Improving stability of biosensor based on covalent immobilization of horseradish peroxidase by γ-aminobutyric acid and application in detection of H2O2.

A novel electrochemical biosensor for the determination of hydrogen peroxide (H2O2) has been fabricated through covalently immobilized horseradish peroxidase (HRP) on modified multi walled carbon nanotube (MWCNT) by γ-aminobutyric acid (GABA) on glassy carbon electrode. Using the electrochemical techniques, the electrochemical properties of the biosensor were specified. Cyclic voltammograms of the enzyme film at pH = 7.0 exhibited a pair of quasi-reversible redox peaks according to Fe(III/II) redox process of HRP. The biosensor exhibited good efficiency for finding H2O2 with a broad linear range from 2.0 × 10-7 M to 2.81 × 10-4 M and a detection limit of 0.13 µM. The surface coverage of active HRP, heterogeneous electron transfer rate constant and Michaelis-Menten constant of immobilized HRP were obtained 3.029 × 10-9 mol cm-2, 1.11 s-1, and 0.23 mM respectively. Similarly, the applicability of the suggested biosensor was examined by detecting H2O2 in human plasma samples and the average recovery was obtained as 100.50 ±â€¯0.25. Moreover, the constructed biosensor presented a high stability, reproducibility and repeatability. Finally using docking and the molecular dynamics calculations, it was determined that GABA interacts with lysine residue and it causes HRP to be placed on the electrode with a specific orientation.

Photoconversion of an anthraquinone derivative in the presence of human serum albumin.

Photconversion of an anthraquinone photochrome (AQP) from Trans to Ana forms were studied by different methods and techniques. Solution of AQP was irradiated under UV light in buffer condition, pH = 7.5, 10 mM phosphate buffer in the absence and presence of human serum albumin at 27 and 37 °C. The results showed that a new peak at higher wavelength was observed that indicative of producing the Ana form. Rate of Trans to Ana conversion increases in the presence of human serum albumin (HSA). Electron transport calculations were carried out from the first principles with a method based on non-equilibrium Green's functions (NEGF) combined with DFT. The results showed that electron transport is easier in Ana form due to increasing the resonance length and electron delocalization. Binding study by docking and spectroscopy showed that Trans form has more tendency to interact with HSA due to higher number of HSA-Trans hydrogen bond. Structural studies by circular dichroism and molecular dynamics results show that at lower concentration of AQP, percentage of helix was increased and then decreases at higher concentration. In addition structural parameters such as RMSD, accessible surface area, hydrogen bond, in associated with experimental results showed that protein folded at low concentration.

Improving of Anticancer Activity and Solubility of Cisplatin by Methylglycine and Methyl Amine Ligands Against Human Breast Adenocarcinoma Cell Line.

Methylglycine, also known sarcosine, is dramatically used in drug molecules and its metal complexes can interact to DNA and also do cleavage. Hence, to study the influence of methylglycine ligand on biological behavior of metal complexes, two water-soluble platinum (II) complexes with the formula cis-[Pt(NH3)2(CH3-gly)]NO3 and cis-[Pt(NH2-CH3)2(CH3-gly)]NO3 (where CH3-gly is methylglycine) have been synthesized and characterized by spectroscopic methods, molar conductivity measurements, and elemental analyzes. The anticancer activity of synthesized complexes was tested against human breast adenocarcinoma cell line of MCF7 using MTT assay and results showed excellent anticancer activity with Cc50 values of 126 and 292 µM after 24 h incubation time, for both complexes of cis-[Pt(NH3)2(CH3gly)]NO3 and cis-[Pt(NH2-CH3)2(CH3gly)]NO3, respectively. Also, the interaction between Pt(II) complexes with calf thymus DNA was extensively studied by means of absorption spectroscopy, fluorescence titration spectra displacement with ethidium bromide (EtBr), and circular dichroism studied in Tris-buffer. The obtained spectroscopic results revealed that two complexes can bind to highly polymerized calf thymus DNA cooperatively and denature at micromolar concentrations. The fluorescence data indicate that quenching effect for cis-[Pt(NH3)2(CH3gly)]NO3 (Ksv = 9.48 mM-1) was higher than that of cis-[Pt(NH2-CH3)2(CH3gly)]NO3 (Ksv = 1.98 mM-1). These results were also confirmed by circular dichrosim spectra. Consequently, docking data showed that cis-[Pt(NH3)2(CH3gly)]NO3 with more interaction energy binds on DNA via groove binding which is more compatible with experimental results. Graphical Abstract ᅟ Two anticancer Pt(II) complexes, cis-[Pt(NH3)2(CH3gly)]NO3 and cis-[Pt(NH2-CH3)2(CH3gly)]NO3, have been synthesized and interacted with calf thymus DNA. Improving solubility of these compounds reduce side effects and increase anticancer activity against human breast cell line. Modes of binding have been studied by electronic absorption, fluorescence, and CD measurements. Results show that both Pt(II) complexes can interact to DNA via groove binding.

Anticancer activity of new imidazole derivative of 1R,2R-diaminocyclohexane palladium and platinum complexes as DNA fluorescent probes.

The aim of this study was synthesis of two new water-soluble fluorescent palladium and platinum complexes with formulas of [Pt(DACH)(FIP)](NO3)2 and [Pd(DACH)(FIP)](NO3)2, respectively, where FIP is 2-(furan-2-yl)-1H-imidazo[4,5-f][1,10] phenanthroline and DACH is 1R,2R-diaminocyclohexane. Fluorescence spectroscopy, circular dichroism (CD), thermal denaturation measurement, ionic strength, and kinetic study displayed groove binding of Pt complex on DNA, while due to binding of Pd complex, B form of DNA convert to Z form. Due to electrostatic interaction of Pd complex with DNA, the DNA form is converted and it provides enough space for Pd complex to insert between base stacking of DNA. UV-vis study shows two complexes could denature the DNA at low concentrations in exothermic process and Pt complex is more active than Pd complex. Finally, the anticancer and growth inhibitory activities of synthesized complexes were investigated against human colon cancer cell line HCT116 after incubation time of 24 h using MTT assay and higher activity was observed for the platinum complex. Interaction of the two metal derivative complexes was studied by molecular docking and molecular dynamics simulation. The results showed that Pt complexes have higher negative docking energy and higher tendency for interaction with DNA, and exert more structural change on DNA.

Spectroscopic, electrochemical, docking and molecular dynamics studies on the interaction of three oxovanadium (IV) Schiff base complexes with bovine serum albumin and their cytotoxicity against cancer.

This study was designed to investigate the interaction of three oxovanadium (IV) Schiff base complexes with bovine serum albumin (BSA) by means of various spectroscopic and electrochemical methods along with molecular docking study and molecular dynamics simulations. Binding constants were estimated by fluorescence and UV-Vis spectroscopy. The results indicated a good affinity of the complexes for BSA in which furyl derivative had more activity. Molecular docking study showed that these complexes have the similar binding modes and located within subdomain IB in site III of BSA. The supporting of molecular docking and molecular dynamics results by experimental data, confirms the validity of the interactions data obtained by these methods. Biological activity against cancer cell showed that furyl derivative has higher activity than other complexes. Pharmaceutical analysis also showed that, these complexes potentially can be used as anticancer agents.

Study on the Interaction of the CpG Alternating DNA with CdTe Quantum Dots.

A novel sensitive method for detection of DNA methylation was developed with thioglycollic acid (TGA)-capped CdTe quantum dots (QDs) as fluorescence probes. Recognition of methylated DNA sites would be useful strategy due to the important roles of methylation in disease occurrence and developmental processes. DNA methylation occurs most often at cytosine-guanine sites (CpG dinucleotides) of gene promoters. The QDs significantly interacted with hybridized unmethylated and methylated DNA. The interaction of CpG rich methylated and unmethylated DNA hybrid with quantum dots as an optical probe has been investigated by fluorescence spectroscopy and electrophoresis assay. The fluorescence intensity of QDs was highly dependent to unmethylated and methylated DNA. Specific site of CpG islands of Adenomatous polyposis coli (APC), a well-studied tumor suppressor gene, was used as the detection target. Under optimum conditions, upon the addition of unmethylated dsDNA, the fluorescence intensity increased in linear range from 1.0 × 10- 10 to 1.0 × 10- 6M with detection limit of 6.2 × 10- 11 M and on the other hand, the intensity of QDs showed no changes with addition of methylated dsDNA. We also demonstrated that the unmethylated and methylated DNA and QDs complexes showed different mobility in electrophoresis assay. This easy and reliable method could distinguish between methylated and unmethylated DNA sequences.

Spectroscopy and computational studies on the interaction of octyl, dodecyl, and hexadecyl derivatives of anionic and cationic surfactants with adenosine deaminase.

Effects of sodium (octyl, dodecyl, hexadecyl) sulfate and their cationic analogous on the structure of adenosine deaminase (ADA) were investigated by fluorescence and circular dichroism spectroscopy as well as molecular dynamics simulation and docking calculation. Root-mean-square derivations, radius of gyration, solvent accessible surface area, and radial distribution function were obtained. The results showed that anionic and cationic surfactants reduce protein stability. Cationic surfactants have more effect on the ADA structure in comparison with anionic surfactants. More concentration and longer surfactants are parallel to higher denaturation. Furthermore, aggregation in the presence of anionic surfactants is more than cationic surfactants. Docking data showed that longer surfactants have more interaction energy and smaller ones bound to the active site.

Detection of Aeromonas hydrophila DNA oligonucleotide sequence using a biosensor design based on Ceria nanoparticles decorated reduced graphene oxide and Fast Fourier transform square wave voltammetry.

A new strategy was introduced for ssDNA immobilization on a modified glassy carbon electrode. The electrode surface was modified using polyaniline and chemically reduced graphene oxide decorated cerium oxide nanoparticles (CeO2NPs-RGO). A single-stranded DNA (ssDNA) probe was immobilized on the modified electrode surface. Fast Fourier transform square wave voltammetry (FFT-SWV) was applied as detection technique and [Ru(bpy)3](2+/3+) redox signal was used as electrochemical marker. The hybridization of ssDNA with its complementary target caused a dramatic decrease in [Ru(bpy)3](2+/3+) FFT-SW signal. The proposed electrochemical biosensor was able to detect Aeromonas hydrophila DNA oligonucleotide sequence encoding aerolysin protein. Under optimal conditions, the biosensor showed excellent selectivity toward complementary sequence in comparison with noncomplementary and two-base mismatch sequences. The dynamic linear range of this electrochemical DNA biosensor for detecting 20-mer oligonucleotide sequence of A. hydrophila was from 1 × 10(-15) to 1 × 10(-8) mol L(-1). The proposed biosensor was successfully applied for the detection of DNA extracted from A. hydrophila in fish pond water up to 0.01 µg mL(-1) with RSD of 5%. Besides, molecular docking was applied to consider the [Ru(bpy)3](2+/3+) interaction with ssDNA before and after hybridization.

Interaction of three new tetradentates Schiff bases containing N2O2 donor atoms with calf thymus DNA.

Interaction of 1,3-bis(2-hydroxy-benzylidene)-urea (H2L1), 1,3-bis(2-hydroxy-3-methoxy-benzylidene)-urea (H2L2) and 1,3-bis(2-hydroxy-3-methoxy-benzylidene)-urea nickel(II) (NiL2) with calf-thymus DNA were investigated by UV-vis absorption, fluorescence emission and circular dichroism (CD) spectroscopy as well as cyclic voltammetry, viscosity measurements, molecular docking and molecular dynamics simulation. Binding constants were determined using UV-vis absorption and fluorescence spectra. The results indicated that studied Schiff-bases bind to DNA in the intercalative mode in which the metal derivative is more effective than non metals. Their interaction trend is further determined by molecular dynamics (MD) simulation. MD results showed that Ni derivative reduces oligonucleotide intermolecular hydrogen bond and increases solvent accessible surface area more than other compounds.

Comparative study of the interaction of meso-tetrakis (N-para-trimethyl-anilium) porphyrin (TMAP) in its free base and Fe derivative form with oligo(dA.dT)15 and oligo(dG.dC)15.

Interaction between a cationic porphyrin and its ferric derivative with oligo(dA.dT)15 and oligo(dG.dC)15 was studied by UV-vis spectroscopy, resonance light scattering (RLS), and circular dichroism (CD) at different ionic strengths; molecular docking and molecular dynamics simulation were also used for completion. Followings are the observed changes in the spectral properties of meso-tetrakis (N-para-trimethyl-anilium) porphyrin (TMAP), as a free-base porphyrin with no axial ligand, and its Fe derivative (FeTMAP) upon interaction with oligo(dA.dT)15 and oligo(dG.dC)15: (1) the substantial red shift and hypochromicity at the Soret maximum in the UV-vis spectra; (2) the increased RLS intensity by increasing the ionic strength; and (3) an intense bisignate excitonic CD signal. All of them are the reasons for TMAP and FeTMAP binding to oligo(dA.dT)15 and oligo(dG.dC)15 with the outside binding mode, accompanied by the self-stacking of the ligands along the oligonucleotide helix. The CD results demonstrated a drastic change from excitonic in monomeric behavior at higher ionic strengths, which indicates the groove binding of the ligands with oligonucleotides. Molecular docking also confirmed the groove binding mode of the ligands and estimated the binding constants and energies of the interactions. Their interaction trend was further confirmed by molecular dynamics technique and structure parameters obtained from simulation. It showed that TMAP reduced the number of intermolecular hydrogen bonds and increased the solvent accessible surface area in the oligonucleotide. The self-aggregation of ligands at lower concentrations was also confirmed.

Effect of two imidazolium derivatives of ionic liquids on the structure and activity of adenosine deaminase.

The effect of two ionic liquids, 1-allyl 3-methyl-imidazolium (IL1) and 1-octhyl 3-methyl-imidozolium chlorides (IL2), on the structure and activity of adenosine deaminase (ADA) were described by UV-vis and fluorescence spectrophotometry in phosphate buffer and results were compared with docking and molecular dynamics (MD) simulation studies. All results showed that inhibition of activity and reduction of enzyme tertiary structure are more for octhyl than allyl derivative due to the more hydrophobic property of it. Finally structure parameters obtained from MD simulation showed that ionic liquid reduces intermolecular hydrogen bond and unfold enzyme structure. Calculation results are in good agreement with spectrophotometric studies.

Tertiary Structure Prediction of a-Glucosidase and Inhibition Properties of N-(Phenoxydecyl) Phthalimide Derivatives.

Due to increasing of population of diabetic patients, identifying factors for disease control has received much attention. α-glucosidase (EC 3.2.1.20) is an essential enzyme that helps to digestion of carbohydrates such as starch and sugar. Carbohydrates are normally converted into simple sugars, which can be absorbed through the intestine. Therefore, α-glucosidase inhibitors can be used to decrease the blood sugar level. We have studied the effect of inhibition of N-(phenoxydecyl) phthalimide derivatives by a computer drug-design protocol involving homology modeling, docking simulation and Quantitative Structure Activity Relationship. The homology modeling of α-glucosidase showed a structure very similar to the crystal structure of oligo-1,6-glucosidase from Saccharomyces cerevisiae. Docking results showed the position of inhibitors binding site is close to active site and the carboxyl oxygen in phthalimide is an effective functional group for binding inhibitors to protein. The equation obtained by QSAR showed that, logIC50 decreases and so inhibition property increases when the size, polarity, geometry and number of halogen factors increase.

Biological evaluation and interaction of a newly designed anti-cancer Pd(II) complex and human serum albumin.

The pharmacokinetics and pharmacodynamics of any drug will depend, largely, on the interaction that has with human serum albumin (HSA), the most abundant plasma protein. The interaction between newly synthesized Pd(II) complexe, 2,2'-bipyridin Butylglycinato Pd(II) nitrate, an anti-tumor component, with HSA was studied at different temperatures by fluorescence, far UV circular dichroism (CD), UV-visible spectrophotometry and theoretical approaches. The Pd(II) complex has a strong ability to quench the intrinsic fluorescence of HSA through a dynamic quenching procedure. The binding parameters and thermodynamic parameters, including δH°, δS° and δG° were calculated by fluorescence quenching method, indicated that hydrophobic forces play a major role in the interaction of Pd(II) complex with HSA. Based on Autodock, FRET (fluorescence resonance energy transfer) and fluorescence quenching data, it may be concluded that one of the binding sites in the complex of HSA is near the only one Trp of HSA (Trp214) in sub domain IIA of the protein. Far-UV-CD results indicated that Pd(II)-complex induced increase in the α-helical content of the protein. The anti-tumor property of the synthesized Pd(II) complex was studied by testing it on human tumor cell line K562. The 50% cytotoxic concentration (Cc50) of complex was determined using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. Also, fluorescence staining with DAPI (4,6-diamidino-2-phenylindole) revealed some typical nuclear changes that are characteristic of apoptosis which is induced at Cc50 concentration of Pd(II) complex in K562 cell line after 24 h incubation. Our results suggest that Pd(II) complex is a promising anti-proliferative agent and should execute its biological effects by inducing apoptosis.

On violations of Le Chatelier's principle for a temperature change in small systems observed for short times.

Le Chatelier's principle states that when a system is disturbed, it will shift its equilibrium to counteract the disturbance. However for a chemical reaction in a small, confined system, the probability of observing it proceed in the opposite direction to that predicted by Le Chatelier's principle, can be significant. This work gives a molecular level proof of Le Chatelier's principle for the case of a temperature change. Moreover, a new, exact mathematical expression is derived that is valid for arbitrary system sizes and gives the relative probability that a single experiment will proceed in the endothermic or exothermic direction, in terms of a microscopic phase function. We show that the average of the time integral of this function is the maximum possible value of the purely irreversible entropy production for the thermal relaxation process. Our result is tested against computer simulations of the unfolding of a polypeptide. We prove that any equilibrium reaction mixture on average responds to a temperature increase by shifting its point of equilibrium in the endothermic direction.

Effects of surfactant, salt and solvent on the structure and activity of adenosine deaminase: molecular dynamic and spectrophotometric studies.

Effects of sodium dodecyl sulfate, dodecyltrimethylammonium bromide, sodium chloride, sodium sulfate, methanol and ethanol, on the structure and activity of adenosine deaminase (ADA) were investigated by UV-Vis, circular dichroism spectrophotometry and molecular dynamics (MDs) studies. Relative activity, experimental and computational helix content, total accessible surface area (ASA) and exposed charged surface area (ECSA) were obtained. The relative activity of ADA in the absence and the presence of denaturants were compared with structural results. It was shown that an increase in the surface area and a decrease in the amount of helicity are associated with a decrease in the activity of ADA.

Kinetic, thermodynamic and statistical studies on the inhibition of adenosine deaminase by aspirin and diclofenac.

The kinetic and thermodynamic effects of aspirin and diclofenac on the activity of adenosine deaminase (ADA) were studied in 50 mM phosphate buffer pH = 7.5 at 27 and 37 degrees C, using UV-Vis spectrophotometry and isothermal titration calorimetry (ITC). Aspirin exhibits competitive inhibition at 27 and 37 degrees C and the inhibition constants are 42.8 and 96.8 microM respectively, using spectrophotometry. Diclofenac shows competitive behavior at 27 degrees C and uncompetitive at 37 degrees C with inhibition constants of 56.4 and 30.0 microM, at respectively. The binding constant and enthalpy of binding, at 27 degrees C are 45 microM, - 64.5 kJ/mol and 61 microM, - 34.5 kJ/mol for aspirin and diclofenac. Thermodynamic data revealed that the binding process for these ADA inhibitors is enthalpy driven. QSAR studies by principal component analysis implemented in SPSS show that the large, polar, planar, and aromatic nucleoside and small, aromatic and polar non-nucleoside molecules have lower inhibition constants.

Kinetics of denaturation of human and chicken hemoglobins in the presence of co-solvents.

The stability of four hemoglobins (Hb) in dimer forms (low concentration) were investigated by the kinetics of denaturation. The rate constants of denaturation were obtained by variation of 280 nm absorption versus time in 10 mM Tris-HCl, 10 mM EDTA, pH 8.0 at 45 degrees C in the absence and presence of 0.5 M ethanol, dimethyl sulfoxide (DMSO), formamide, and glycerol. The results show the trend of rate constants in different co-solvents in the following order: chicken hemolysate < human hemolysate and chicken Hb D < chicken Hb A. The buried surface area was calculated for Hb samples in the absence of co-solvents. Accordingly, the trend points out that: chicken Hb D > chicken Hb A > human Hb A. These results suggest that both chicken hemolysate and chicken Hb D are relatively more stable than human and chicken Hb A, respectively. However, the denaturation rate constants of Hb in different co-solvents have designated the following order: ethanol > DMSO > formamide > glycerol. As a matter of fact, this phenomenon is an indication of an increase in the denaturation capacity (DC) and hydrophobicity, and a decrease in the surface tension of the solution in the preceding co-solvents.

Comparative structural and functional studies of avian and mammalian hemoglobins.

Thermal stabilities of chicken, grey lag goose (Anser anser), turkey as avian hemoglobins (Hbs); and human, bovine, sheep and horse as mammalian Hbs in hemolysate form were investigated and compared with oxygen affinities taken from literature. The thermal stability was obtained from thermal profiles using temperature scanning spectrophotometry. The buffer conditions were 50 mM Tris, pH 7.2, and 1 mM EDTA. The average of the inverse temperature transitions, average hydrophobicity, total van der Waals volume, partial molal volume and hydration potential were calculated by computational methods. The hemolysed avian Hbs have a lower oxygen affinity, higher thermal stability and higher self association than the mammalian Hbs. These observations are based on amino-acid composition, influence of ionic effectors, and the presence of Hb D in several avian Hbs. The results indicate that the avian Hbs have a more tense (T) conformation than the mammalian Hbs.