Influence of millimeter range electromagnetic waves on bovine serum albumin interaction with acridine orange.

The effect of non-ionizing millimeter range electromagnetic waves (MM EMW) (30-300 GHz) on the bovine serum albumin (BSA) interaction peculiarities with acridine orange (AO) has been studied in vitro. The frequencies 41.8 and 50.3 GHz were chosen, since the first one is nonresonant frequency for the water, while the second one is resonant for water. The binding constant and number of binding sites were calculated at both irradiation presence and absence. AO was revealed to bind to BSA, while after the protein irradiation the interaction force strengthens. However, it was also shown that there are differences of the interaction parameters while irradiating by 41.8 or 50.3 GHz. AO binds to BSA, irradiated by MM EMW with the frequency 41.8 GHz much more weaker, than to that, irradiated by MM EMW with the frequency 50.3 GHz.

The manuscript is devoted to the study of the effect of millimeter range electromagnetic waves with the frequencies 41.8 and 50.3 GHz on the model biological system, being on molecular level of organization. Nowadays millimeter range electromagnetic waves compose a significant part of electromagnetic pollution in the environment and affect biological material, besides these waves are used in extremely high frequencies-therapy ((30­300 GHz), which are millimeter range electromagnetic waves (1­10 mm)). On the other hand, the problem of their effect mechanism is mainly connected to water, since the resonant frequencies for water molecules are in the interval of millimeter waves. In the present study, as such biological molecule, the bovine serum albumin has been chosen, which interacts with acridine orange. Serum albumins are known to carry and transport various endogenous and exogenous agents, including drugs throughout circulatory system. In turn, acridine orange has been extensively used for biological staining to differentiate DNA from RNA by fluorescence emission for years, Nowadays, it is considered as a promising agent for antitumorous treatment and diagnosis.The data obtained show that the interaction between bovine serum albumin and acridine orange changes, when the solution of albumin is irradiated by the millimeter waves with the frequencies 41.8 and 50.3 GHz. However, the interaction alteration depends on the frequency as well. Thus, the irradiation with the frequency 41.8 GHz makes insignificant changes, while that with the frequency 50.3 GHz induces significant changes of measured parameters. The studies were conducted by absorption, fluorescence and CD spectroscopy methods.

Binding of flavonoids to yeast aldehyde dehydrogenase: a molecular mechanism and computational approach.

The interaction of three flavonoids, apigenin, fisetin and quercetin with yeast aldehyde dehydrogenase, ALDH was studied by spectroscopic and molecular docking methods. A combination of both static and dynamic processes interaction mechanism for the binding of flavonoids with ALDH was found. The interaction takes place with moderate binding and the interaction was driven by hydrophobic contacts. The microenvironments of the fluorescent amino acids changed upon flavonoids binding. The distances between ALDH and flavonoids determined by Förster Resonant Energy Transfer (FRET) confirmed the results obtained by fluorescence. The structure of ALDH against thermal denaturation was stabilized by apigenin and destabilized by fisetin and quercetin. Molecular docking simulation showed that all flavonoids bind to the same site of ALDH and confirmed the moderate binding straight found in fluorescence.Communicated by Ramaswamy H. Sarma.

Interaction of MnTOEtPyP4 porphyrin with DNA.

The binding of water-soluble meso-tetra-(4N-oxyethylpyridyl) porphyrin (H2TOEtPyP4) and its manganese (III) derivative (MnTOEtPyP4) with calf thymus DNA have been quantitatively studied using UV/Vis spectrophotometry, Circular Dichroism (CD), thermal melting curves and viscometry. The results show, that porphyrins interact with DNA via one binding mode at low relative concentrations (r) and two binding modes at high values of r. The binding constant (Kb) and stoichiometry (n) were determined from binding isotherms for both porphyrin-DNA complexes. The thermal melting analysis indicates that the double-helical structure of DNA molecules is stabilizing in presence of studied porphyrins. At certain concentrations of porphyrin, two-stage melting curves were observed, which indicates the existence of two different binding modes. Obtained results show that MnTOEtPyP4 associates with DNA duplex via outside binding mode.Communicated by Ramaswamy H. Sarma.

Interaction of doxorubicin delivered by superparamagnetic iron oxide nanoparticles with DNA.

We studied the interaction of superparamagnetic iron oxide nanoparticles (SPIONs), covered by trisodium citrate, with doxorubicin (DOX) and DNA using the spectrophotometric method. We calculated the binding parameters in the binary (DOX-SPION and SPION-DNA) and the ternary (DOX-SPION-DNA) systems. Our studies showed that the nanoparticles do not interact with DNA. We also observed that one nanoparticle loads rather a large number of DOX molecules with a quite high binding constant value (kDOX-SPION = 1.2 × 104 M-1). The DNA addition to the DOX-SPION system induces DOX release from the SPION surface and the formation of DOX-DNA complexes. The presence of nanoparticles has almost no effect on the constant of doxorubicin binding to DNA (kDOX-DNA ≈ 3 × 104 M-1). At high DNA concentrations, almost all DOX molecules bind to DNA. Accordingly, the use of SPIONs as DOX carriers does not require an increased drug dose to achieve a therapeutic effect. Thus, SPIONs are perspective nanocarriers for DOX delivery.

Determination of protein-protein interactions at the single-molecule level using optical tweezers.

Biomolecular interactions are at the base of all physical processes within living organisms; the study of these interactions has led to the development of a plethora of different methods. Among these, single-molecule (in singulo) experiments have become relevant in recent years because these studies can give insight into mechanisms and interactions that are hidden for ensemble-based (in multiplo) methods. The focus of this review is on optical tweezer (OT) experiments, which can be used to apply and measure mechanical forces in molecular systems. OTs are based on optical trapping, where a laser is used to exert a force on a dielectric bead; and optically trap the bead at a controllable position in all three dimensions. Different experimental approaches have been developed to study protein­protein interactions using OTs, such as: (1) refolding and unfolding in trans interaction where one protein is tethered between the beads and the other protein is in the solution; (2) constant force in cis interaction where each protein is bound to a bead, and the tension is suddenly increased. The interaction may break after some time, giving information about the lifetime of the binding at that tension. And (3) force ramp in cis interaction where each protein is attached to a bead and a ramp force is applied until the interaction breaks. With these experiments, parameters such as kinetic constants (koff, kon), affinity values (KD), energy to the transition state ΔG≠, distance to the transition state Δx≠ can be obtained. These parameters characterize the energy landscape of the interaction. Some parameters such as distance to the transition state can only be obtained from force spectroscopy experiments such as those described here.

Microscale thermophoresis as a powerful growing analytical technique for the investigation of biomolecular interaction and the determination of binding parameters.

Thein vitropanel of technologies to address biomolecular interactions are in play, however microscale thermophoresis is continuously increasing in use to represent a key player in this arena. This review highlights the usefulness of microscale thermophoresis in the determination of molecular and biomolecular affinity interactions. This work reviews the literature from January 2016 to January 2022 about microscale thermophoresis. It gives a summarized overview about both the state-of the art and the development in the field of microscale thermophoresis. The principle of microscale thermophoresis is also described supported with self-created illustrations. Moreover, some recent advances are mentioned that showing application of the technique in investigating biomolecular interactions in different fields. Finally, advantages as well as drawbacks of the technique in comparison with other competing techniques are summarized.

Comparison of mobility shift affinity capillary electrophoresis and capillary electrophoresis frontal analysis for binding constant determination between human serum albumin and small drugs.

In this study, two capillary electrophoresis-based ligand binding assays, namely, mobility shift affinity capillary electrophoresis (ms-ACE) and capillary electrophoresis-frontal analysis (CE-FA), were applied to determine binding parameters of human serum albumin toward small drugs under similar experimental conditions. The substances S-amlodipine (S-AML), lidocaine (LDC), l-tryptophan (l-TRP), carbamazepine (CBZ), ibuprofen (IBU), and R-verapamil (R-VPM) were used as the main binding partners. The scope of this comparative study was to estimate and compare both the assays in terms of their primary measure's precision and the reproducibility of the derived binding parameters. The effective mobility could be measured with pooled CV values between 0.55% and 7.6%. The precision of the r values was found in the range between 1.5% and 10%. Both assays were not universally applicable. The CE-FA assay could successfully be applied to measure the drugs IBU, CBZ, and LDC, and the interaction toward CBZ, S-AML, l-TRP, and R-VPM could be determined using ms-ACE. The average variabilities of the estimated binding constants were 64% and 67% for CE-FA and ms-ACE, respectively.

Analytical methods for obtaining binding parameters of drug-protein interactions: A review.

The study of drug-protein interactions can reveal the corresponding binding mechanisms, providing valuable information for the early phase drug development and development of new drugs. This article reviews the methods used for obtaining the binding parameters of drug-protein systems. The methods include equilibrium dialysis, high-performance affinity chromatography, capillary electrophoresis, spectroscopy, calorimetry, competition and displacement, mass spectrometry, fluorescence resonance energy transfer, and thermal stability shift analysis. Relevant parameters include the association constant, number of binding sites, thermodynamic properties, binding force types, binding site types, binding distances, changes in protein conformation, and changes in protein stability. In addition, the review also summarizes the principles, advantages, and limitations of each method in detail. The comparison of parameter information can not only guide method selection but also provide valuable reference information for in-depth exploration of drug-protein interaction mechanisms.

Dengue fusion peptide in Langmuir monolayers: A binding parameter study.

Membrane fusion is known to be the primary mechanism of entry of flaviviruses into host cells. Several studies reported the investigation of the membrane fusion mechanism mediated by the fusion peptide, a component of the membrane protein surrounding the flaviviruses. In this study, we investigated the interaction of Dengue fusion peptide (FLAg) with Langmuir monolayers to uncover the role of membrane charges and organization in its membrane binding. Binding parameters of FLAg were obtained by measuring its adsorption onto Langmuir monolayers of different types of individual lipids, as well as their mixtures. Specific peptide binding was observed in the presence of charged lipid monolayers at different pHs, revealing that the lipid composition of the membrane modulates peptide interaction, and the preference of the peptide for negatively charged lipids.

Studies to explore the UVA photosensitizing action of 9-phenylacridine in cells by interaction with DNA.

Acridine and its derivatives are well known for their DNA binding properties. In this report, we present our findings on evaluating different binding parameters of the interaction of 9-phenylacridine (ACPH) with DNA. Absorption spectroscopic studies including standard and reverse titration, the effects of ionic strength and temperature on titration, and Job plot analysis were done to calculate the binding constant and determine the different thermodynamic parameters and stoichiometry of the binding. Spectrofluorimetry and circular dichroism (CD) spectral titration were also utilized to confirm these findings. The results indicated that ACPH binds to DNA reversibly through non-electrostatic interactions by hydrogen bonding and van der Waals interactions. The binding constant and the number of binding sites were of the order 103 M-1 and ≈2, respectively with a binding stoichiometry of 1:4. The binding of ACPH with DNA was spontaneous, exothermic and enthalpy-driven. The extent of uptake of ACPH in B16 melanoma cells was estimated. As this compound absorbs in the UVA region, the effect of treatment with ACPH prior to UVA exposure was assessed to evaluate its phototoxicity in these cells. Our results indicated that the binding to DNA enhanced damage to sensitize cells to killing through apoptosis. Our findings indicated its potential to act as a photosensitizer.

pH-dependent complex formation of Zn-meso-tetra(4-N-hydroxyethylpyridyl) porphyrin with cancer DNA.

The complex formation between the synthetic water-soluble Zn-meso-tetra(4-N-hydroxyethylpyridyl) porphyrin (ZnTOEPyP4) and cancer DNA in comparison to healthy DNA was investigated using the UV/VIS spectrophotometry method in phosphate-buffered saline at different pHs. The increasing of DNA/porphyrin ratio leads to hypochromicity and red shift in the Soret band, which indicate the complexation of the ZnTOEPyP4 with DNA. The results show that the binding constant (Kb) and the exclusion parameter (n) of ZnTOEPyP4 with DNA strongly depend upon the pH. The Kbof ZnTOEPyP4 with cancer DNA is higher than with normal DNA.Communicated by Ramaswamy H. Sarma.

New aryl Schiff bases of thiadiazole derivative of ibuprofen as DNA binders and potential anticancer drug candidates.

The work presented in this paper describes the synthesis of two new aryl Schiff bases [(E)-N-(4-(benzyloxy)-3-methoxybenzylidene)-5-(1-(4-isobutylphenyl)ethyl)-1,3,4-thiadiazol-2-amine] (ASB-1) and [(E)-N-(4-(benzyloxy)benzylidene)-5-(1-(4-isobutylphenyl)ethyl)-1,3,4-thiadiazol-2-amine] (ASB-2). These compounds were characterized by different analytical techniques and then studied for DNA binding. Binding studies were carried out at neutral pH (7.0) and at 37 °C by theoretical and experimental methods including DFT, molecular docking, spectroscopy (UV-visible, fluorescence), cyclic voltammetry (CV) and viscometry. Further investigations of these compounds were done on hepatocellular carcinoma; Huh-7 cancer cell line. Binding constant, free energy change and binding site size, i.e. Kb, ΔG and n were evaluated which indicated that both ASB-1 and ASB-2 bind significantly and spontaneously with the DNA. However, data revealed relatively greater binding of ASB-1 with DNA. Spectral and voltammetric results were found supportive of each other. Binding site sizes and viscosity measurements verified the mixed binding mode of interactions as observed in molecular docking analysis, i.e. intercalation with groove binding. DNA binding studies were very well correlated with the in-vitro studies performed on Huh-7 cell line as well as normal HEK-293 cell lines. The compound ASB-1 not only showed greater binding affinity toward DNA but also showed greater anticancer potency with least IC50 value as compared to ASB-2.

Studies of eosin Y - DNA interaction using a competitive binding assay.

The interaction between xanthene dye eosin Y and double stranded DNA has been studied by spectrophotometry. The conventional titration study does not show the interaction in the eosin Y - DNA system. Therefore, the competitive binding assay was carried out. The DNA-targeted ligands proflavine and methylene blue were used as competitors. Multivariate curve resolution - alternative least squares method (MCR-ALS) was applied to analyze the spectrophotometric titration data. The experimental binding isotherms were fitted by Scatchard and McGee equations. The binding constant of eosin Y with DNA was found to be 1.7·104 M-1. It is shown that the competitive binding assay requires consideration of heteroassociation for the correct determination of ligand-DNA binding parameters.

Biophysical Characterization of Aptamer-Target Interactions.

Aptamers are single-stranded nucleic acid molecules forming well-defined 3D structures. Aptamers typically bind to their ligands with high affinity and specificity. They are capable of interacting with various kinds of ligands: ions, small molecules, peptides, proteins, viruses, bacteria, and even cells. Therefore, aptamers are in widespread use as sensor molecules or as targeting agents in diagnostics and pharmaceutics. As a prerequisite for their use in these economic high-value areas, aptamers must be studied in detail with respect to different biophysical characteristics. Of central importance are basic binding parameters of the aptamer-target interaction, such as binding affinity and kinetics. Numerous biophysical methods with different features, characteristics, and capabilities are used in the field today for this purpose.This chapter provides an overview of the current state-of-the-art technologies for studying interactions between aptamers and targets and discusses their advantages as well as drawbacks. Furthermore, essential aspects influencing any aptamer characterization strategy will be presented. Finally, issues of comparability of binding data between different aptamer characterization technologies will be discussed. Graphical Abstract.

Binding analysis between monomeric ß-casein and hydrophobic bioactive compounds investigated by surface plasmon resonance and fluorescence spectroscopy.

ß-Casein, a phosphoprotein representing 37% of the bovine milk caseins, has specific features promoting its application as a nanocarrier for hydrophobic bioactives. In this study, the interactions of ß-casein with curcumin and vitamin D3 under the same physico-chemical conditions were investigated. The interaction kinetics have been studied by surface plasmon resonance (SPR) and fluorescence spectroscopy. The KD value for curcumin-ß-casein interaction has been successfully evaluated (4.1 ±â€¯0.7 × 10-4 M) using SPR by fitting data to a 1:1 Langmuir interaction model. Conversely, the SPR responses obtained for vitamin D3 show that the interactions between this hydrophobic compound and the ß-casein immobilized on the sensor chip were below the sensitivity of the SPR apparatus. Moreover, the fluorescence quenching data show that curcumin has higher affinity to ß-casein (KA = 23.5 ±â€¯1.9 × 104 M-1) than vitamin D3 (KA = 5.8 ±â€¯1.1 × 104 M-1).

Structural Features of Amyloid Fibrils Formed from the Full-Length and Truncated Forms of Beta-2-Microglobulin Probed by Fluorescent Dye Thioflavin T.

The persistence of high concentrations of beta-2-microglobulin (ß2M) in the blood of patients with acute renal failure leads to the development of the dialysis-related amyloidosis. This disease manifests in the deposition of amyloid fibrils formed from the various forms of ß2M in the tissues and biological fluids of patients. In this paper, the amyloid fibrils formed from the full-length ß2M (ß2m) and its variants that lack the 6 and 10 N-terminal amino acids of the protein polypeptide chain (ΔN6ß2m and ΔN10ß2m, respectively) were probed by using the fluorescent dye thioflavin T (ThT). For this aim, the tested solutions were prepared via the equilibrium microdialysis approach. Spectroscopic analysis of the obtained samples allowed us to detect one binding mode (type) of ThT interaction with all the studied variants of ß2M amyloid fibrils with affinity ~104 M-1. This interaction can be explained by the dye molecules incorporation into the grooves that were formed by the amino acids side chains of amyloid protofibrils along the long axis of the fibrils. The decrease in the affinity and stoichiometry of the dye interaction with ß2M fibrils, as well as in the fluorescence quantum yield and lifetime of the bound dye upon the shortening of the protein amino acid sequence were shown. The observed differences in the ThT-ß2M fibrils binding parameters and characteristics of the bound dye allowed to prove not only the difference of the ΔN10ß2m fibrils from other ß2M fibrils (that can be detected visually, for example, by transmission electron microscopy (TEM), but also the differences between ß2m and ΔN6ß2m fibrils (that can not be unequivocally confirmed by other approaches). These results prove an essential role of N-terminal amino acids of the protein in the formation of the ß2M amyloid fibrils. Information about amyloidogenic protein sequences can be claimed in the development of ways to inhibit ß2M fibrillogenesis for the treatment of dialysis-related amyloidosis.

Investigation of α-Synuclein Amyloid Fibrils Using the Fluorescent Probe Thioflavin T.

In this work, α-synuclein amyloid fibrils-the formation of which is a biomarker of Parkinson's disease-were investigated using the fluorescent probe thioflavin T (ThT). The experimental conditions of protein fibrillogenesis were chosen so that a sufficient number of continuous measurements could be performed to characterize and analyze all stages of this process. The reproducibility of fibrillogenesis and the structure of the obtained aggregates (which is a critical point for further investigation) were proven using a wide range of physical-chemical methods. For the determination of ThT-α-synuclein amyloid fibril binding parameters, the sample and reference solutions were prepared using equilibrium microdialysis. By utilizing absorption spectroscopy of these solutions, the ThT-fibrils binding mode with a binding constant of about 104 M-1 and stoichiometry of ThT per protein molecule of about 1:8 was observed. Fluorescence spectroscopy of the same solutions with the subsequent correction of the recorded fluorescence intensity on the primary inner filter effect allowed us to determine another mode of ThT binding to fibrils, with a binding constant of about 106 M-1 and stoichiometry of about 1:2500. Analysis of the photophysical characteristics of the dye molecules bound to the sites of different binding modes allowed us to assume the possible localization of these sites. The obtained differences in the ThT binding parameters to the amyloid fibrils formed from α-synuclein and other amyloidogenic proteins, as well as in the photophysical characteristics of the bound dye, confirmed the hypothesis of amyloid fibril polymorphism.

Effect of N- and C-Terminal Amino Acids on the Interfacial Binding Properties of Phospholipase D from Vibrio parahaemolyticus.

The effects of N-terminal (1⁻34 amino acids) and C-terminal (434⁻487 amino acids) amino acid sequences on the interfacial binding properties of Phospholipase D from Vibrio parahaemolyticus (VpPLD) were characterized by using monomolecular film technology. Online tools allowed the prediction of the secondary structure of the target N- and C-terminal VpPLD sequences. Various truncated forms of VpPLD with different N- or C-terminal deletions were designed, based on their secondary structure, and their membrane binding properties were examined. The analysis of the maximum insertion pressure (MIP) and synergy factor "a" indicated that the loop structure (1⁻25 amino acids) in the N-terminal segment of VpPLD had a positive effect on the binding of VpPLD to phospholipid monolayers, especially to 1,2-dimyristoyl-sn-glycero-3-phosphoserine and 1,2-dimyristoyl-sn-glycero-3-phosphocholine. The deletion affecting the N-terminus loop structure caused a significant decrease of the MIP and synergy factor a of the protein for these phospholipid monolayers. Conversely, the deletion of the helix structure (26⁻34 amino acids) basically had no influence on the binding of VpPLD to phospholipid monolayers. The deletion of the C-terminal amino acids 434⁻487 did not significantly change the binding selectivity of VpPLD for the various phospholipid monolayer tested here. However, a significant increase of the MIP value for all the phospholipid monolayers strongly indicated that the three-strand segment (434⁻469 amino acids) had a great negative effect on the interfacial binding to these phospholipid monolayers. The deletion of this peptide caused a significantly greater insertion of the protein into the phospholipid monolayers examined. The present study provides detailed information on the effect of the N- and C-terminal segments of VpPLD on the interfacial binding properties of the enzyme and improves our understanding of the interactions between this enzyme and cell membranes.

Frontal analysis capillary electrophoresis: recent advances and future perspectives.

The developments on frontal analysis capillary electrophoresis (FACE) from 2012 to 2017 to study interactions of simple and complex systems are reviewed. Most research papers focused on therapeutic drug-related studies; however, other studies include chemical sensing, drug delivery, inhibitor screening and capillary coating. New ligand-substrate systems such as template-molecularly imprinted polymer systems were reported. Comparison of FACE with other analytical techniques used to investigate binding interaction, and the determination of binding parameters using different isotherm models are also covered. In 2017, eight research papers were reported including new detection by ESI-MS. Future research direction of FACE may include high sensitivity detection and throughput screening of drugs, natural products and biomarkers for clinical diagnosis.

Mechanical properties of BiP protein determined by nano-rheology.

Immunoglobulin Binding Protein (BiP) is a chaperone and molecular motor belonging to the Hsp70 family, involved in the regulation of important biological processes such as synthesis, folding and translocation of proteins in the Endoplasmic Reticulum. BiP has two highly conserved domains: the N-terminal Nucleotide-Binding Domain (NBD), and the C-terminal Substrate-Binding Domain (SBD), connected by a hydrophobic linker. ATP binds and it is hydrolyzed to ADP in the NBD, and BiP's extended polypeptide substrates bind in the SBD. Like many molecular motors, BiP function depends on both structural and catalytic properties that may contribute to its performance. One novel approach to study the mechanical properties of BiP considers exploring the changes in the viscoelastic behavior upon ligand binding, using a technique called nano-rheology. This technique is essentially a traditional rheology experiment, in which an oscillatory force is directly applied to the protein under study, and the resulting average deformation is measured. Our results show that the folded state of the protein behaves like a viscoelastic material, getting softer when it binds nucleotides- ATP, ADP, and AMP-PNP-, but stiffer when binding HTFPAVL peptide substrate. Also, we observed that peptide binding dramatically increases the affinity for ADP, decreasing it dissociation constant (KD ) around 1000 times, demonstrating allosteric coupling between SBD and NBD domains.