A novel biological role for nsLTP2 from Oriza sativa: Potential incorporation with anticancer agents,nucleosides and their analogues

Development of a protein-based drug delivery system has major impact on the efficacy and bioavailability of unstable and water insoluble drugs. In the present study, the binding modes of a nonspecific lipid transfer protein (nsLTP2) from Oryza sativa with various nucleosides and analogous molecules were identified. The 3-D structure of the protein was designed and validated using modeler 9.13, Molegro virtual docker and procheck tool, respectively. The binding affinity and strength of interactions, key contributing residues and specificity toward the substrates were accomplished by computational docking and model prediction. The protein presented high affinity to acyclovir and vidarabine as purine-analogous drugs. Binding affinity is influenced by the core template and functional groups of the ligands which are structurally different cause the variation of interaction energies with nsLTP2. Nonetheless, all the evaluated analogous drugs occupy the proximity space at the nsLTP active site with high similarity in their binding modes. Our findings hold great promise for the future applications of nsLTPs in various aspects of pharmaceutical science and molecular biology.     

Characterization of binding sites for sulfadimethoxine and its major metabolite, N4-acetylsulfadimethoxine, on human and rabbit serum albumin

In order to gain an understanding of protein binding of sulfadimethoxine (SDM) and its major metabolite, N4-acetylsulfadimethoxine (N4-AcSDM), the binding of SDM and N4-AcSDM to human and rabbit serum albumin (HSA and RSA) was investigated using circular dichroism (CD), fluorescence and dialysis techniques. The CD spectral characteristics of the compounds bound to the albumins suggested that the drug-binding sites on the HSA and RSA had somewhat different asymmetries. The binding constants for SDM-HSA and -RSA interaction were smaller than those for N4-AcSDM. Two specific drug-binding sites were found on RSA, similarly to HSA, from the results of competitive displacement using fluorescence probes. Moreover, SDM and N4-AcSDM were found to share the same first binding site on the albumins. It can be presumed from the displacement data with a series of p-aminobenzoates that the characteristics of the binding sites (such as depth and width of the hydrophobic cleft) for SDM and N4-AcSDM on RSA may be almost the same, but the characteristics of these drug-binding sites on HSA may be somewhat different.     

Disaggregation of amphotericin B by sodium deoxycholate micellar aggregates

Amphotericin B (AmB) is present as aggregates in aqueous media. Sodium deoxycholate (NaDC) is known to cause disaggregation of AmB and to form monomers, as is observed by the increase of absorbance at 414 nm. By following the fluorescence intensity at 472 nm (lambda(ex) 335 nm), it was found that with increasing NaDC concentration there is a progressive disaggregation of AmB leading to increase in both monomeric and dimeric forms. Although the presence of dimeric AmB form in NaDC micellar media is known, observation of the growth of the dimeric species is novel. Studies in fluorescence lifetime and steady state fluorescence anisotropy also support this conclusion. As NaDC (at ca. 10 mM) is the preferred choice of medium for AmB delivery and AmB aggregation has been implicated for its toxicity, the observation of dimeric AmB at these NaDC concentrations assumes significance.     

Binding Mechanism of Bioactive Cetirizine Hydrochloride to Sudlow’s Site I of Serum Albumins

The mechanism of interaction of an anti-allergic drug, cetirizine dihydrochloride, with the proteins bovine and human serum albumins has been investigated by spectrofluorimetry, FTIR, UV-vis absorption and circular dichroism methods at physiological conditions. The drug was found to quench the fluorescence intensity of the protein through a dynamic quenching mechanism. Various binding parameters were evaluated based on fluorescence quenching studies carried out at 293 K, 301 K and 308 K. The nature of the binding force operating between the drug and protein was proposed, based on the measured thermodynamic parameters. A change in the secondary structure of the protein was evident from the circular dichroism measurements, wherein the α-helicity decreased from 65.69% to 51.23% and from 42.7% to 38.55% for the CTZ–BSA and CTZ–HSA systems, respectively. Based on Förster’s theory of non-radiative energy transfer, the distance between the protein and drug was determined. The common blood plasma ions K⁺, Cu²⁺, Ni²⁺, Mn²⁺ and Co²⁺ were found to influence the binding of the drug to the protein. Displacement experiments were carried out to determine the binding site for drug on the protein. These studies, coupled with other spectroscopic results, revealed that the drug was bound to the hydrophobic pocket located in sub domain IIA of site I.     

Evaluation of gel electrophoresis techniques and laser ablation–inductively coupled plasma-mass spectrometry for screening analysis of Zn and Cu-binding proteins in plankton

The determination of metal-binding proteins in plankton is important because of their involvement in photosynthesis, which is fundamental to the biogeochemical cycle of the oceans and other ecosystems. We have elaborated a new strategy for screening of Cu and Zn-containing proteins in plankton on the basis of separation of proteins by use of Blue-Native PAGE (BN-PAGE), which entails use of a non-denaturing Tris–tricine system and detection of metals in the proteins by laser ablation inductively coupled plasma mass spectrometry (LA–ICP–MS). For comparison, denaturing PAGE based on Tris–glycine and Tris–tricine systems and Anodic-Native PAGE have also been investigated. A large number of protein bands with MW between 20 and 75 kDa were obtained by use of Tris–glycine PAGE but detection of metals by LA–ICP–MS was unsuccessful because of loss of metals from the proteins during the separation process. Different protein extraction, purification, and preconcentration methods were evaluated, focussing on both issues—achieving the best extraction and characterization of the proteins while maintaining the integrity of metal–protein binding in the plankton sample. Use of 25 mmol?L^?1 Tris–HCl and a protease inhibitor as extraction buffer with subsequent ultrafiltration and acetone precipitation was the most efficient means of sample preparation. Two Cu and Zn proteins were detected, a protein band corresponding to a MW of 60 kDa and another poorly resolved band with a MW between 15 and 35 kDa.     

Interaction Between Ginkgolic Acid and Human Serum Albumin by Spectroscopy and Molecular Modeling Methods

The interaction of ginkgolic acid (15:1, GA) with human serum albumin (HSA) was investigated by FT–IR, CD and fluorescence spectroscopic methods as well as molecular modeling. FT–IR and CD spectroscopic showed that complexation with the drug alters the protein’s conformation by a major reduction of α-helix from 54 % (free HSA) to 46–31 % (drug–complex), inducing a partial protein destabilization. Fluorescence emission spectra demonstrated that the fluorescence quenching of HSA by GA was by a static quenching process with binding constants on the order of 10⁵ L·mol^?1. The thermodynamic parameters (ΔH = ?28.26 kJ·mol^?1, ΔS = 11.55 J·mol^?1·K^?1) indicate that hydrophobic forces play a leading role in the formation of the GA–HSA complex. The ratio of GA and HSA in the complex is 1:1 and the binding distance between them was calculated as 2.2 nm based on the Förster theory, which indicates that the energy transfer from the tryptophan residue in HSA to GA occurs with high probability. On the other hand, molecular docking studies reveal that GA binds to Site II of HSA (sub-domain IIIA), and it also shows that several amino acids participate in drug–protein complexation, which is stabilized by H-bonding.     

Studies on the Antagonistic Behavior Between Cyclophosphamide Hydrochloride and Aspirin with Human Serum Albumin: Time-Resolved Fluorescence Spectroscopy and Isothermal Titration Calorimetry

The interactions between cyclophosphamide hydrochloride (CYC) and aspirin (ASA) with human serum albumin (HSA) were investigated by measuring fluorescence anisotropy, poly-dispersity index, and time-resolved fluorescence. Also, isothermal titration calorimetry (ITC) was performed. The fluorescence spectra of the drugs exhibited an appreciable hypsochromic shift along with an enhancement in the fluorescence intensity. The gradual addition of HSA led to a marked increase in fluorescence anisotropy (r), and from this value it is argued that the drugs were located in a restricted environment of the protein. The binding constants for the ASA–HSA and CYC–HSA complexes were found to be 1.27 × 10⁸ and 4.23 × 10⁸ mol·L^?1, respectively, as calculated from the relevant fluorescence data. The polydispersity index and size distribution of the protein–drug complex were measured at several concentrations of the drugs by the zeta potential technique, which confirmed the already obtained experimental results. From the analysis of the steady-state and time-resolved fluorescence quenching of the drugs in aqueous solutions in the presence of HSA, it was found that the quenching is static in nature. ITC experiments revealed that, in the absence of drugs, the dominant forces are electrostatic, whereas hydrophobic and weak electrostatic forces became significant in the presence of the drug. The primary binding pattern between the drugs and HSA was interpreted as a combined effect of hydrophobic association and electrostatic interactions.     

Electrochemical Study of the Lipid‐Transfer Protein

Electrochemical study of barley grain lipid‐transfer protein (LTP) revealed that it may belong to the metal‐binding protein class. Using differential pulse polarography the presence of Cu(II) and Zn(II) ions in the native LTP structure was proved, as well as its affinity for binding Ni(II) ion. Application of a more sensitive electroanalytical technique, such as anodic stripping voltammetry with analyte preconcentration, revealed the presence of Pb(II) and Cd(II) ions and also enabled the following Hg(II) ion binding. Possible biological role of LTP in plant stress response and its contribution to barley phytoextraction potential are discussed.     

Spectroscopic Investigation of the Interactions of Cryptotanshinone and Icariin with Two Serum Albumins

The interactions of two drugs, cryptotanshinone (CTS) and icariin, with bovine serum albumin (BSA) and human serum albumin (HSA) have been investigated using multiple spectroscopic techniques under imitated physiological conditions. CTS and icariin can quench the fluorescence intensity of BSA/HSA by a static quenching mechanism with complex formation. The binding constants of CTS–BSA, CTS–HSA, icariin–BSA and icariin–HSA complexes were observed to be 1.67 × 10⁴, 4.04 × 10⁴, 4.52 × 10⁵ and 4.20 × 10⁵ L·mol^?1, respectively at 298.15 K. The displacement experiments suggested icariin/CTS are primarily bound to tryptophan residues of the proteins within site I and site II. The thermodynamic parameters calculated on the basis of the temperature dependence of the binding constants revealed that the binding of CTS–BSA/HSA mainly depends on van der Waals interaction and hydrogen bonds, and yet the binding of icariin–HSA/BSA strongly relies on the hydrophobic interactions. The binding distances between BSA/HSA and CTS/icariin were evaluated by the Föster non-radiative energy transfer theory. The results of synchronous fluorescence, 3D fluorescence, FT-IR and CD spectra indicates that the conformations of proteins were altered with the addition of CTS or icariin. In addition, the effects of some common ions on the binding constants of CTS/icariin to proteins are also discussed.     

Microseparation techniques for the study of the enantioselectivity of drug–plasma protein binding

Stereoselectivity in protein binding can have a significant effect on the pharmacokinetic and pharmacodynamic properties of chiral drugs. The investigation of enantioselectivity of drugs in their binding with human plasma proteins and the identification of the molecular mechanisms involved in the stereodiscrimination by the proteins represent a great challenge for clinical pharmacology. In this review, the separation techniques used for enantioselective protein binding experiments are described and compared. An overview of studies on enantiomer–protein interactions, enantiomer–enantiomer interactions as well as chiral drug–drug interactions, including allosteric effects, is presented. The contribution of individual plasma proteins to the overall enantioselective binding and the animal species variability in drug–plasma protein binding stereoselectivity are reviewed. Copyright © 2008 John Wiley & Sons, Ltd.     

A comparison of the inclusion behavior of human serum albumin and holo transferrin with fluoxymesterone in the presence of three different cyclodextrins

This article describes the interaction of fluoxymesterone (Flu) with HSA and HTF in the absence and presence of cyclodextrins (CDs) (α, β and γ). According to fluorescence data, the binding of Flu to the proteins caused strong static quenching in the binary and ternary systems. The fluorescence quenching results demonstrated that HSA and HTF had two and one class of apparent binding sites with a distinct binding constant in the presence of the CDs, respectively. The effects of Flu on the structure of HSA and HTF were analyzed using synchronous fluorescence spectroscopy, which showed that the interaction of Flu with both proteins in the binary and ternary systems altered the microenvironment around the Trp and Tyr residues. The distance, r, between Flu and the proteins was obtained according to FRET which pointed at a successful formation of a drug-protein complex. Far-UV CD spectra indicated that the binding of the drug to both proteins induced changes in the secondary structure of HSA and HTF in the binary and ternary systems. Finally, molecular modeling provided possible binding sites of Flu within the proteins for the binary and ternary systems and also confirmed the experimental results. The obtained data can be useful for determining usage drug doses in drug delivery.     

Design and construction of glutamine binding proteins with a self-adhering capability to unmodified hydrophobic surfaces as reagentless fluorescence sensing devices

The chemically and genetically remodeling of proteins with ligand binding specificities can be utilized to synthesize various protein-based microsensors for detecting single biomolecules. Here, we describe the construction and characterization of fluorophore-labeled glutamine binding proteins (QBP) and derivatives coupled to the independently designed hydrophobic polypeptide (E12) that can adhere onto solid surfaces via hydrophobic interactions. The single cysteine mutant (N160C QBP) modified with the three environmentally sensitive fluorescent dyes (IAANS, acrylodan, and IANBD ester) showed increased changes in fluorescence intensity induced by glutamine binding. The use of these conjugates as reagentless fluorescence sensors enables us to determine the glutamine concentrations (0.1-50 microM) in homogeneous solution. The fusion of N160C QBP with E12, (Gly4-Ser)n spacers (GSn), and IANBD resulted in the novel fluorescence sensing elements having an adhering capability to hydrophobic surfaces of unmodified microplates. In ELISA and fluorescence experiments for the microplates treated with a series of the conjugates, IANBD-labeled N160C QBP-GS1-E12 displayed the best reproducibility in adhesion onto the hydrophobic surfaces and the precise correlation between fluorescence changes and glutamine concentrations. The performance of the biosensor-attached microplate for glutamine titrations demonstrated that the hydrophobic interaction of E12 with solid surfaces is useful for effective immobilization of proteins that need specific conformational movements in recognizing particular biomolecules. Therefore, the technique using E12 as a surface-linking domain for protein adhesion onto unmodified substrates could be applied effectively to prepare microplates/arrays for a wide variety of high-throughput assays on chemical and biological samples.     

Use of entrapment and high-performance affinity chromatography to compare the binding of drugs and site-specific probes with normal and glycated human serum albumin

Protein entrapment and high-performance affinity chromatography were used with zonal elution to examine the changes in binding that occurred for site-specific probes and various sulfonylurea drugs with normal and glycated forms of human serum albumin (HSA). Samples of this protein in a soluble form were physically entrapped within porous silica particles by using glycogen-capped hydrazide-activated silica; these supports were then placed into 1.0 cm × 2.1 mm inner diameter columns. Initial zonal elution studies were performed using (R)-warfarin and l-tryptophan as probes for Sudlow sites I and II (i.e., the major drug binding sites of HSA), giving quantitative measures of binding affinities in good agreement with literature values. It was also found for solutes with multisite binding to the same proteins, such as many sulfonylurea drugs, that this method could be used to estimate the global affinity of the solute for the entrapped protein. This entrapment and zonal approach provided retention information with precisions of ±0.1–3.3% (± one standard deviation) and elution within 0.50–3.00 min for solutes with binding affinities of 1?×?10⁴–3?×?10⁵ M^?1. Each entrapped-protein column was used for many binding studies, which decreased the cost and amount of protein needed per injection (e.g., the equivalent of only 125–145 pmol of immobilized HSA or glycated HSA per injection over 60 sample application cycles). This method can be adapted for use with other proteins and solutes and should be valuable in high-throughput screening or quantitative studies of drug–protein binding or related biointeractions.

Carbohydrate Binding and Unfolding of Spatholobus parviflorus Lectin: Fluorescence and Circular Dichroism Spectroscopic Study

Biophysical and carbohydrate binding studies have been carried out on a lectin of Spatholobus parviflorus (SPL) seeds isolated by affinity chromatography on cross-linked guar gum. It agglutinated erythrocytes of all ABO blood groups. SDS-PAGE, both in reducing and non-reducing conditions, showed two bands with MW of 29 and 31 kDa. MALDI TOF analysis revealed two peaks at 60 and 120 kDa, indicating that SPL is a hetero-dimeric tetramer. Temperature and pH stability studies revealed that SPL is a thermostable protein and its lectin activity is unaffected in the temperature range of 0–70 °C. Its activity was maximal in the pH range of 7–8. Unfolding studies with different denaturants like urea and guanidine hydrochloride indicated its globular nature and the presence of tryptophan in the highly hydrophobic area, which could be correlated to the results of fluorescence spectroscopic studies. The effect of carbohydrate binding on the lectin, shown by circular dichroism spectra, indicated the changes in its secondary and tertiary structures. SPL exerted anti-fungal activity against Aspergillus sp.     

Gel electrophoresis of native actin and the actin-deoxyribonuclease I complex

Electrophoresis of monomeric actin (G-actin) on 8-25% acrylamide Pharmacia PhastGels was carried out using gels and agarose buffer strips preequilibrated in buffer containing adenosine triphosphate (ATP), calcium ions (Ca2+) and dithiothreitol. On these gels G-actin ran as a sharp band at an apparent molecular mass of 45 kDa relative to standard proteins which is slightly greater than its actual molecular mass of 42 kDa. Electrophoresis in the absence of these solutes led to denaturation and aggregation of the protein, as reflected by a long streak. Filamentous actin (F-actin) did not enter the gel. The actin monomer-binding protein, deoxyribonuclease I, (DNase I) forms a binary complex with G-actin. The purity and apparent molecular mass 74 kDa of this complex were determined by native gel electrophoresis. By the simple procedure of preequilibrating both gel and buffer strips with appropriate ligands, this technique could be extended to investigate interactions between actin and other G-actin-binding proteins and other proteins whose stability is ligand dependent.     

Molecular encapsulation of Valganciclovir by β-cyclodextrin influences its binding to bovine serum albumin: a spectroscopic study

We report, in this paper, the stoichiometry, the binding constant and the structure of the β-cyclodextrin complex of the famous drug Valganciclovir. We investigate the influence of the complex formation of Valganciclovir with β-cyclodextrin, in the binding strength of the drug to the model carrier protein bovine serum albumin. Based on the electronic absorption, fluorescence and 2D rotating-frame Overhauser effect spectroscopy (ROESY) NMR spectral data, it follows that Valganciclovir forms a 1:1 complex with β-cyclodextrin. The β-CD molecule encapsulates the aliphatic chain of the substituted ester molecule. The association constant value of the drug-protein binding in the presence of β-cyclodextrin decreases from that in the absence of β-cyclodextrin, i.e., from that in the case of free drug, i.e. from 3.99 × 10⁴ M^?1 to 5.21 × 10³ M^?1. We compare the results of the binding of the drug to bovine serum albumin in free- and β-cyclodextrin-complex forms.     

Single-molecule nanopore sensing of actin dynamics and drug binding

Actin is a key protein in the dynamic processes within the eukaryotic cell. To date, methods exploring the molecular state of actin are limited to insights gained from structural approaches, providing a snapshot of protein folding, or methods that require chemical modifications compromising actin monomer thermostability. Nanopore sensing permits label-free investigation of native proteins and is ideally suited to study proteins such as actin that require specialised buffers and cofactors. Using nanopores, we determined the state of actin at the macromolecular level (filamentous or globular) and in its monomeric form bound to inhibitors. We revealed urea-dependent and voltage-dependent transitional states and observed the unfolding process within which sub-populations of transient actin oligomers are visible. We detected, in real-time, filament-growth, and drug-binding at the single-molecule level demonstrating the promise of nanopore sensing for in-depth understanding of protein folding landscapes and for drug discovery.

Nanopipettes were used for real-time investigation into actin dynamics and drug binding at single-molecule resolution, showing promise for a better understanding of the mechanism of protein–protein interactions and drug discovery.     

Calorimetric and spectroscopic studies on the interaction of anticancer drug mitoxantrone with human serum albumin

Binding of the anticancer drug mitoxantrone with the protein human serum albumin (HSA) has been studied by using isothermal titration calorimetry (ITC), in combination with fluorescence, UV–visible, and circular dichroism spectroscopy. The thermodynamic parameters of binding have been evaluated from ITC and spectroscopic results and compared. The ITC results demonstrate that the binding of mitoxantrone with HSA occurs according to two sets of binding sites on the protein as opposed to the fluorescence and UV–visible spectroscopic results. Blockage of one binding site on HSA for mitoxantrone in the presence of NaCl indicates strong involvement of electrostatic interactions in the binding of the drug with the protein. An insignificant temperature dependence of the association constant observed in fluorescence measurements suggests a very low enthalpy of binding which is in close agreement with the results obtained from ITC measurements. Fluorescence life time measurements suggest formation of a static complex between mitoxantrone and HSA. The discrepancies in the ITC and fluorescence results suggest that one of the binding sites on the protein for mitoxantrone does not contain tryptophan residue in its immediate vicinity. The calorimetric and spectroscopic results have provided quantitative information on the binding of mitoxantrone with HSA and suggest that the binding is dominated by electrostatic interactions.     

Multi-spectroscopic and HPLC Studies of the Interaction Between Estradiol and Cyclophosphamide With Human Serum Albumin: Binary and Ternary Systems

Drug resistance is a phenomenon that frequently impairs a proper treatment of infections and cancer with chemotherapy. Multidrug efflux transporters extrude structurally dissimilar organic compounds often providing resistance to multiple toxic chemotherapeutic agents. The quantitative analysis of drug efflux requires measuring the affinity of ligands. In this work, the interaction between cyclophosphamide (Cyc) and estradiol (ES) with human serum albumin (HSA) was studied by fluorescence polarization, circular dichroism and high-performance liquid chromatography (HPLC) under physiological conditions (pH = 7.4). Gradual addition of HSA led to a marked increase in fluorescence polarization. Our assays indicated that the protein was bound to these drugs with different K _d. Also, the Hill coefficient showed a simple drug binding process with no cooperativity. Circular dichroism results revealed the occurrence of conformational changes in HSA molecules by the binding of Cyc and ES. The protein binding of the drug was studied by HPLC. Our results indicated that the drug was bound to the protein and that the presence of a second drug affected the interaction and resistance between the first drug and the protein.     

Studies on the interaction of cinnamic acid with bovine serum albumin

The interaction between cinnamic acid and bovine serum albumin (BSA) have been studied at three temperatures, 296, 303 and 310 K. Fluorescence quenching spectra in combination with Fourier transform infrared (FT-IR) spectroscopy and circular dichroism (CD) spectroscopy was used to investigate the drug-binding mode, the binding constant and the protein structure changes in the presence of cinnamic acid in aqueous solution at pH 7.40. The fluorescence quenching constant K(q), K(sv) and the binding constant K were calculated according to Stern-Volmer equation based on the quenching of the fluorescence of BSA in the presence of cinnamic acid. The thermodynamic parameters, the enthalpy (DeltaH) and the entropy change (DeltaS) were estimated to be -16.457 kJ mol(-1) and 38.028 J mol(-1) K(-1) according to the van't Hoff equation. The displacement experiment shows that cinnamic acid can bind to the subdomain IIA (corresponding to Sudlow's drug binding site I). The distance between the tryptophan residues in BSA and cinnamic acid bound to site I was estimated to be 1.63 nm using F?ster's equation on the basis of fluorescence energy transfer. The decreased binding constant in the presence of common ions indicates that common ions have effect on drug-BSA system.