The interaction of CuFe2 O4 NP with BSA decreased the esterase activity in the BSA assay, revealing the affinity of copper ferrite towards the active site of BSA.