Impact of Sinapic Acid on Bovine Serum Albumin Thermal Stability.

The thermal stability of bovine serum albumin (BSA) in Tris buffer, as well as the effect of sinapic acid (SA) on protein conformation were investigated via calorimetric (differential scanning microcalorimetry-µDSC), spectroscopic (dynamic light scattering-DLS; circular dichroism-CD), and molecular docking approaches. µDSC data revealed both the denaturation (endotherm) and aggregation (exotherm) of the protein, demonstrating the dual effect of SA on protein thermal stability. With an increase in ligand concentration, (i) protein denaturation shifts to a higher temperature (indicating native form stabilization), while (ii) the aggregation process shifts to a lower temperature (indicating enhanced reactivity of the denatured form). The stabilization effect of SA on the native structure of the protein was supported by CD results. High temperature (338 K) incubation induced protein unfolding and aggregation, and increasing the concentration of SA altered the size distribution of the protein population, as DLS measurements demonstrated. Complementary information offered by molecular docking allowed for the assessment of the ligand binding within the Sudlow's site I of the protein. The deeper insight into the SA-BSA interaction offered by the present study may serve in the clarification of ligand pharmacokinetics and pharmacodynamics, thus opening paths for future research and therapeutic applications.

Gelation Behaviour of Pluronic F127/Polysaccharide Systems Revealed via Thioflavin T Fluorescence.

Fast, reliable methods for characterizing the micelle-to-gel transition in emerging Pluronic F127/polysaccharide materials are essential for tailoring their applications as in situ gelling delivery systems. This study describes a simple fluorimetric method based on the response to gelation of the molecular probe thioflavin T (ThT). The techniques employed are (second derivative) steady-state and synchronous fluorescence. The capabilities of ThT as gelation reporter are tested for three model systems: Pluronic F127 (P16.6%), Pluronic F127/alginate (P16.6%ALG2%) and Pluronic F127/hyaluronic acid (P16.6%HA0.5%). We demonstrate that the changes in the short and long wavelength emissions of ThT allow accurate determination of the critical gelation temperatures in the investigated systems. The spectroscopic data providing information at molecular level are complemented with differential scanning microcalorimetric results revealing additional macroscopic insight into the micellization process. The gelation study is preceded by a solvatochromic analysis of ThT.

Trametes versicolor laccase activity modulated by the interaction with gold nanoparticles.

In this study, the impact of gold nanoparticles (AuNPs) on the structure and activity of laccase from Trametes versicolor (Lc) was described. Fluorescence experiments revealed that AuNPs efficiently quench Lc's tryptophan fluorescence by a static and dynamic process. By using differential scanning microcalorimetry and circular dichroism spectroscopy, it was determined how the concentration of nanoparticles and the composition of the medium affected the secondary structure of Lc. The data revealed that upon binding with AuNPs, conformational changes take place mainly in presence of high amounts of nanoparticles. The complex kinetic analysis unveiled the Lc activity enhancement at low concentrations of AuNPs as opposed to the concentrated regime, where it can be reduced by up to 55%. The Michaelis-Menten tests highlighted that the activity of the biocatalyst is closely related to the concentration of AuNPs, while the Selwyn analysis demonstrated that even in a concentrated regime of Lc it is not deactivated regardless of the amount of AuNPs added. The thermal parameters improved by twofold in the presence of low AuNPs concentration, whereas the activation energy increased with AuNPs content, implying that not all collisions are beneficial to the enzyme structure. The effect of AuNPs on the decomposition of a recalcitrant dye (naphthol green B, NG) by Lc was also evaluated, and the Michaelis-Menten model revealed that only the high AuNPs content influenced negatively the Lc activity. The isothermal titration calorimetry revealed that hydrogen bonds are the main intermolecular forces between Lc and AuNPs, while electrostatic interactions are responsible for NG adsorption to AuNPs. The results of the docking analysis show the binding of NG near the copper T1 site of Lc with hydrogen bonds, electrostatic and hydrophobic interactions. The findings of this work provide important knowledge for laccase-based bio-nanoconjugates and their use in the field of environmental remediation.

Physico-Chemical Changes Induced by Gamma Irradiation on Some Structural Protein Extracts.

In this study, the effect of gamma irradiation (10 kGy) on proteins extracted from animal hide, scales, and wool was evidenced by calorimetric (µDSC) and spectroscopic (IR, circular dichroism, and EPR) methods. Keratin was obtained from sheep wool, collagen and bovine gelatin from bovine hide, and fish gelatin from fish scales. The µDSC experiments evidenced that gamma irradiation influences the thermal stability of these proteins differently. The thermal stability of keratin decreases, while a resistance to thermal denaturation was noticed for collagen and gelatins after gamma irradiation. The analysis of the IR spectra demonstrated that gamma irradiation determines changes in the vibrational modes of the amide groups that are associated with protein denaturation, most meaningfully in the case of keratin. As evidenced by circular dichroism for all proteins considered, exposure to gamma radiation produces changes in the secondary structure that are more significant than those produced by UV irradiation. Riboflavin has different effects on the secondary structure of the investigated proteins, a stabilizing effect for keratin and fish gelatin and a destabilizing effect for bovine gelatin, observed in both irradiated and non-irradiated samples. The EPR spectroscopy evidences the presence, in the gamma-irradiated samples, of free radicals centered on oxygen, and the increase in their EPR signals over time due to the presence of riboflavin.

Revising Protein Corona Characterization and Combining ITC and Nano-DSC to Understand the Interaction of Proteins With Porous Nanoparticles.

The exposure of nanoparticles (NPs) to biological fluids leads to the formation of a protein coating that is known as protein corona (PC). Since PC formation is influenced by the physicochemical properties of the nanoparticles, the understanding of the interplay of the factors that participate in this process is crucial for the development of nanomaterials as cell-targeted delivery vehicles. In general, it is accepted that the PC formation is a complex and dynamic process, which depends on the composition of the medium and the properties of the NP mainly size, shape, and superficial charge. Interestingly, although the interaction between the protein and the NP is essentially a superficial phenomenon, the influence of the roughness of the nanoparticle surface has been scarcely studied. In this work, the influence of superficial roughness and porosity has been studied with the aid of nanodifferential scanning calorimetry (nano-DSC) and isothermal titration calorimetry (ITC) using mesoporous silica nanoparticles (MSNs) as an NP model. The interaction process of the proteins with the NP surface was analyzed by ITC measurements, while the stability and denaturation of the proteins was monitored by nano-DSC. Thanks to the complementarity of these two techniques, a more complete insight into the PC formation on the pores has been accomplished.

Towards FAIR nanosafety data.

Nanotechnology is a key enabling technology with billions of euros in global investment from public funding, which include large collaborative projects that have investigated environmental and health safety aspects of nanomaterials, but the reuse of accumulated data is clearly lagging behind. Here we summarize challenges and provide recommendations for the efficient reuse of nanosafety data, in line with the recently established FAIR (findable, accessible, interoperable and reusable) guiding principles. We describe the FAIR-aligned Nanosafety Data Interface, with an aggregated findability, accessibility and interoperability across physicochemical, bio-nano interaction, human toxicity, omics, ecotoxicological and exposure data. Overall, we illustrate a much-needed path towards standards for the optimized use of existing data, which avoids duplication of efforts, and provides a multitude of options to promote safe and sustainable nanotechnology.

Model Systems for Evidencing the Mediator Role of Riboflavin in the UVA Cross-Linking Treatment of Keratoconus.

Riboflavin under UVA radiation generates reactive oxygen species (ROS) that can induce various changes in biological systems. Under controlled conditions, these processes can be used in some treatments for ocular or dermal diseases. For instance, corneal cross-linking (CXL) treatment of keratoconus involves UVA irradiation combined with riboflavin aiming to induce the formation of new collagen fibrils in cornea. To reduce the damaging effect of ROS formed in the presence of riboflavin and UVA, the CXL treatment is performed with the addition of polysaccharides (dextran). Hyaluronic acid is a polysaccharide that can be found in the aqueous layer of the tear film. In many cases, keratoconus patients also present dry eye syndrome that can be reduced by the application of topical solutions containing hyaluronic acid. This study presents physico-chemical evidence on the effect of riboflavin on collagen fibril formation revealed by the following methods: differential scanning microcalorimetry, rheology, and STEM images. The collagen used was extracted from calf skin that contains type I collagen similar to that found in the eye. Spin trapping experiments on collagen/hyaluronic acid/riboflavin solutions evidenced the formation of ROS species by electron paramagnetic resonance measurements.

Thermodynamic Parameters at Bio-Nano Interface and Nanomaterial Toxicity: A Case Study on BSA Interaction with ZnO, SiO2, and TiO2.

Understanding nanomaterial (NM)-protein interactions is a key issue in defining the bioreactivity of NMs with great impact for nanosafety. In the present work, the complex phenomena occurring at the bio/nano interface were evaluated in a simple case study focusing on NM-protein binding thermodynamics and protein stability for three representative metal oxide NMs, namely, zinc oxide (ZnO; NM-110), titanium dioxide (TiO2; NM-101), and silica (SiO2; NM-203). The thermodynamic signature associated with the NM interaction with an abundant protein occurring in most cell culture media, bovine serum albumin (BSA), has been investigated by isothermal titration and differential scanning calorimetry. Circular dichroism spectroscopy offers additional information concerning adsorption-induced protein conformational changes. The BSA adsorption onto NMs is enthalpy-controlled, with the enthalpic character (favorable interaction) decreasing as follows: ZnO (NM-110) > SiO2 (NM-203) > TiO2 (NM-101). The binding of BSA is spontaneous, as revealed by the negative free energy, ΔG, for all systems. The structural stability of the protein decreased as follows: TiO2 (NM-101) > SiO2 (NM-203) > ZnO (NM-110). As protein binding may alter NM reactivity and thus the toxicity, we furthermore assessed its putative influence on DNA damage, as well as on the expression of target genes for cell death (RIPK1, FAS) and oxidative stress (SOD1, SOD2, CAT, GSTK1) in the A549 human alveolar basal epithelial cell line. The enthalpic component of the BSA-NM interaction, corroborated with BSA structural stability, matched the ranking for the biological alterations, i.e., DNA strand breaks, oxidized DNA lesions, cell-death, and antioxidant gene expression in A549 cells. The relative and total content of BSA in the protein corona was determined using mass-spectrometry-based proteomics. For the present case study, the thermodynamic parameters at bio/nano interface emerge as key descriptors for the dominant contributions determining the adsorption processes and NMs toxicological effect.

Interaction of piroxicam with bovine serum albumin investigated by spectroscopic, calorimetric and computational molecular methods.

The binding of drugs to serum proteins is governed by weak non-covalent forces. In this study, the nature and magnitude of the interactions between piroxicam (PRX) and bovine serum albumin (BSA) was assessed using spectroscopic, calorimetric and computational molecular methods. The fluorescence data revealed an atypical behavior during PRX and BSA interaction. The quenching process of tryptophan (Trp) by PRX is a dual one (approximately equal static and dynamic quenched components). The FRET results indicate that a non-radiative transfer of energy occurred. The association constant and the number of binding sites indicate moderate PRX and BSA binding. The competitive binding study indicates that PRX is bound to site I from the hydrophobic pocket of subdomain IIA of BSA. The synchronous spectra showed that the microenvironment around the BSA fluorophores and protein conformation do not change considerably. The Trp lifetimes revealed that PRX mainly quenches the fluorescence of Trp-213 situated in the hydrophobic domain. The CD and DSC investigation show that addition of PRX stabilizes the protein structure. ITC results revealed that BSA-PRX binding involves a combination of electrostatic, hydrophobic and hydrogen interactions. The analysis of the computational data is consistent with the experimental results. This thorough investigation of the PRX-BSA binding may provide support for other studies concerning moderate affinity drugs with serum protein.Communicated by Ramaswamy H. Sarma.

Quercetin Influence on Thermal Denaturation of Bovine Serum Albumin.

The effect of quercetin (QUER) binding on bovine serum albumin (BSA) thermal denaturation was systematically investigated by means of differential scanning calorimetry (DSC). Additional information concerning thermodynamic and structural binding parameters was provided by isothermal titration calorimetry (ITC) and molecular docking. The most relevant effect of QUER is manifested in the modification of the two-step thermal fingerprint of protein denaturation. Higher QUER concentrations result in a single-step denaturation thermogram, ascribed to the interplay between specific and nonspecific binding and enhancement of the solvent unfolding action. Analysis of ITC data indicate sequential binding of two molecules of QUER occurring spontaneously at different binding sites of BSA involving hydrophobic, electrostatic and hydrogen binding forces. Identification of QUER binding sites was possible through corroboration of DSC runs in the presence of site markers and molecular docking. Modeling of ligand-protein interaction confirmed the experimental data. On one hand, a neutral form of QUER binds in a nonplanar conformation to Sudlow's site I, a large hydrophobic cavity of subdomain IIA of BSA and decreases its thermal stability. On the other hand, a second molecule of QUER, the anionic form, is bound in planar conformation to Sudlow's site II, situated in the subdomain IIIA of the folded protein, and increases the thermal stability of the corresponding structural domain of the protein.

Interaction between Albumin and Pluronic F127 Block Copolymer Revealed by Global and Local Physicochemical Profiling.

The interaction of human serum albumin (HSA) with amphiphilic block copolymer Pluronic F127 has been investigated by several physical methods. Interest in studying this system stems from a broad range of bioactivities involving both macromolecules. Serum albumins constitute a significant class of proteins in the circulatory system, acting as carriers for a wide spectrum of compounds or assemblies. Pluronic block copolymers have revealed their capacity to ferry a variety of biologically active compounds. Circular dichroism, rheological measurements, and differential scanning microcalorimetry (µDSC) were employed to get insight into the interaction betweeen the two macromolecules. The results reveal that Pluronic F127 induces conformational changes to albumin if it is organized in a micellar form, while albumin influences the self-assembly of Pluronic F127 into micelles or gels. F127 micelles, however, induce smaller conformational changes compared to ionic surfactants. The µDSC thermograms obtained for HSA and/or F127 show that HSA shifts the critical micellar temperature (cmt) to lower values, while concurrently the HSA denaturation behavior is influenced by F127, depending on its concentration. Rheological measurements on solutions of F127 17% have shown that a sol-to-gel transition occurs at higher temperatures in the presence of HSA and the resulting gel is weaker. The global profile on HSA/F127 systems was complemented by local information provided by EPR measurements. A series of X-band EPR experiments was performed with spin probes 4-(N,N'-dimethyl-N-hexadecyl)ammonium-2,2',6,6'-tetramethylpiperidine-1-oxyl iodide (CAT16) and 5-doxyl stearic acid (5-DSA). These spin probes bind to albumin sites and are sensitive to phase transformations in Pluronic block copolymer solutions. For a given F127 concentration, the spin probe binds only to HSA below cmt and migrates to the F127 micelles above cmt. The collective data suggest soft interactions between the macromolecules, with the emerging results projecting potential applications linked to reaching optimal conditions for certain drug formulations.