Characterization of interaction between scoparone and bovine serum albumin: spectroscopic and molecular docking methods

Scoparone is a major biological active substance derived from the traditional Chinese herbal medicine called Artemisia capillaris. It has been confirmed that scoparone has anti-inflammatory, anti-tumor, hepatoprotective and antioxidant effects. However, the binding interaction of scoparone with bovine serum albumin (BSA) still remains unknown. Therefore, the present study was conducted to clarify the binding interaction of scoparone with BSA under simulated physiological conditions (pH = 7.4) by utilizing spectroscopic and molecular docking methods. The formation of the scoparone–BSA complex was identified by UV-vis absorption spectroscopy experiment results. The fluorescence experiment results revealed that the quenching mechanism was static quenching and the binding procedure was spontaneous mainly driven by hydrophobic interaction. At 310 K, the number of binding sites was approximately equal to 1 and the binding constant was 6.79 × 10⁵ mol L⁻¹. The binding distance (4.81 nm) between scoparone and BSA was determined by Förster''s non-radiative energy transfer theory. Molecular docking and site marker competitive experiment results verified that scoparone was more likely to be located in site I of BSA. In addition, the results of synchronous fluorescence spectroscopy and circular dichroism spectroscopy experiments proved that scoparone slightly changed the conformation of BSA by binding interaction with BSA. These findings would be useful for understanding the pharmacokinetics of scoparone in vivo, including scoparone transport, distribution, metabolism and excretion.

The interaction of scoparone with bovine serum albumin (BSA) was studied by utilizing spectroscopic and molecular docking methodologies.     

Characterizing the binding interaction of astilbin with bovine serum albumin: a spectroscopic study in combination with molecular docking technology

Astilbin (ASN) is a flavonoid compound isolated from the rhizome of Smilax china L. (Smilacaceae). It has many bioactivities, such as selective immunosuppression, antioxidant, anti-hepatic injury, etc., and is widely used in traditional Chinese medical treatments. The interaction of ASN with bovine serum albumin (BSA) was studied in a physiological buffer (pH = 7.40) using multi-spectroscopic techniques in combination with molecular docking methods. UV-vis absorption measurements proved that a ASN–BSA complex could be formed. Fluorescence data revealed that ASN could strongly quench the intrinsic fluorescence of BSA in terms of a static quenching procedure. The process of binding was spontaneous and the binding occurred mainly through hydrogen bonding and van der Waals forces. The distance r between donor (BSA) and acceptor (ASN) was calculated to be 4.80 nm based on Förster''s non-radiative energy transfer theory. The binding constant (K_a = 7.31 × 10⁴ mol L⁻¹) and the number of binding sites (n ≈ 1) at 298 K suggested that ASN only occupied one site in BSA with high affinity. Moreover, the results of molecular docking indicated that ASN was more likely to be located in site I (sub-domain IIA) of BSA. The results of synchronous fluorescence and three-dimensional fluorescence spectra showed that ASN induced conformational changes of BSA. The findings would be beneficial for research on the transportation, distribution and some important bioactivities of ASN in the human body.

The interaction of astilbin with bovine serum albumin was confirmed by multi-spectroscopic techniques and molecular docking methods.     

Synthesis of novel genistein amino acid derivatives and investigation on their interactions with bovine serum albumin by spectroscopy and molecular docking

Genistein amino acid derivatives 4a–4d were synthesized and evaluated for their cytotoxic activities against MCF-7, Hela, MGC-803 and HCT-116 cell lines by MTT assays in vitro. The results revealed that compounds 4a–4d showed better activity than the parent compound genistein. Particularly, compound 4b displayed the most significant anticancer activity against MGC-803 with an IC₅₀ value of 12.08 μM. In addition, the mechanisms of interaction between genistein, compounds 4a–4d and BSA were investigated via multi-spectroscopic techniques such as ultraviolet (UV) spectroscopy, fluorescence, circular dichroism (CD), and molecular docking under physiological conditions. The results suggested that endogenous fluorescence of BSA could be quenched by genistein and compounds 4a–4dvia forming BSA-compound complex, which meant a static quenching mechanism was involved. The negative values of enthalpy (ΔH) and entropy (ΔS) indicated that interactions between BSA and the ligands were spontaneous, and hydrogen bonding and van der Waals interactions were involved in the BSA-compound complexion formation. The UV, synchronous and 3D fluorescence results revealed that the micro-environment of tryptophan and conformation of BSA were changed after binding to ligands. CD analysis demonstrated the variation in the secondary structure and that the α-helix content of BSA decreased. Eventually, molecular docking was executed to forecast the binding forces and binding sites between BSA and compounds 4a–4d.

Introducing amino acid into genistein can not only improve its antitumor activity, but also enhance its binding affinity to BSA.     

Combined spectroscopy methods and molecular simulations for the binding properties of trametinib to human serum albumin

Trametinib is a novel anticancer drug for treating metastatic cutaneous melanoma. The present study probed into the binding of trametinib to human serum albumin (HSA) through spectroscopy methods and molecular simulations. Trametinib could quench the fluorescence of HSA through static quenching which could be probed via fluorescence spectroscopy and time-resolved fluorescence. Thermodynamic parameters and docking results indicated that hydrogen bonds and van der Waals forces play crucial roles in this binding process, which exerts almost no effect on the HSA conformation under synchronous fluorescence, three-dimensional fluorescence, circular dichroism spectra, and molecular dynamics simulations. Site marker displacement experiments and molecular docking reveal that trametinib primarily binds to Sudlow site I of HSA. In addition, the trametinib–HSA interaction was hardly influenced by varying amino acid (glutamine, alanine, glycine, and valine) concentrations. This study can provide useful information for the pharmacokinetic properties of trametinib.

Probing the binding properties of trametinib to human serum albumin.     

Insights into the interaction mechanism between tiagabine hydrochloride and two serum albumins

Tiagabine hydrochloride (TGB) is a widely used anticonvulsive drug for the treatment of epilepsy. To better understand the interactions of TGB with plasma proteins, human serum albumin (HSA) and bovine serum albumin (BSA) were selected as model proteins. TGB slightly increased thermal stability of the proteins as confirmed by VP-capillary differential scanning calorimetric (DSC) measurements. Isothermal titration calorimeter (ITC) results showed that TGB could be combined with HSA and BSA moderately, which was also corroborated by fluorescence analysis. Besides, the thermodynamic parameters (ΔH > 0, ΔS > 0) indicated that hydrophobic forces played a major role in the formulation of TGB–HSA and TGB–BSA complexes. Moreover, the main binding sites of TGB to HSA and BSA were also examined by classical fluorescent probe (dansylsarcosine and dansylamide) experiments, showing that TGB and dansylsarcosine competitively interacted with HSA and BSA at the same binding sites. Additionally, TGB had no obvious effect on the conformation change of HSA and BSA as indicated by spectroscopic analyses. This study provides useful information about the interaction mechanism of TGB and serum albumins, which could help to better utilize TGB in biomedical field.

Tiagabine could bind in Sudlow site II of HSA and BSA.     

Characterizing the interaction between methyl ferulate and human serum albumin by saturation transfer difference NMR

Methyl ferulate (MF) is an alkyl ferulate ester that widely exists in edible plants and has application value in the food and medicine industries. Thus, its effect on biological macromolecules should be considered. In this study, we exploit saturation transfer difference NMR (STD-NMR) to characterize the interaction of all protons of MF with human serum albumin (HSA) at the molecular level. STD-NMR and K_a (1.298 × 10³ M⁻¹) revealed that protons H1–6 and H8 bound to HSA with a medium affinity. Binding epitope mapping further showed that the aromatic ring played a key role in the HSA–MF interaction. STD-NMR site-marker-displacement experiments and circular dichroism spectroscopy revealed that MF prefered to bind to site II of HSA without changing the basic skeleton of HSA. Computer simulations confirmed these experimental results. Overall, this work elucidates the molecular level interaction of MF with HSA and provides new insights into the possibility of the potential applications of MF in the food and medicine industries.

STD-NMR technique characterized the recognition mechanism of methyl ferulate and human serum albumin qualitatively and quantitatively.     

Interaction of indomethacin with adult human albumin and neonatal serum

The binding of indomethacin to albumin was investigated at 37 degrees C, pH 7.4. The first stoichiometric binding constant is 2.5 X 10(5) M-1. Indomethacin utilizes both the bilirubin and diazepam binding functions equally. The effect on bilirubin binding to albumin is negligible at therapeutic indomethacin blood levels. Oleic acid does not affect indomethacin binding until the oleate/albumin molar ratio exceeds 2. A method for measuring the reserve albumin equivalent for binding indomethacin is described. Measurement of this reserve binding equivalent in 33 neonatal serum samples shows no significant correlation of the reserve binding to the albumin level. This methodology may be useful in studying the variable response of infants with patent ductus arteriosus to indomethacin.     

Insights from a combination of theoretical and experimental methods for probing the biomolecular interactions between human serum albumin and clomiphene

In this study, the interaction of clomiphene (CLO), a non-steroidal and ovulatory stimulant drug employed in the treatment of infertility, with human serum albumin (HSA), the most abundant plasma transport protein, was investigated using spectrofluorometric, FT-IR, UV-Vis, and molecular modeling methods. The obtained results indicated that the binding of CLO to HSA led to intense fluorescence quenching of HSA via a static quenching mechanism, and that the process of CLO binding to HSA was enthalpy driven. By using experimental and theoretical methods, it was confirmed that as a result of binding CLO, slight conformational changes in HSA occurred. Also, the negative ΔH of interaction indicated that the binding of CLO with HSA was mainly enthalpy driven. The experimental and computational results suggested that hydrogen bonds and van der Waals interactions played a major role in the binding, with overall binding constants of K = 3.67 × 10⁹ M⁻¹ at 286 K and 6.52 × 10⁵ mol L⁻¹ at 310 K. Moreover, the results of molecular modeling showed that Asp234, Phe228, Leu327, and Arg209 in HSA had the highest interaction energies with the ligand.

In this study, the interaction of clomiphene with human serum albumin (HSA), the most abundant plasma transport protein, was investigated using spectrofluorometric, FT-IR, UV-Vis, and molecular modeling methods.     

Insights into the molecular inactivation mechanism of human activated thrombin‐activatable fibrinolysis inhibitor

See also Gils A. Hot spots in TAFIa. This issue, pp 1054–5. Summary. Background: Thrombin‐activatable fibrinolysis inhibitor (TAFI) is a validated target for thrombotic diseases. TAFI is converted in vivo to activated TAFI (TAFIa) by removal of its pro‐domain. Whereas TAFI is stable and persists in the circulation, possibly in complex with plasminogen, TAFIa is unstable and poorly soluble, with a half‐life of minutes.Objectives: In order to study the molecular determinants of this instability, we studied the influence of protein inhibitors on human TAFIa. Results: We found that protein inhibitors significantly reduced the instability and insolubility of TAFIa. In addition, we solved the 2.5‐Å resolution crystal structure of human TAFIa in complex with a potent protein inhibitor, tick‐derived carboxypeptidase inhibitor, which gives rise to a stable and soluble TAFIa species. The structure revealed a significant reduction in the flexibility of dynamic segments when compared with the structures of bovine and human TAFI. We also identified two latent hotspots, loop Lβ2β3 and segment α5–Lα5β7–β7, where conformational destabilization may begin. These hotspots are also present in TAFI, but the pro‐domain may provide sufficient stabilization and solubility to guarantee protein persistence in vivo. When the pro‐domain is removed, the free TAFIa moiety becomes unstable, its activity is suppressed, and the molecule becomes insoluble. Conclusions: The present study corroborates the function of protein inhibitors in stabilizing human TAFIa and it provides a rigid and high‐resolution mold for the design of small molecule inhibitors of this enzyme, thus paving the way for novel therapy for thrombotic disorders.     

Normal and low molecular weight heparins: interaction with human platelets

Porcine mucosal heparin was chemically depolymerized. The depolymerization was stopped at different steps to obtain two low molecular weight (LMW) heparins with a molecular weight of 10 000 and 6000, respectively. The LMW heparins were tested in vitro for anti-clotting activities and for platelet serotonin release in different systems in comparison with normal heparins, dermatan and heparan sulphate. After addition of various amounts of heparin preparations to washed platelets, no significant release was observed for all tested heparins. On the contrary, different heparins showed an inhibition of serotonin-release induced by collagen in platelet rich plasma, whereas the ADP-induced release was increased. The effect on the platelet release appears related to the molecular weight. In fact, it is significant only for normal heparins whereas it is not for LMW heparins. A good relation was observed, also, between anti-activated factor X activity/antiglobal clotting activity (Xa/APTT) ratio of different heparins and the effect on platelet release.     

Thermodynamic investigation of the interaction between ionic liquid functionalized gold nanoparticles and human serum albumin for selective determination of glutamine

The excellent biocompatible and monodispersed gold nanoparticles (AuNPs) functionalized by amino based ionic liquid (IL) have been synthesized for the demonstration of their interaction with human serum albumin (HSA). Amino based IL stabilizes the surface of AuNPs and provides a colorimetric sensor platform. The size of synthesized IL–AuNPs was identified by use of transmission electron microscopy (TEM) and dynamic light scattering (DLS) techniques. Molecular interaction of functionalized AuNPs with HSA have been investigated using multispectroscopic techniques, such as UV-Vis, fluorescence and Fourier transform infra-red (FT-IR) spectroscopy. The fluorescence and synchronous fluorescent intensity together indicated that IL–AuNPs exhibits a strong ability to quench the intrinsic fluorescence of HSA via a dynamic quenching mechanism. Moreover, the binding constant (K_a), Stern–Volmer quenching constant (K_SV) and different thermodynamic parameters, namely Gibb''s free energy (ΔG), enthalpy (ΔH) and entropy (ΔS) have been evaluated at different temperatures. This interactive study focuses on the nature of surface modification of IL–AuNPs via HSA for selective detection of glutamine (Glu) with a lower limit of detection of 0.67 nM in the linear range of 10–100 nM for Glu.

The excellent biocompatible and monodispersed gold nanoparticles (AuNPs) functionalized by amino based ionic liquid (IL) have been synthesized for the demonstration of their interaction with human serum albumin (HSA).     

The presence of halide salts influences the non‐covalent interaction of MRI contrast agents and human serum albumin

The rationale and objectives of the study were to evaluate the influence of the experimental conditions (buffer, salt, etc.) on the data characterizing the non‐covalent interaction between MRI contrast agents and human serum albumin and hence their in vivo relaxivity. The interaction of three gadolinium contrast agents (Gd‐EOB‐DTPA, Gd‐BOPTA and MP‐2269) with human serum albumin was assessed through the measurement of proton relaxation rate enhancement in various experimental conditions. The data show the negative effect of halide salts on the paramagnetic relaxation enhancement of the three contrast agents. The presence of halide salts can thus have a negative effect on the efficacy of MRI contrast agents interacting with HSA. In addition, careful attention must be paid to comparisons of the binding parameters of various contrast agents reported in different studies since the composition of the medium can greatly influence the non‐covalent interaction. Copyright © 2007 John Wiley & Sons, Ltd.     

Pyrogen reactions associated with the infusion of normal serum albumin (human)

In November, 1974, eight patients in three hospitals had pyrogen reactions associated with the infusion of 25% Normal Serum Albumin from the same lot. The reactions were recognized because the same physician or nurse observed several patients having recurrent reactions or because a single patient receiving several vials had consecutive reactions. The remaining albumin in three vials associated with reactions had apparent endotoxin concentrations of 4, 16, and 32 ng/ml and that 22 vials from recalled supplies had a median concentration of 4 ng/ml (range: 2 to 64) as determined by the Limulus amebocyte lysate test, but the lot again passed the rabbit pyrogen test. In a prospective study to determine the efficacy of the Limulus test in quality control, patients had their temperatures taken hourly during albumin infusions and the remaining fluid was tested by the Limulus assay. The albumin in 443 of the 662 vials infused (65%) gave a positive test and 311 of these vials (45%) had apparent endotoxin concentrations of 4 to 64 ng/ml, but no patient had a reaction. Because of the limitations of both the rabbit pyrogen and Limulus test, the detection of some pyrogenic lots continues to depend on hospital surveillance and reporting os suspect reactions.     

Studies on the coumarin anticoagulant drugs: interaction of human plasma albumin and warfarin sodium

In studies by continuous flow electrophoresis the coumarin anticoagulant drug warfarin sodium was found to be bound solely to the albumin fraction of the plasma proteins. The interaction was studied in detail by equilibrium dialysis of solutions of crystalline human plasma albumin and warfarin sodium. Analysis of the data showed that albumin possesses a single strong binding site for warfarin with an association constant of 154,000 at 3 degrees C and secondary classes of several sites with a much lower affinity. The free energy of binding for the first anion determined at 3 degrees and 37 degrees C was -6.54 and -7.01 kcal per mole, respectively. The standard enthalpy change for the interaction was -3.48 kcal per mole, and the entropy change was +11.2 U.The negative enthalpy change was surprisingly large and the positive entropy change small for an anion-albumin interaction, suggesting significant nonionic binding. The inability to saturate the albumin binding sites, even when high concentrations of warfarin were used, is consistent with a reversible configurational alteration of the albumin molecule during the binding process. The thermodynamic data indicate that the albumin binding sites for warfarin sodium are formed during the process of binding, rather than being performed as in antigen-antibody reactions. The strength of the binding process suggests that many of the pharmacodynamic characteristics of warfarin sodium in man are determined by its strong interaction with plasma albumin. Such correlations of the physicochemical interactions and biologic effects of the coumarin anticoagulant drugs should lead to a better understanding of their mechanisms of action.     

Perfusion of the intervillous space of the human placenta measured with 99mtechnetium labelled human serum albumin

Summary. The perfusion of the intervillous space of the human placenta was investigated with ^99mTc-Human Serum Albumin and external counting in 45 pregnant women between the 34th and 38th week of gestation. The pregnancy was normal in 34 women, while there was placental insufficiency in 11. Two variables were calculated from the placenta curve: the placenta flow index (PFZ) and the mean transit time (t) for plasma through the intervillous space. From double determinations, the coefficient of variation for PFI was calculated to 1·6%. For t it was 20%. PFZ was significantly higher in the controls than in the patients with placental dysfunction (P < 0·05). There was a significant correlation between PFZ and i (r_s= -0·36, P<0·05). The plasma oestriol level and the PFZ correlated significantly, whereas the correlation between i and the plasma oestriol level was not significant. The placenta flow index can be used to measure the physiological effect of a pharmacological treatment, bedrest or other treatments.