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The demand for bioactive molecules, such as ß- carotene (ß-car), has increased, but some characteristics such as low water solubility and low photo stability limit its application in many formulations. The bioactive entrapment into milk proteins may overcome this barrier. Thus, the aim of this work was to study the interaction between ß-car and bovine serum albumin (BSA) or ß-casein and the photo stability of this bioactive in the presence of the proteins. Fluorescence spectroscopy showed that at pH 7.0, increasing concentrations of ß-carotene reduced the fluorescence intensity of both proteins, and the fluorescence-quenching mechanism is mainly static. The stoichiometry of the ß-car/protein complex varied between proteins, being 1:1 to native BSA, 1:3 to denatured BSA (d-BSA), and 1:2 for ß-casein. The standard Gibbs-free energy (ΔG°) of complex formation was negative for all systems studied and followed the order ΔG°BSA <ΔG°ß-casein <ΔG°d-BSA. The formation of ß-car/protein complex was driven by entropy increasing in all studied conditions. Both proteins improved ß-car photo stability, but ß-casein micelle was more efficient, reducing and increasing four times, respectively, the bioactive degradation constant and the halftime of ß-car. The overall results pointed to the strategic use of milk proteins, especially ß-casein micelles as nanovehicle for ß-car in food and other systems. [ABSTRACT FROM AUTHOR]

Caffeine is a molecule with bioactive properties related to increased mental performance. Its use in food products is limited by its bitterness and self-association behavior. The complex formed by β-lactoglobulin and caffeine was characterized by fluorescence spectroscopy, isothermal titration calorimetry, and molecular docking. Fluorescence spectroscopy revealed that β-lactoglobulin interacted with caffeine to form a 1:1 stoichiometric complex. Isothermal titration calorimetry characterization of β-lactoglobulin-caffeine binding revealed a different stoichiometry (1:3). Molecular docking analysis confirmed the information obtained by the two thermodynamic evaluation techniques, showing that the best interaction occurred in the hydrophobic cavity of β-lactoglobulin. The effect of protein conformation was also evaluated by fluorescence spectroscopy. The complex stoichiometry and binding constants were from the same order as those obtained for native β-lactoglobulin. However, in contrast to the complex formation with native protein, the flexibility increase observed in the thermal unfolded protein promoted a great temperature effect on the standard enthalpy and entropy changes of interaction, indicating the presence of a balance between hydrophobic and hydrophilic forces. • β-lactoglobulin complex with caffeine in its native or denatured form. • β-lactoglobulin conformation influences in the thermodynamics of interaction with caffeine. • Caffeine binds predominantly on β-lactoglobulin hydrophobic cavity for both conformations. [ABSTRACT FROM AUTHOR]

The complexation between protein and polyphenol affects their biological functions. A complete understanding of such interactions requires comprehensive thermodynamic and kinetic characterizations. Surface plasmon resonance (SPR) and fluorescence spectroscopy (FS) described similarly the thermodynamic of interaction between bovine lactoferrin (bLF) and epigallocatechin-3-gallate (EGCG). The formation of the bLF-EGCG complex is spontaneous (Δ G S P R o ≈ -29.00 kJ mol−1, Δ G F S o ≈ -26.00 kJ mol−1) and entropically driven (Δ H S P R o = 14.26, Δ H F S o = 10.20 kJ mol−1 and T Δ S S P R o ≈ 43.00, T Δ S F S o ≈ 36.00 kJ mol−1). The kinetic parameters obtained by SPR showed that the reaction occurs through an activated complex, whose energetic formation parameters from the association of free molecules ( E a c t (a) = 49.5 kJ mol−1, Δ H a ‡ = 47.0 kJ mol−1, and T Δ S a ‡ = −2.10 kJ mol−1) were higher than those in the opposite direction (namely the dissociation of the stable complex, E a c t (d) = 17.4 kJ mol−1, Δ H d ‡ = 32.8 kJ mol−1, and T Δ S d ‡ = −45.10 kJ mol−1), except for Δ G ‡ (Δ G a ‡ = 49.1 kJ mol−1 and Δ G d ‡ = 77.9 kJ mol−1). This study provides useful information for optimizing the use of bLF – EGCG complex as a bioactive compound in different systems, such as medical, food, cosmetic, and pharmaceutical formulations. Image 1 • Epigallocatechin-3-gallate (EGCG) and bovine lactoferrin (bLF) form a stable complex. • bLF – EGCG complex formation was driven by enthalpic and entropic interactions. • bLF – EGCG complex formation proceeded through an intermediate complex. • Intermediate formation from free EGCG and bLF was accompanied by an entropy decrease. • Fluorescence spectroscopy supported SPR thermodynamic binding parameters. [ABSTRACT FROM AUTHOR]

Here, investigation was made of the interaction between lactoferrin (Lf) and the anionic surfactants sodium dodecyl sulfate (SDS), sodium dodecylbenzene sulfonate (SDBS), and sodium decyl sulfate (DSS), using isothermal titration calorimetry, Nano differential scanning calorimetry (NanoDSC), and fluorescence spectroscopy. The Lf-surfactant interaction was enthalpically favorable (the integral enthalpy change ranged from −5.99 kJ mol −1 , for SDS at pH 3.0, to −0.61 kJ mol −1 , for DSS at pH 12.0) and promoted denaturation of the protein. The Lf denaturation efficiency followed the order DSS < SDS < SDBS. The adsorption capacity of the protein with respect to surfactant strongly depended on pH and the surfactant structure, reaching a maximum value of 505 SDBS molecules per gram of Lf at pH 3.0. The different efficiencies of the surfactants in denaturing Lf were attributed to the balance of hydrophobic and electrostatic interactions, which also depended on pH and the surfactant structure, highlighting the SDBS-tryptophan residue specific interaction, where SDBS acted as a quencher of fluorescence. Interestingly, the NanoDSC and fluorescence measurements showed that the ferric ion bound to Lf increased its stability against denaturation induced by the surfactants. The results have important implications for understanding the influence of surfactants on structural changes in metalloproteins. [ABSTRACT FROM AUTHOR]

Cinnamic acid (CA) and methyl cinnamate (MC) have attracted interest of researchers because of their broad therapeutic functions. Here, we investigated the interaction of CA and MC with bovine serum albumin (BSA) at pH 3.5, 5.0, and 7.4 using fluorescence spectroscopy, differential scanning nanocalorimetry, and measurements of interfacial tension, size, and zeta potential. BSA formed a complex with the ligands with stoichiometry of approximately 1.0. At pH 7.4, CA-BSA complex formation was entropically driven. The interaction between MC and BSA was more favorable than with CA and was enthalpically driven under the same conditions. The pH played an important role in BSA conformation, which altered the manner in which it interacts with the ligands. Interestingly, both CA and MC had no effect on the surface tension of BSA/air interfaces. These data contribute to the knowledge of CA/MC-BSA interactions and provide important data for application in the food industry. [ABSTRACT FROM AUTHOR]

Ponceau 4R (P4R) and bovine serum albumin (BSA) may interact changing food properties. We compared fluorescence spectroscopy and surface plasmon resonance (SPR) for studying, in vitro , the interactions between BSA and P4R at pH 7.4 and 3.5 in different temperatures. Fluorescence data pointed to the formation of a complex where P4R was bound on site I or II of BSA, with a stoichiometry around one and a binding constant (K b ) ranging from 1.37 × 10 5 to 20.15 × 10 6 L mol −1 . The complex formation at both pH was enthalpically driven (standard enthalpy change, ΔH° F = −60.69 and −63.06 kJ mol −1 , for pH 7.4 and 3.5, respectively). Using SPR, we also found the formation of 1:1 BSA-P4R complexes, but the calculated K b values were much smaller, on the order of 10 3 L mol −1 . Again, we found that the formation of BSA-P4R complex was driven by enthalpy decreasing; however the standard enthalpy change was less negative than that found by fluorescence (ΔH° SPR = −15.05 and −40.55 kJ mol −1 , at pH 7.4 and 3.5, respectively). Our results show that these distinct techniques provided different thermodynamic binding parameters for the BSA-P4R interaction, especially regarding ΔH° values, indicating that BSA-P4R binding was a multisite phenomenon, and that sites far from tryptophan residues were the main responsible by electrostatic interaction. Thus, this work clearly shows the importance of using complementary techniques for a complete thermodynamic characterization of complexes formed between azo-colorants and proteins; which is directly related to physicochemical properties of systems containing both molecules together. [ABSTRACT FROM AUTHOR]

[Display omitted] β-Carotene (β C A R) is a pro-vitamin A carotenoid with powerful antioxidant properties. However, it has low aqueous solubility, light and heat stability, and bioavailability. An interesting strategy to improve β C A R incorporation in food matrices while preserving its properties is the use of proteins as nanocarriers. Lysozyme (LYS) is a protein capable of carrying bioactive molecules and has antitumor, antiviral, and antimicrobial activities. To enable the use of LYS with β C A R , it is important to understand the dynamics of LYS - β C A R binding. Here, a thermodynamic and kinetic investigation of LYS - β C A R ° complex formation at physiological pH was carried out using fluorescence spectroscopy (FS), differential scanning nanocalorimetry (nanoDSC), molecular docking, and surface plasmon resonance (SPR). The binding constants were found to be on the order of 105 to 103 L mol−1 by FS and SPR, respectively, revealing that the interaction between LYS and β C A R is entropically driven. In addition, kinetic parameters related to intermediate complex formation were obtained, and we found that the rate of intermediate complex formation was higher on the free molecules association than on the LYS - β C A R ° dissociation based on the lower Δ G a ‡ values than Δ G d ‡. In addition, the occurrence of an isokinetic relationship indicates that intermediate complex formation results in changes in LYS binding sites, and both desolvation of the free molecules and conformational changes are temperature dependent. Thus, we have elucidated the dynamics of LYS - β C A R ° complex formation and provided important evidence that LYS can be used to carry β C A R , which will improve its application and functionality in food. [ABSTRACT FROM AUTHOR]

The incorporation of bioactive molecules is important to improve food functionality. Despite the low solubility of some bioactive molecules, e.g., flavonoids, their binding with proteins contribute to their incorporation. There are some studies regarding globular proteins as flavonoid nanocarriers, but binding between flavonoids and βCN monomers, which are flexible proteins, is less reported. Here, the thermodynamics and kinetics of naringin–β-casein (NR–βCN) binding were determined by fluorescence spectroscopy (FS) and surface plasmon resonance (SPR). FS indicated that NR binds with βCN close to Trp 143 with K b 24 ° C = 2.71 × 104 m −1 and a key role of desolvation. SPR revealed that NR also binds far from Trp 143 (K b 24 ° C = 7.01 × 103 m −1) with small enthalpic and entropic temperature dependencies. We also present the kinetic parameters related to an intermediate complex formation, which was formed with minimal conformation and desolvation changes. Our results demonstrate βCN monomers as potential nanocarriers for NR. [ABSTRACT FROM AUTHOR]

The thermodynamics of curcumin (CUR) binding with bovine lactoferrin (bLF) was investigated by employing fluorescence spectroscopy (FS) and surface plasmon resonance (SPR) and comparing the results. Moreover, the kinetics of bLF-CUR binding was studied using SPR. FS data showed that with an increase in temperature (from 293.15 to 306.15 K), more complexes were formed (K b ranged from 2.07 × 104 to 6.68 × 104 M−1) owing to an increase in the entropy (Δ H ° = 66.70 kJ mol−1; T Δ S ° ranged from 90.93 to 94.98 kJ mol−1), demonstrating the role of hydrophobic interactions. However, the bLF-CUR complex formation detected through the SPR technique was entropically and enthalpically driven below and above 293.75 K, respectively. SPR also demonstrated that an enthalpy increase is compensated by an entropy increase for both stable and activated complex formation, characterizing enthalpy-entropy compensation and iso-kinetic compensation, respectively. These results are helpful to better understand the dynamics of complex formation between proteins and small bioactive molecules. Image 1 • Curcumin (CUR) and bovine lactoferrin (bLF) formed a stable complex. • SPR showed that bLF has more sites for CUR binding than those close to tryptophan. • The stable bLF-CUR complex formation passes through an intermediate state. • The bLF-CUR interaction occurred with enthalpy-entropy compensation. • bLF-CUR binding is driven mainly by the conformational and desolvation processes. [ABSTRACT FROM AUTHOR]