Probing the Interaction of Human Serum Albumin With Bilirubin in the Presence of Aspirin by Multi-Spectroscopic,Molecular Modeling and Zeta Potential Techniques: Insight on Binary and Ternary Systems

Abstract

Here, we report on the effect of aspirin (ASA), on the binding parameters with regard to bilirubin (BR) to human serum albumin (HSA). Two different classes of binding sites were detected. Binding to the first and second classes of the binding sites was dominated by hydrophobic forces in the case of HSA-BR, whereas in the case of the ternary system, binding to the first and second classes of the binding sites was achieved by electrostatic interaction. The binding constant (K_a) and number of binding site (n) obtained were 1.6 × 10⁶ M^?1 and 0.98, respectively, for the primary binding site in the case of HSA-BR, and 3.7 × 10⁶ M^?1 and 0.84, respectively, in the presence of ASA (ternary complex) at δ_ex = 280 nm. The progressive quenching of the protein fluorescence as the BR concentration increased indicated an arrangement of the domain IIA in HSA. Changes in the environment of the aromatic residues were also observed by synchronous fluorescence spectroscopy (SFS). Changes of the secondary structure of HSA involving a decrease of α-helical and β-sheet contents and increased amounts of turns and unordered conformations were mainly found at high concentrations of BR. For the first time, the relationship between the structural parameters of HSA-BR by RLS for determining the critical induced aggregation concentration (C_CIAC) of BR in the absence and presence of ASA was investigated, and there was a more significant enhancement in the case of the ternary mixture as opposed to the binary one. Changes in the zeta potential of HSA and the HSA-ASA complex in the presence of BR demonstrated a hydrophobic adsorption of this anionic ligand onto the surface of HSA in the binary system as well as both electrostatic and hydrophobic adsorption in the case of the ternary complex. By performing docking experiments, it was found that the acting forces between BR and HSA were mainly hydrophobic > hydrogen bonding > electrostatic interactions, and consequently BR had a long storage time in blood plasma, especially in the presence of ASA. This was due to the electrostatic interaction force between the BR and HSA being stronger in (HSA-ASA) BR than in the HSA-BR complex. In addition, it was demonstrated that, in the presence of ASA, the first binding site of BR on HSA was altered, but the parameters of binding did not become significantly modified, and thus the affinity of BR barely changed with and without ASA.     

Investigating the antagonistic action between aspirin and tamoxifen with HSA: identification of binding sites in binary and ternary drug-protein systems by spectroscopic and molecular modeling approaches

The combination of several drugs is often necessary, especially during long-term therapy. A competitive binding of the drugs can cause a decrease of the amount of drugs actually bound to the protein and increase the biologically active fraction of the drug. The aim of this study has been to analyze the interactions of tamoxifen (TMX) and aspirin (ASA) with human serum albumin (HSA) and to evaluate the mechanism of a simultaneous binding of TMX and ASA to the protein. Fluorescence analysis was used to estimate the effect of the drugs on the protein fluorescence and to define the binding and quenching properties of drug-HSA complexes. The binding sites for TMX and ASA were identified in ternary structures of HSA by means of spectrofluroscence. The analysis of the fluorescence quenching of HSA in binary and ternary systems pointed at TMX and ASA having an effect on the HSA-ASA and HSA-TMX complexes. Furthermore, the results of synchronous fluorescence, resonance light scattering and circular dichroism of the binary and ternary systems showed that the binding of TMX and ASA to HSA could induce conformational changes in HSA. Moreover, the simultaneous presence of TMX and ASA during binding to HSA should be taken into account in multi-drug therapy, as it induces the necessity of a monitoring therapy owing to the possible increase of uncontrolled toxic effects. Competitive site marker experiments demonstrated that the binding site of ASA and TMX to HSA differed in the binary system as opposed to in its ternary counterpart. Finally, molecular modeling of the possible binding sites of TMX and ASA in binary and ternary systems to HSA confirmed the experimental results.     

Investigations with spectroscopy, zeta potential and molecular modeling of the non-cooperative behaviour between cyclophosphamide hydrochloride and aspirin upon interaction with human serum albumin: binary and ternary systems from multi-drug therapy

The interaction between cyclophosphamide hydrochloride (CYC) and aspirin (ASA) with human serum albumin (HSA) was studied by various kind of spectroscopic, ζ potential and molecular modeling under physiological conditions. The fluorescence data showed that the binding of drugs to proteins caused strong static fluorescence quenching. The analysis of the fluorescence quenching of HSA in the binary and ternary systems displayed that ASA was affected by the complex formed between CYC and HSA. Moreover, CYC was influenced by the HSA-ASA complex. The inherent binding information, including the quenching mechanism, binding constants, number of binding sites, effective quenching constant, fraction of the initial fluorescence and thermodynamic parameters were measured by the fluorescence quenching technique at various temperatures. In addition, according to the synchronous fluorescence spectra of HSA, the results showed that the fluorescence quenching of HSA originated from the Trp and Tyr residues, and indicated a conformational change of HSA with the addition of the drugs. Far-UV CD spectra of HSA were recorded before and after the addition of ASA and CYC as binary and ternary systems. An increase in intensity of the positive CD peak of HSA was observed in the presence of the drugs. The results were interpreted by excited interactions between the aromatic residues of the HSA binding sites and the drugs bound to them. The distance r between donor and acceptor was obtained by the Forster energy according to fluorescence resonance energy transfer (FRET) and found to be 2.35 nm and 1.78 nm for CYC and ASA, respectively. This confirmed the existence of static quenching for proteins in the presence of CYC and ASA. Furthermore, docking studies pointed at a reduction of the affinity of each of the drug compounds to the protein in the presence of the other in meaningful amounts. Pre-binding of any of the said compounds forced the second to bind in a non-optimized location and orientation. The potential at the electrokinetic shear surface of the protein-drug solution were measured at several concentrations of the drugs by the ζ potential technique, which confirmed experimental and theoretical results.     

Study of the interaction between fisetin and human serum albumin: a biophysical approach

The binding of fisetin with human serum albumin (HSA) has been studied at different pH using UV-Vis, FTIR, CD and fluorescence spectroscopic techniques. The binding constants were found to increase with the rise in pH of the media. The negative ΔH° (kJ mol-1) and positive ΔS° (J mol-1 K-1) indicate that fisetin binds to HSA via electrostatic interactions with an initial hydrophobic association that result in a positive ΔS° . In presence of potassium chloride (KCl) the binding constants were found to be decrease. The α-helical content of HSA increased after binding with fisetin as analyzed from both CD and FTIR methods. The site marker displacement studies using fluorescence anisotropy suggest that fisetin binds to the hydrophobic pocket (Site 1, subdomain IIA) of HSA which is in good accordance with the molecular docking study. The change in accessible surface area (ASA) of residues of HSA was calculated to get a better insight into the binding.     

Human serum albumin–malathion complex study in the presence of silver nanoparticles at different sizes by multi spectroscopic techniques

The binding of malathion to human serum albumin (HSA) in the presence of silver nanoparticles (AgNPs) was investigated for the first time by multiple spectroscopic methods such as fluorescence quenching, fluorescence resonance energy transfer (FRET), circular dichroism, red-edge excitation shift (REES), synchronous fluorescence and three dimensional fluorescence spectroscopy under physiological conditions .The results indicated that binding of malathion to HSA induced fluorescence quenching through static mechanism. The number of binding sites was calculated by double logarithmic equation. Changes in the micro-environment of the fluorophore residues were also probed by synchronous fluorescence spectroscopy and REES. Changes of secondary structure of HSA in HSA–malathion complex was verified by circular dichroism approach in the presence of AgNPs that showed the electrostatic interaction changes in the protein structure. The binding average distance (r) between the donor (HSA) and the acceptor (malathion) was measured and found to be 1.63?nm according to the Forster’s theory of non-radiation energy transfer which was <7?nm confirmed the existence of static quenching in the presence of AgNPs. The conformational changes of HSA by three-dimensional fluorescence spectroscopy were studied. By comparing the resonance light scattering in the binary and ternary systems, we could estimate the effect of AgNPs on the precipitation of the malathion on the HSA. Generally we have discussed the toxicity reduction effect of malathion in food industrial by the results of spectroscopy techniques.     

A fluorescent fatty acid probe, DAUDA, selectively displaces two myristates bound in human serum albumin

11-(Dansylamino) undecanoic acid (DAUDA) is a dansyl-type fluorophore and has widely used as a probe to determine the binding site for human serum albumin (HSA). Here, we reported that structure of HSA-Myristate-DAUDA ternary complex and identified clearly the presence of two DAUDA molecules at fatty acid (FA) binding site 6 and 7 of HSA, thus showing these two sites are weak FA binding sites. This result also show that DAUDA is an appropriate probe for FA site 6 and 7 on HSA as previous studied, but not a good probe of FA binding site 1 that is likely bilirubin binding site on HSA.     

Comparison of the binding behavior of FCCP with HSA and HTF as determined by spectroscopic and molecular modeling techniques

The interaction of carbonylcyanide p‐(trifluoromethoxy) phenylhydrazone (FCCP) with human serum albumin (HSA) and human transferrin (HTF) was investigated using multiple spectroscopy, molecular modeling, zeta‐potential and conductometry measurements of aqueous solutions at pH 7.4. The fluorescence, UV/vis and polarization fluorescence spectroscopy data disclosed that the drug–protein complex formation occurred through a remarkable static quenching. Based on the fluorescence quenching, two sets of binding sites with distinct affinities for FCCP existed in the two proteins. Steady‐state and polarization fluorescence analysis showed that there were more affinities between FCCP and HSA than HTF. Far UV‐CD and synchronous fluorescence studies indicated that FCCP induced more structural changes on HSA. The resonance light scattering (RLS) and zeta‐potential measurements suggested that HTF had a greater resistance to drug aggregation, whereas conductometry measurements expressed the presence of free ions improving the resistance of HSA to aggregation. Thermodynamic measurements implied that a combination of electrostatic and hydrophobic forces was involved in the interaction between FCCP with both proteins. The phase diagram plots indicated that the presence of second binding site on HSA and HTF was due to the existence of intermediate structures. Site marker competitive experiments demonstrated that FCCP had two distinct binding sites in HSA which were located in sub‐domains IIA and IIIA and one binding site in the C‐lobe of HTF as confirmed by molecular modeling. The obtained results suggested that both proteins could act as drug carriers, but that the HSA potentially had a higher capacity for delivering FCCP to cancerous tissues. Copyright © 2013 John Wiley & Sons, Ltd.     

Bilirubin,model membranes and serum albumin interaction: The influence of fatty acids

Electronic circular dichroism (ECD), absorption and fluorescence spectroscopy were used to study the enantioselective interactions which involved bilirubin (BR), liposomes, human serum albumin of two different purities, pure (HSA) and non-purified of fatty acids (FA-HSA), and individual fatty acids.The application of the ECD technique to such a complex problem provided a new perspective on the BR binding to liposomes. Our results demonstrated that in the presence of pure HSA, BR preferred to bind to the protein over the liposomes. However, in the presence of FA-HSA, BR significantly bound to the liposomes composed either of DMPC or of sphingomyelin and bound only moderately to the primary and secondary binding sites of FA-HSA even at high BR concentrations. For the DMPC liposomes, even a change of BR conformation upon binding to the primary binding site was observed. The individual saturated fatty acids influenced the BR binding to HSA and liposomes in a similar way as fatty acids from FA-HSA. The unsaturated fatty acids interacted with BR alone and prevented it from interacting with either 99-HSA or the liposomes. In the presence of arachidonic acid, BR interacted enantioselectively with the liposomes and only moderately with 99-HSA.Hence, our results show a substantial impact of the liposomes on the BR binding to HSA. As a consequence of the existence of fatty acids in the blood plasma and in the natural structure of HSA, BR may possibly bind to the cell membranes even though it is normally bound to HSA.     

Calorimetric and Binding Dissections of HSA Upon Interaction with Bilirubin

The interactions between bilirubin and human serum albumin (HSA) were studied by isothermal titration calorimetry (ITC) and UV–vis spectrophotometry at 27°C in 100 mM phosphate buffer pH 7.4 containing 1 mM EDTA. The biphasic shape of the HSA–bilirubin binding curve depicted the existence of two bilirubin binding sets on the HSA structure which had distinct binding interactions. Each binding set contained one or more bilirubin binding site. The first binding set at subdomain IIA included one binding site and had a more hydrophobic microenvironment than the other two binding sites in the second bilirubin binding set (subdomain IIIA). With our method of analysis, the calculated dissociation constant of the first binding site is 1.28×10⁻⁶ M and 4.80×10⁻⁴ M for the second and third binding sites. Here, the typical Boltzmann’s equation was used with a new approach to calculate the dissociation constants as well as the standard free energy changes for the HSA–bilirubin interactions. Interestingly, our calculations obtained using the Wyman binding potential theory confirmed that our analysis method had been correct (especially for the second binding phase). The molar extinction coefficient determined for the first bound bilirubin molecule depicted that the bilirubin molecules (in low concentrations) should interact with the nonpolar microenvironment of the first high affinity binding site. Binding of the bilirubin molecules to the first binding site was endothermic (ΔH^o>0) and occurred through the large increase in the binding entropy established when the hydrophobic bilirubin molecules escaped from their surrounding polar water molecules and into the hydrophobic medium of the first binding site. On the other hand, the calculated molar extinction coefficient illustrated that the microenvironment of the second binding set (especially for the third binding site) was less hydrophobic than the first one but still more hydrophobic than the buffer medium. The binding of the third bilirubin molecule to the HSA molecule was established more through exothermic (electrostatic) interactions.     

Binding analysis of glycyrrhetinic acid to human serum albumin: fluorescence spectroscopy, FTIR, and molecular modeling

Fluorescence spectroscopy, Fourier transform infrared spectroscopy (FTIR), and molecular modeling methods were employed to analyze the binding of glycyrrhetinic acid (GEA) to human serum albumin (HSA) under physiological conditions with GEA concentrations from 4.0x10(-6) to 4.5x10(-5) mol L(-1). The binding of GEA to HSA was via two types of sites: the numbers of binding site for the first type was near 0.45 and for the second type it was approximately 0.75. The binding constants of the second type binding site were lower than those of the first type binding site at corresponding temperatures, the results suggesting that the first type of binding site had high affinity and the second binding site involved other sites with lower binding affinity and selectivity. The fluorescence titration results indicated that GEA quenched the fluorescence intensity of HSA through static mechanism. The FTIR spectra evidence showed that the protein secondary structure changed with reduction of alpha-helices about 26.2% at the drug to protein molar ratio of 3. Thermodynamic analysis showed that hydrogen bonds were the mainly binding force in the first type of binding site, and hydrophobic interactions might play a main role in the second type of binding site. Furthermore, the study of computational modeling indicated that GEA could bind to the site I of HSA and hydrophobic interaction was the major acting force for the second type of binding site, which was in agreement with the thermodynamic analysis.     

The role of electrostatic interactions in human serum albumin binding and stabilization by halothane

Electrostatic interactions have been proposed as a potentially important force for anesthetics and protein binding but have not yet been tested directly. In the present study, we used wild-type human serum albumin (HSA) and specific site-directed mutants as a native protein model to investigate the role of electrostatic interactions in halothane binding. Structural geometry analysis of the HSA-halothane complex predicted an absence of significant electrostatic interactions, and direct binding (tryptophan fluorescence and zonal elution chromatography) and stability experiments (hydrogen exchange) confirmed that loss of charge in the binding sites, by charged to uncharged mutations and by changing ionic strength of the buffer, generally increased both regional (tryptophan region) and global halothane/HSA affinity. The results indicate that electrostatic interactions (full charges) either do not contribute or diminish halothane binding to HSA, leaving only the more general hydrophobic and van der Waals forces as the major contributors to the binding interaction.     

Photo-isomerization and oxidation of bilirubin in mammals is dependent on albumin binding

The bilirubin (BR) photo-conversion in the human body is a protein-dependent process; an effective photo-isomerization of the potentially neurotoxic Z,Z-BR as well as its oxidation to biliverdin in the antioxidant redox cycle is possible only when BR is bound on serum albumin. We present a novel analytical concept in the study of linear tetrapyrroles metabolic processes based on an in-depth mapping of binding sites in the structure of human serum albumin (HSA). A combination of fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and molecular modeling methods was used for recognition of the binding site for BR, its derivatives (mesobilirubin and bilirubin ditaurate), and the products of the photo-isomerization and oxidation (lumirubin, biliverdin, and xanthobilirubic acid) on HSA. The CD spectra and fluorescent quenching of the Trp–HSA were used to calculate the binding constants. The results of the CD displacement experiments performed with hemin were interpreted together with the findings of molecular docking performed on the pigment–HSA complexes. We estimated that Z,Z-BR and its metabolic products bind on two independent binding sites. Our findings support the existence of a reversible antioxidant redox cycle for BR and explain an additional pathway of the photo-isomerization process (increase of HSA binding capacity; the excess free [unbound] BR can be converted and also bound to HSA).     

Molecular interaction of some cardiovascular drugs with human serum albumin at physiological‐like conditions: A new approach

In the present study, the interaction of human serum albumin (HSA) with some cardiovascular drugs (CARs) under physiological conditions was investigated via the fluorescence spectroscopic and Fourier transform infrared spectroscopy. The CAR included Captopril, Timolol, Propranolol, Atenolol, and Amiodarone. Cardiovascular drugs can effectively quench the endogenous fluorescence of HSA by static quenching mechanism. The fluorescence quenching of HSA is mainly caused by complex formation of HSA with CAR. The binding reaction of CAR with HSA can be concluded that hydrophobic and electrostatic interactions are the main binding forces in the CAR‐HSA system. The results showed that CAR strongly quenched the intrinsic fluorescence of HSA through a static quenching procedure, and nonradiation energy transfer happened within molecules. Fourier transform infrared spectroscopy absorption studies showed that the secondary structure was changed according to the interaction of HSA and CAR. The binding reaction of CAR with HSA can be concluded that hydrophobic and electrostatic interactions are the main binding forces in the CAR‐HSA system. The results obtained herein will be of biological significance in pharmacology and clinical medicines.     

Binding of the Promen fluorescent probe to human serum albumin: a fluorescence spectroscopic study.

The binding of Promen (6-propionyl-2-methoxynapthalene) to human serum albumin (HSA) was measured by fluorescence spectroscopy, finding only one class of binding sites on the protein. Hydrophobic interactions play an important role to stabilize the complex. Attempts were made to characterize its binding site using as competitors warfarin, phenylbutazone and diazepam, which bind in a specific site or region on the HSA. Fluorescence polarization measurements and spectrofluorimetric results suggest that diazepam and Promen bind at different but interacting binding sites on the HSA. The changes in the fluorescence emission of the bound Promen in the presence of these drugs, allow to use Promen to detect unspecific interactions with the site II on the HSA.     

Binding of the bioactive component jatrorrhizine to human serum albumin

The interaction between Jatrorrhizine with human serum albumin (HSA) were studied by fluorescence quenching technique, circular dichroism (CD) spectroscopy, and Fourier transform infrared (FT-IR) spectroscopy. Fluorescence data revealed the presence of a single class of binding site on HSA and its binding constants (K) are 7.278 x 10(4), 6.526 x 10(4), and 5.965 x 10(4) L.mol(-1) at 296, 303, and 310 K, respectively. The CD spectra and FT-IR spectra have proved that the protein secondary structure changed in the presence of Jatrorrhizine in aqueous solution. The effect of common ions on the binding constants was also investigated. In addition, the thermodynamic functions standard enthalpy (DeltaH(0)) and standard entropy (DeltaS(0)) for the reaction were calculated to be -10.891 kJ.mol(-1) and 56.267 J.mol(-1) K(-1), according to the van't Hoff equation. These data indicated that hydrophobic and electrostatic interactions played a major role in the binding of Jatrorrhizine to HSA. Furthermore, the displacement experiments indicated that Jatrorrhizine could bind to the site I of HSA, which was also in agreement with the result of the molecular modeling study.     

Multi-spectroscopic and molecular modeling studies of interaction between two different angiotensin I converting enzyme inhibitory peptides from gluten hydrolysate and human serum albumin

The present study was carried out to characterize Angiotensin-converting enzyme (ACE) inhibitory peptides which are released from the trypsin hydrolysate of wheat gluten protein. The binding of two inhibitory peptide (P₄ and P₆) to human serum albumin (HSA) under physiological conditions has been investigated by multi-spectroscopic in combination with molecular modeling techniques. Time-resolved and quenching fluorescence spectroscopies results revealed that the quenching of HSA fluorescence by P₄ and P₆ in the binary and ternary systems caused HSA-peptides complexes formation. The results indicated that both peptides quenched the fluorescence intensity of HSA through a static mechanism. The binding affinities and number of binding sites were obtained for the HSA-peptides complexes. The circular dichroism (CD) data revealed that the presence of both peptides increased the α-helix content of HSA and induced the remarkable folding of the polypeptide of the protein. Therefore, the CD data determined that the protein structure has been stabilized in the percent of ACE inhibitory peptides in binary and ternary systems. The binding distances between HSA and both peptides were estimated by the Forster theory, and it was revealed that nonradiative energy transfer from HSA to peptides occurred with a high probability. ITC experiments reveal that, in the absence and presence of P₆, the dominant forces are electrostatic in binary and ternary systems. Furthermore, molecular modeling studies confirmed the experimental results. Molecular modeling investigation suggested that P₄ bound to the site IA and IIA of HSA in binary and ternary systems, respectively. This study on the interaction of peptides with HSA should prove helpful for realizing the distribution and transportation of food compliments and drugs in vivo, elucidating the action mechanism and dynamics of food compliments and drugs at the molecular level. It should moreover be of great use for understanding the pharmacokinetic and pharmacodynamic mechanism of the food compliments and drugs.     

Spectroscopic investigation of the anticancer alkaloid piperlongumine binding to human serum albumin from the viewpoint of drug delivery

Piperlongumine (PL) is a very promising natural agent with a high potential for cancer treatment. To overcome the poor water solubility of PL, there is a need to develop a novel water‐soluble formulation in which PL is non‐covalently bound to human serum albumin (HSA). PL binding to HSA was studied by various spectroscopic techniques under simulated physiological conditions. Spectroscopic evidence showed that the interaction of PL with HSA could form a PL–HSA complex. The binding constant (K_a) values increased with increasing temperature, and a similar dependence was observed for the number of binding sites (n) values. The number of PL molecules bound to HSA reached 8.1 when the temperature was raised to 308 K. Thermodynamic calculation results suggested that the binding reaction occurred spontaneously but was an entropy‐driven process, and hydrophobic forces played a major role in stabilizing the complex. Furthermore, PL binding induced conformational and microenvironmental changes in HSA. Displacement studies indicated that PL and warfarin had separate binding regions in site I. Therefore, it would be possible to develop a novel water‐soluble formulation involving PL and HSA. This study may provide some valuable information in terms of improving the poor water solubility of PL.     

A new drug binding subsite on human serum albumin and drug-drug interaction studied by X-ray crystallography

3'-Azido-3'-deoxythymidine (AZT) is the first clinically effective drug for the treatment of human immunodeficiency virus infection. The drug interaction with human serum albumin (HSA) has been an important component in understanding its mechanism of action, especially in drug distribution and in drug-drug interaction on HSA in the case of multi-drug therapy. We present here crystal structures of a ternary HSA-Myr-AZT complex and a quaternary HSA-Myr-AZT-SAL complex (Myr, myristate; SAL, salicylic acid). From this study, a new drug binding subsite on HSA Sudlow site 1 was identified. The presence of fatty acid is needed for the creation of this subsite due to fatty acid induced conformational changes of HSA. Thus, the Sudlow site 1 of HSA can be divided into three non-overlapped subsites: a SAL subsite, an indomethacin subsite and an AZT subsite. Binding of a drug to HSA often influences simultaneous binding of other drugs. From the HSA-Myr-AZT-SAL complex structure, we observed the coexistence of two drugs (AZT and SAL) in Sudlow site 1 and the competition between these two drugs in subdomain IB. These results provide new structural information on HSA-drug interaction and drug-drug interaction on HSA.     

Exploration of the binding of proton pump inhibitors to human P450 2C9 based on docking and molecular dynamics simulation

Human P450 protein CYP2C9 is one of the major drug-metabolizing isomers, contributing to the oxidation of 16% of the drugs currently in clinical use. To examine the interaction mechanisms between CYP2C9 and proton pump inhibitions (PPIs), we used molecular docking and molecular dynamics (MD) simulation methods to investigate the conformations and interactions around the binding sites of PPIs/CYPP2C9. Results from molecular docking and MD simulations demonstrate that nine PPIs adopt two different conformations (extended and U-bend structures) at the binding sites and position themselves far above the heme of 2C9. The presence of PPIs changes the secondary structures and residue flexibilities of 2C9. Interestingly, at the binding sites of all PPI–CYP2C9 complexes except for Lan/CYP2C9, there are hydrogen-bonding networks made of PPIs, water molecules, and some residues of 2C9. Moreover, there are strong hydrophobic interactions at all binding sites for PPIs/2C9, which indicate that electrostatic interactions and hydrophobic interactions appear to be important for stabilizing the binding sites of most PPIs/2C9. However, in the case of Lan/2C9, the hydrophobic interactions are more important than the electrostatic interactions for stabilizing the binding site. In addition, an interesting conformational conversion from extended to U-bend structures was observed for pantoprazole, which is attributed to an H-bond interaction in the binding pocket, an internal π–π stacking interaction, and an internal electrostatic interaction of pantoprazole.     

Investigation of the Interaction Between Human Serum Albumin and Antitumor Palladium(II) Complex Containing 1,10-Phenanthroline and Dithiocarbamate Ligands

The interaction between [Pd(But-dtc)(phen)]NO₃ (where But-dtc = butyldithiocarbamate and phen = 1,10-phenanthroline) with HSA (Human Serum Albumin) was investigated by applying fluorescence, UV–Vis and circular dichroism techniques under physiological conditions. The results of fluorescence spectra indicated that the Pd(II) complex could effectively quench the fluorescence intensity of HSA molecules via static mechanism. The number of binding sites and binding constant of HSA–Pd(II) complex were calculated. Analysis of absorption titration data on the interaction between Pd(II) complex and HSA revealed the formation of HSA–Pd(II) complex with high-binding affinity. Thermodynamic parameters indicated that hydrophobic forces play a major role in this interaction. Furthermore, CD measurements were taken to explore changes in HSA secondary structure induced by the Pd(II) complex.