RESUMEN
Isonicotinic acid hydrazide (Isoniazid, INH) is one of the most commonly used first-line anti-tuberculosis drugs, which has been reported that the high concentration of INH in human body can lead to epilepsy, liver function failure, and even death. Therefore, studying the potential binding effects of INH on the structure and activity of human serum albumin (HSA) and catalase (CAT) is very essential for evaluating its toxicity and side effect. In this paper, multi-spectroscopic and molecular docking methods were used to elucidate the patterns of INH to HSA and CAT under imitated physiological conditions. The inner filter effect of all fluorescence data in the paper was eliminated to get accurate binding parameters. The Stern-Volmer quenching constants (KSV) of both HSA-INH system and CAT-INH system inversely correlated with temperatures, demonstrating that INH quench the intrinsic fluorescence of HSA and CAT via static quenching. The conformational investigation of HSA and CAT through UV-visible absorption spectroscopy, synchronous fluorescence and circular dichroism (CD) showed that INH could change the micro-environment of tryptophan residues and reduced the α-helix content of protein. These results demonstrated that the binding of INH may lead to the loosening of protein skeleton, which which may affect its physiological function. The results of molecular docking revealed that the INH was located in Sudlow's site I of HSA. And INH bound to CAT at a cavity among the wrapping domain helical domain and ß-barrel, which resulted in the inhibition of CAT activity. In addition, Levofloxacin (LVFX) is a new effective and safe second-line anti-tuberculosis drugs and can improve the curative effect on anti-TB by using with other anti-TB drugs, the result of Hill's coefficients (nH) about synergy between INH and proved that LVFX promoted the interaction of HSA with INH. Moreover, according to the CD spectra, synergy between INH and LVFX changed the conformation of HSA and the amount of α-helix decreased about 7.9%. This work will provide important insights into the binding and toxicity mechanism of INH to HSA and CAT in vivo and is expected to be helpful in evaluating the essential information for using the INH safely.
Asunto(s)
Simulación del Acoplamiento Molecular , Sitios de Unión , Catalasa , Dicroismo Circular , Fluorescencia , Humanos , Isoniazida , Unión Proteica , Albúmina Sérica , Albúmina Sérica Humana , Espectrometría de Fluorescencia , TermodinámicaRESUMEN
Perfluorononanoic acid (PFNA) is the third most frequently detected in serum among all perfluoroalkyl acids (PFAAs) which is a kind of toxic emerging environmental contaminant. The influence of PFNA on the conformation and even function of human serum albumin (HSA) is still just at the beginning of research. The attempt of this paper was to completely elucidate the interaction mechanism of PFNA with HSA by means of multi-spectroscopic, molecular docking and isothermal titration calorimetry (ITC) techniques. The inner filter effect of all fluorescence data in the paper was eliminated to get accurate binding parameters. The results showed that the fluorescence of HSA was quenched by PFNA through a combined quenching procedure of dynamic and static quenching. Through site marker competitive experiments, subdomain IIA of HSA had been assigned to possess the high-affinity binding site of PFNA. Furthermore, molecular docking reconfirmed that PFNA was bound in subdomain IIA mainly through polar force, hydrophobic interaction and halogen-bond, and the calculated free energy was -26.54 kJ·mol(-1) which indicated that the PFNA molecule exhibited large binding affinity towards HSA. The thermodynamic characterizations of two different classes of binding sites by ITC displayed that the first class with a higher affinity constant was dominated by an enthalpic contribution due to electrostatic interactions and halogen-bond, whereas the second class with a lower affinity constant was preponderated by hydrophobic interaction. The three-dimensional fluorescence revealed that the conformation of HSA was changed and the hydrophobicity of the Trp and Tyr residues microenvironment increased after formation of PFNA-HSA complex. The alterations of the protein secondary structure were quantitatively calculated from circular dichroism (CD) spectroscopy with reduction of α-helix content about 14.3%, ß-sheet 5.3%, ß-turn 3.5%, and augment in random content from 14.4% to 37.5%. Above results revealed that the binding of PFNA with HSA can alter the secondary structure of HSA, further probably affecting HSA physiological function. The results can provide insights with the binding mechanism of PFNA with HSA and salient biophysical and biochemical clues on elucidating the transport and distribution of PFNA in vivo.
Asunto(s)
Simulación del Acoplamiento Molecular , Sitios de Unión , Calorimetría , Dicroismo Circular , Ácidos Grasos , Fluorescencia , Fluorocarburos , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Unión Proteica , Estructura Secundaria de Proteína , Albúmina Sérica , Albúmina Sérica Humana , Espectrometría de Fluorescencia , Electricidad Estática , TermodinámicaRESUMEN
Perfluorododecanoic acid (PFDoA) is the most toxic emerging environmental contaminant among the 8ï½12 carbon chain perfluoroalkyl acids (PFAAs). A large amount of knowledge in the field of environmental PFAAs has been accumulated so far, while we are still just at the beginning of research into the interaction between PFDoA and human serum albumin (HSA). The goal of this study was to comprehensively determinate the binding mechanism of PFDoA with HSA by using fluorescence quenching technique in combination with molecular modeling and circular dichroism (CD) spectroscopy under the simulative physiological conditions. The quenching of HSA fluorescence by PFDoA was found to be a result of the combination of dynamic quenching and the formation of PFDoA-HSA complex. The calculated binding distance (r=3.65 nm) indicated that the non-radioactive energy transfer came into being in the interaction between PFDoA (acceptor) and HSA (donor). By performing displacement measurements, the specific binding of PFDoA in the vicinity of site I of HSA was clarified. Furthermore, the binding details between PFDoA and HSA were further confirmed by molecular docking studies, which revealed that PFDoA was bound at subdomain IIA by multiple interactions, such as the interaction between O1 of PFDoA with Arg 257 and Ser 287 predominately through polar force. And the best calculated docking energy is -25.87 kJ·mol-1, this high negative value indicated that the PFDoA molecule exhibited large binding affinity towards HSA. The effects of PFDoA on the conformation of HSA were analyzed by synchronous fluorescence spectra and three-dimensional fluorescence spectra, and the results exhibited that the hydrophobicity of the microenvironment around tryptophan residue was increased and the conformation of HSA was altered after binding PFDoA. The CD spectra quantitatively calculated the protein secondary structure, which suggested a loss of helical stability after the PFDoA-HSA complex formation. The binding research presented in this paper enriches our knowledge of the interaction dynamics of perfluoroalkyl acids to the HSA and reveals the chemical essence of the interaction between biomacromolecule and ligand.
RESUMEN
Acetylisovaleryltylosin tartrate (ATLL) is a new macrolide veterinary antibiotic, it is necessary to study the binding of ATLL to protein, which will directly correlate with the efficiency in vivo. Bovine serum albumin (BSA) is structure homologous with human serum albumin (HSA), and is commonly chosen as a model to investigate drug-protein interaction.There are many metal ions in plasma, as yet, the studies on mainly focus on single metal ion. In this study, the multiple systems formed by ATLL and BSA without or with Zn2+ and Cu2+ have been studied by mult-spectroscopy. The results showed that, the fluorescence of BSA was quenched by ATLL through a static quenching mechanism. The effective quenching constant (Ka) of ATLL to BSA decreased with Zn2+ and increased with Cu2+. Thermodynamic parameters revealed that hydrogen bonds and hydrophobic forces played significant roles in the binding of ATLL to BSA. The polarity of tryptophan and tyrosine residues changed when adding ATLL with or without Zn2+ and Cu2+. FT-IR spectra showed that ATLL changed α-helix and ß-sheet of BSA into ß-turn and random structure. The UV-Vis spectra indicated that the effects of Zn2+ on ATLL binding to BSA may cause by a competition binding, and Cu2+ possibly formed Cu2+-ATLL complex via metal ion bridge. All the knowledge obtained in this work will be helpful to understand the transport mechanism of ATLL with BSA and the effect of metal ions on the interaction of drug-protein in vivo.
RESUMEN
Perfluorocarboxylic acids (PFCAs) have been widespread used for over half century as surfactants in commercial and industrial products because of their hydrophilic and hydrophobic peculiarity. Perfluoroundecanoic acid (PFUnA) and perfluorotridecanoic acid (PFTriA) are two representatives of long-chain PFCAs, and they were detected more frequently in human body recent years, however, the two PFCAs were found to express endocrine disruption effects, developmental toxicity and teratogenicity. In this study, we established a new strategy to probe the binding modes of PFUnA (PFTriA) with the most abundant protein human serum albumin (HSA) based on spectroscopic and molecular docking methods. Results showed that both PFUnA and PFTriA can quench the intrinsic fluorescence of HSA with one binding site by means of dynamic and static quenching procedure with a strong affinity and the order is PFUnA>PFTriA. On the basis of thermodynamic results, we knew that the main driving force of the interaction between PFUnA and HSA was electrostatic force (ΔH=-26.32 kJ·mol-1, ΔS=21.76 J·mol-1·K-1), while van der Waals interaction and halogen-bond played major roles in complexation process of PFTriA-HSA (ΔH=-39.69 kJ·mol-1, ΔS=-25.66 J·mol-1·K-1). The binding distance (r<8 nm) indicated that the non-radioactive energy transfer came into being from HSA to PFUnA (PFTriA). The binding process of PFUnA (PFTriA) with HSA caused conformational and some micro-environmental changes of HSA, but also led to a loss of helical stability through three-dimensional fluorescence and circular dichroism spectra (CD). Furthermore, site markers competitive experiments and molecular docking revealed that PFUnA and PFTriA had a high affinity into hydrophobic pocket of subdomain IIA in HSA through polar force, hydrophobic interaction and halogen-bond and so on, and the fluorophore Trp residues was located in the binding position which proved further the quenching of PFUnA and PFTriA on HSA fluorescence. The accurate and full basic data in the work are beneficial to clarify the binding mechanism of long-chain perfluorocarboxylic acids with serum protein in vivo, and provide essential theoretical clues for their toxicity assessment and toxicologic research.
Asunto(s)
Unión Proteica , Albúmina Sérica Humana/química , Sitios de Unión , Dicroismo Circular , Transferencia de Energía , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Simulación del Acoplamiento Molecular , Albúmina Sérica , Espectrometría de Fluorescencia , Electricidad Estática , TermodinámicaRESUMEN
To know the interaction of cefodizime (CEF) with human serum albumin (HSA), techniques of different spectroscopies and molecular modeling were used. The inner filter effects were eliminated to get accurate binding parameters. Steady state fluorescence suggested a static type for CEF-HSA interaction, and the complex formation had a high affinity of 10(5) L mol(-1). On the basis of the thermodynamic results and site marker competitive experiments, it was considered that CEF was bound to site I (subdomain IIA) of HSA mainly by hydrogen bonds and van der Waals force. The calculated binding distance (r) indicated that the non-radioactive energy transfer came into being in the interaction between CEF and HSA. Furthermore, molecular modeling was applied to further define that CEF interacted with the Trp214, Lys199, Phe211, Leu238 residues of HSA. In addition, three-dimensional fluorescence and circular dichroism (CD) results showed that the binding of CEF can cause conformational and some microenvironmental changes of HSA. This paper provides reasonable models helping us further understand the transportation and distribution of CEF when it spreads into human blood serum which is of great importance in pharmacology and pharmacodynamics.
Asunto(s)
Cefotaxima/análogos & derivados , Albúmina Sérica/química , Sitios de Unión/fisiología , Cefotaxima/química , Dicroismo Circular , Transferencia de Energía , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Simulación del Acoplamiento Molecular/métodos , Unión Proteica/fisiología , Espectrometría de Fluorescencia/métodos , TermodinámicaRESUMEN
The present study was designed to investigate the interaction of doxycycline (DC) with human serum albumin (HSA) by the inner filter effects, displacement experiments and molecular docking methods, based on classic multi-spectroscopy. With fluorescence quenching method at 298 and 310 K, the binding constants Ka, were determined to be 2. 73 X 10(5) and 0. 74X 10(5) L mol-1, respectively, and there was one binding site between DC and HSA, indicating that the binding of DC to HSA was strong, and the quenching mechanism was a static quenching. The thermodynamic parameters (enthalpy change, AH and enthropy change, delta S) were calculated to be -83. 55 kJ mol-1 and -176. 31 J mol-1 K-1 via the Vant' Hoff equation, which indicated that the interaction of DC with HSA was driven mainly by hydrogen bonding and van der Waals forces. Based on the Föster's theory of non-radiation energy transfer, the specific binding distance between Trp-214 (acceptor) and DC (donor) was 4. 98 nm, which was similar to the result confirmed by molecular docking. Through displacement experiments, sub-domain IIA of HSA was assigned to possess the high-affinity binding site of DC. Three-dimensional fluorescence spectra indicated that the binding of DC to HSA induced the conformation change of HSA and increased the disclosure of some part of hydrophobic regions that had been buried before. The results of FTIR spectroscopy showed that DC bound to HSA led to the slight unfolding of the polypeptide chain of HSA. Furthermore, the binding details between DC and HSA were further confirmed by molecular docking methods, which revealed that DC was bound at sub-domain IIA through multiple interactions, such as hydrophobic effect, polar forces and pi-pi interactions. The experimental results provide theoretical basis and reliable data for the study of the interaction between small drug molecule and human serum albumin