Kinetic and thermodynamic characterization of the supramolecular structures formation: lactoferrin-phenothiazine dyes and lactoferrin-polysaccharides

A manifestação da vida é baseada principalmente no reconhecimento molecular. Consequentemente, elucidar a cinética e a termodinâmica das interações proteína-ligante é estratégico para compreensão e modulação dos sistemas biológicos. Além disso, a formação de estruturas supramoleculares é muito importante em aplicações industriais, como processos de formação e estabilização de emulsões e encapsulamento. Assim, neste trabalho avaliou-se a termodinâmica e a cinética de interação entre a lactoferrina (BLF) e dois grupos de ligantes: corantes fenotiazínicos (azul de metileno (MB) e azure A (AZA)) e polissacarídeos (carboximetilcelulose (CMC) e kappa-carragena (κCG)), combinando dados de ressonância plasmônica de superfície (SPR), calorimetria de titulação isotérmica (ITC) e espectroscopia de fluorescência (EF). O trabalho foi dividido em três artigos: no primeiro, foi feito um estudo comparativo da interação entre a BLF e os corantes MB e AZA, utilizando múltiplas técnicas (SPR, EF e ITC), visando elucidar o mecanismo de ligação desses corantes à BLF e às contribuições dos grupos CH 3 para essa interação. No segundo, investigou-se a cinética e a termodinâmica da interação BLF-CMC, utilizando EF e SPR. Por fim, no terceiro artigo foi estudado a energética e a dinâmica de formação dos complexos BLF-κCG, na ausência e presença de KCl, por meio das técnicas de EF e SPR. Acerca da interação BLF-corantes fenotiazínicos, constatou-se que apesar das semelhanças estruturais entre os corantes, os parâmetros termodinâmicos de ligação BLF-AZA foram maiores em magnitude comparados ao sistema BLF-MB. Os resultados obtidos por SPR e EF indicaram que os fatores entrópicos eram as únicas forças motrizes da interação. No entanto, a ITC mostrou que há uma forte contribuição entálpica associada à interação com a holo-BLF. Embora a formação dos complexos de transição BLF-MB e BLF-AZA a partir da associação dos ligantes livres exigiu parâmetros energéticos muito semelhantes (∆ ‡a,MB= 51,84 e ∆‡ a,AZA= 50,7 kJ mol -1 ), na dissociação estes parâmetros foram dependentes do número de grupos CH 3 na estrutura dos corantes (∆ ‡d,MB= 81,4 e ∆‡ d,AZA= 74,93 KJ mol-¹). Quanto à interação BLF-CMC, o estudo cinético sugeriu que a formação do complexo ativado a temperaturas ≤ 289,2 K foi dominada pela energia liberada pelas interações específicas entre a proteína e o polissacarídeo. Embora os dados de SPR tenham demonstrado que a formação dos complexos BLF-CMC foi conduzida entropicamente (57 ≤ ∆° ≤ 60 kJ mol-¹) os ensaios de EF revelaram valores negativos ∆° até 293,2 K e positivos em temperaturas ≥ 298,2 Ke -58 ≤ ∆° ≤ 230 KJ mol-¹. Por fim, o estudo da interação BLF-κCG mostrou que a formação do complexo foi entalpicamente dirigida (∆° = −81 kJ mol −1 ) e a comparação entre os dados termodinâmicos obtidos por SPR e EF revelou que a interação BLF-κCG ocorreu em vários sítios da BLF. Já os parâmetros cinéticos mostraram que a interação ocorreu através da formação de um complexo de transição e que esse processo foi mais rápido a partir da associação das moléculas livres. A mudança na força iônica alterou a forma como a BLF e a κCG interagem, tornando a ligação um processo de uma única etapa. Palavras-chave: Lactoferrina. Corantes fenotiazínicos. Polissacarídeos.Life manifestation is based mainly on molecular recognition. Consequently, the elucidation of the kinetics and thermodynamics of the protein-ligand interactions is strategic for the comprehension and modulation of biological systems. Besides that, the formation of supramolecular structures is very important to industrial applications, such as, formation and stabilization of emulsions, and encapsulation. Therefore, in this work, it was evaluated the thermodynamics and kinetics of the interaction between lactoferrin (BLF) and two groups of ligands: phenothiazine dyes (methylene blue (MB) and azure A (AZA)) and polysaccharides (carboxymethylcellulose (CMC) and κ-carrageenan (κCG)); combining surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), and fluorescence spectroscopy (FS) data. The work was divided into three articles: in the first one, the comparative study of the interaction between the MB and AZA dyes were made, using multiple techniques (SPR, FS, and ITC), aiming the elucidation of the binding mechanism between these dyes and BLF, and contributions of the CH 3 groups to this interaction. In the second one, the kinetics and thermodynamics of the BLF-CMC interaction were investigated, using FS, and SPR. Finally, in the third article, it was studied the energetics and dynamics of the formation of BLF-κCG complexes, in the absence and presence of KCl, via FS and SPR techniques. Concerning the BLF-phenothiazine dyes interaction, it was verified that, although the similarity between the dyes structures, the thermodynamic parameters for the BLF-AZA binding was higher in magnitude when compared to the BLF-MB system. The results obtained from SPR and FS indicated the entropic factors were the only driving forces of the interaction. However, the ITC showed there is a strong enthalpic contribution associated with the interaction with the holo- BLF. Although the formation of the BLF-MB and BLF-AZA transition complexes, from the association of free ligands, requires similar energetic parameters (∆ ‡a,MB= 51,84 and ∆‡ a,AZA= 50,7 kJ mol -1 ), in the dissociation, these parameters depended on the number of CH 3 groups in the dye structure ∆ ‡d,MB= 81,4 and ∆‡ d,AZA= 74,93 KJ mol-¹). As for the BLF-CMC interaction, the kinetic study suggested the formation of the activated complex at temperatures ≤ 289.2 K was dominated by the energy released by specific interactions between the protein and polysaccharide. Although the SPR data have shown the formation of the BLF-CMC complex was entropy-driven (57 ≤ ∆° ≤ 60 kJ mol-¹), the FS assays revealed negative ∆° até 293,2 K and positive values in temperatures ≥298.2 K, and - 58 ≤ ∆° ≤ 230 KJ mol-¹. Finally, the study of the BLF-κCG interaction showed the complex formation was enthalpy-driven (∆° = −81 kJ mol −1 ) and the comparison between the thermodynamic data obtained by SPR and FS revealed the interaction occurs in various sites of BLF. While the kinetic parameters showed the interaction occurs through the formation of a transition complex and this process is faster from the association of the free molecules. The change in the ionic strength altered the way BLF and κCG interact, making the binding a one- step process. Keywords: Lactoferrin. Phenothiazine dyes. Polysaccharides

New insights about the partition of phenothiazines dyes in aqueous two-phase systems

Sistemas aquosos bifásicos (SABs) têm provado ser um método eficiente e ambientalmente seguro para a extração de muitas espécies químicas. No entanto, a termodinâmica de partição de solutos nesses sistemas ainda é pouco compreendida. Há poucos estudos que trazem um enfoque termodinâmico que contribua para compreensão das forças motrizes que regem o processo de transferência de solutos em SABs. Uma abordagem estratégica para estudar as forças que determinam a partição é a utilização de compostos químicos com estruturas químicas semelhantes, como sondas moleculares, para através da termodinâmica compreender a relação existente entre a estrutura e a partição destes compostos. O azul de metileno (AM), azure A (AZA), azure B (AZB) e acetato de tionina (AT) são corantes catiônicos que apresentam o mesmo grupo central fenotiazínico, se diferenciando apenas no número de substituintes metila. Essas características podem fornecer informações úteis sobre os fatores que governam a partição destes compostos em SABs. Neste trabalho, um estudo termodinâmico completo do comportamento de partição dos corantes (MB), (AZB), (AZA) e (TA) foi investigado utilizando os SABs: PEO 1500+MgSO 4 +H 2 O; PEO 1500+Na 2 SO 4 +H 2 O; PEO1500+Li 2 SO 4 +H 2 O. Os parâmetros termodinâmicos de transferência, tais como a variação da energia livre de Gibbs padrão de transferência ), a variação da entalpia padrão de transferência ( padrão de transferência ) e a variação da entropia ), e sua dependência com a estrutura do corante, natureza do eletrólito e comprimento da linha de amarração (CLA) foram avaliados. Além disso, as energias entálpicas de interação corante-PEO e corante-SAL foram determinadas. Pela primeira vez, foi descoberto que cátions se concentram preferencialmente na fase enriquecida em polímero dos SABs, com valores de K entre 6,16 e 225, 60 e valores de compreendidos entre -4,16 kJ mol -1 a -13,47 kJ mol -1 . O processo de transferência dos corantes fenotiazínicos foi entalpicamente dirigido, -11,40 kJ mol -1 < < -51,24 kJ mol -1 , resultante das interações específicas atrativas corante-PEO e interações repulsivas corante-Sal, e ocorreram mesmo com um decréscimo entrópico do sistema (-4.93 kJ mol -1 K -1 < < -38.78 kJ mol -1 K -1 ). A diferença na estrutura química dos corantes teve um efeito pouco pronunciado sobre os valores das energias entálpicas de interação corante-PEO e corante-Sal, mostrando que essa interação é promovida principalmente pelo anel condensado do corante. Também foi observado que o processo de partição dos corantes fenotiazínicos é dependente da linha de amarração e natureza do ânion, entretanto pouco influenciado pela natureza do cátion.Aqueous two-phase systems (ATPSs) have proven to be an efficient and environmentally safe method for extraction of chemical species. However, the solutes partition phenomenon in these systems is still little understood. There are few studies carrying a thermodynamic focus that contributes for the understanding of the driving forces governing solute transfer in ATPSs. A strategic approach for studying it is to use chemical compounds with similar chemical structures, as molecular probes, for comprehending thermodynamically the relationship between the structures and partition these compounds. The methylene blue (MB), azure A (AZA), azure B (AZB) and thionine acetate (TA) are cationic dyes that have the same phenothiazine core group and differ only in the number of methyl substituents. These characteristics may provide useful information about the factors that govern the partition process of these compounds in ATPSs. In this paper, the thermodynamic partition behavior of MB, AZA, AZB and TA dyes was investigated using the following ATPSs: PEO 1500 + MgSO 4 + H 2 O; PEO 1500 + Na 2 SO 4 + H 2 O; PEO1500 + Li 2 SO 4 + H 2 O. Standard transfer parameters such as free energy change of transfer ( transfer ( ), entropy change of transfer ), enthalpy change of ) and its dependence on the dye structure, electrolyte nature and tie line length (TLL) were evaluated. Besides, the dye- PEO enthalpy energy interaction and dye-salt interaction were determined. For the first time cations were discovered to concentrate in the polymer enriched phase, with K values ranging from 6.16 to 225.60, and values ranging from 4.16 to -13.47 kJ mol -1 . The transfer process of phenothiazine dyes was enthalpy driven, with -11.40 kJ mol -1 < < -51.24 kJ mol -1 , resulting from specific attractive dye-PEO interactions and repulsive dye-salt interactions, occurring even with a system entropy decrease (-4.93 kJ mol -1 K -1 < < -38.78 kJ mol -1 K -1 ). The dyes chemical structure differences had insignificant effect on the dye-PEO and dye-salt enthalpy interaction energy values, showing that interactions in these systems are mainly promoted by the dye condensed ring. It was also observed that the phenothiazine dyes partition process is dependent of tie line and nature of the anion, however, little influenced by the cation nature.Conselho Nacional de Desenvolvimento Científico e Tecnológic

Chromium speciation using aqueous biphasic systems: Development and mechanistic aspects

The two principal oxidation states of chromium diverge significantly in their biological and toxicological properties, and it is very important to distinguish between them. Develop procedures that are efficient, fast, easier and cheaper for chromium speciation is an analytical challenge. Chromium speciation was investigated using aqueous biphasic systems (ABS) formed by polyethylene oxide (PEO1500) or a triblock copolymer (L64), an electrolyte (Li2SO4, Na2SO4, MgSO4, C6H5Na3O7, or C4H4Na2O6) and water, without the presence of any extractant. Cr(III) was favorably partitioned to the electrolyte-rich phase (ERP) of the ABS, whereas the Cr(VI) was preferentially concentrated in the macromolecule-rich phase (MRP) in most analyzed ABS. The fluorescence spectroscopy data revealed that the Cr(III) and Cr(VI) separation process is governed by specific interactions between the ethylene oxide segments in the macromolecules and the Cr(VI) species, which do not occur for Cr(III) species. The extraction percentage (%E) of chromium species from the ERP to the MRP was affected by pH, tie-line length (TLL), electrolyte nature, and macromolecule hydrophobicity. For the ABS comprising of PEO1500 + Na2SO4 + H2O, TLL = 50.89% (m/m), 25 °C and pH 2.0, the %E values for Cr(III) and Cr(VI) were 0.0100 and 99.9%, respectively. The speciation of Cr(III) and Cr(VI) that was achieved in this ABS resulted in an excellent separation factor (SCr(VI),Cr(III)) of 1.00 × 10 8

Aggregation of sodium dodecylbenzene sulfonate : weak molecular interactions modulated by imidazolium cation of short alkyl chain length.

Ionic liquids (ILs) can modify cooperative process in aqueous solutions to a large extent, including anionic surfactant aggregation. Here, the micellization of sodium dodecylbenzene sulfonate (SDBS) was evaluated in low concentrations of 1-alkyl-3-methylimidazolium chloride (CnmimCl, n = 0, 2, and 4) aqueous solutions through fluorescence spectroscopy, isothermal titration calorimetry, dynamic light scattering, and conductometry. The thermodynamic stability of SDBS aggregates strongly depended on the IL structure and concentration, following the order C4mim+ > C0mim+ ? C2mim+. At 1.0 mmol L?1 of the ILs, the increase of the hydrophobicity of the imidazolium cation decreased the enthalpic favorableness, changing from ?3.75 ? 0.07 kJ mol?1, for C0mim+, to ?2.69 ? 0.01 kJ mol?1, for C4mim+. On the other hand, the entropic feasibility showed an opposite trend, i.e., the higher hydrophobicity of C4mim+ overcame the kosmotropic effect of IL cations in the bulks. We suggested that the imidazolium cations interact with the SDBS monomers on the micellar surface, mainly through hydrophobic, ?-?, and electrostatic interactions for C4mim+ and C2mim+, and through electrostatic interactions and hydrogen bonds for C0mim+

Solvophobic effect of 1-alkyl-3-methylimidazolium chloride on the thermodynamic of complexation between ?-cyclodextrin and dodecylpyridinium cation.

Preferential solvation participate in various supramolecular self-assembly processes, whose thermodynamic properties can be modulated by the addition of ionic liquids (ILs). However, the effects of these liquids on the thermodynamics of the host-guest complexation process remain unexplored. In this study, the thermodynamic properties of the complexation between 1-dodecylpyridinium cations (C12Py+) and ?-cyclodextrin (?CD) species in aqueous solutions with different concentrations of 1-alkyl-3-methylimidazolium halides (CnmimX) were investigated by isothermal titration calorimetry. In water, C12Py+ and ?CD form a 1:1 inclusion complex, which is enthalpically ( ?9.2 ? 0.1?kJ mol?1) and entropically ( 16.1 ? 0.2?kJ mol?1) favorable. However, in IL aqueous solutions, all the ?CD?C12Py+ thermodynamic parameters of the complexation change and this IL effect is dependent on the carbon chain length of Cnmim+ cations. ILs with shorter alkyl chains (Cnmim+, n ? 4) decreases the system entropy, while ILs with longer alkyl chains (Cnmim+, n ? 6) reduce the enthalpy values. These effects are attributed to i) preferential solvation of surfactant tails by ILs; ii) ability of the ILs to modify the 3D water structure and iii) inclusion of IL molecules into the inner cavities of ?CD

Kinetics and thermodynamics of bovine serum albumin interactions with Congo red dye

To optimize the therapeutic applications of Congo red (CR), a potential inhibitor of protein aggregation, the kinetics and thermodynamics of the interactions between CR and a model protein need to be understood. We used surface plasmon resonance (SPR) and fluorescence techniques to determine the dynamics and thermodynamic parameters for the formation of complexes between CR and bovine serum albumin (BSA). CR interacts with BSA through a transition complex; the activation energy for association (Eact(a)) was determined to be 35.88 kJ mol−1, while the activation enthalpy (ΔH‡), entropy (ΔS‡), and Gibbs free energy (ΔG‡) are 33.41 kJ mol−1, 0.18 J mol−1 K−1, and 33.35 kJ mol−1, respectively. When this intermediate transforms into the final CR-BSA complex, the entropy of the system increases and part of the absorbed energy is released; this process is associated with a reverse activation energy (Eact(d)) of 20.17 kJ mol−1, and values of ΔH‡, ΔS‡, and ΔG‡ of 17.69 kJ mol−1, −162.86 J mol−1 K−1, and 66.25 kJ mol−1, respectively. A comparison of the SPR and fluorescence results suggests that there is more than one site where BSA interacts with CR