Interaction of glucose oxidase with alkyl-substituted sepharose 4B

Glucose oxidase (GOD) is often used in immobilized forms for determination of glucose. To examine the possibility of its adsorption by hydrophobic interactions, palmityl-substituted Sepharose 4B (Sepharoselipid) was employed as an adsorptive matrix. Various conditions were used in tests to improve the limited immobilization of the enzyme observed under normal (native) conditions, including use of high concentrations of denaturing agents. Of the denaturants used, only the cationic detergent dodecyl trimethyl ammonium bromide was effective in denaturing the protein and exposing its hydrophobic sites for interaction with alkyl residues on the support. This, followed by the process of renaturation, provided catalytically active immobilized preparations. The apoenzyme, prepared by treatment of the holoenzyme with acidified (NH4)2SO4 or thermal denaturation, was totally immobilized on the support. Furthermore, it was shown that either flavin adenine dinucleotide (FAD) or the alkyl residues, not both, may interact with the nucleotide site at any given time. Results are discussed in terms of high rigidity of GOD molecule and limited exposure of hydrophobic sites in its native structure. The observations are in accord with suggestions in the literature that the FAD pocket is a very narrow channel of hydrophobic properties, adapted to accept its natural coenzyme.     

Microcalorimetry, energetics and binding studies of DNA-dimethyltin dichloride complexes

The interaction of dimethyltin dichloride (Me₂SnCl₂) with calf thymus DNA was studied at 27 °C, pH 7.6 using various techniques including isothermal titration calorimetry (ITC) and UV-Vis, fluorescence and IR spectrophotometries. The binding isotherm and enthalpy curve for Me₂SnCl₂-DNA interaction was a biphasic transition process. This was determined by the analysis of the binding data with the Hill equation. The first phase of the enthalpy curve (exothermic process) was consistent with the first set of binding site, the second phase (endothermic process, less exothermicity) was consistent with second set of binding site from the cited interactions. Our results showed that the first set of binding sites is occupied by one mole of ligand bound per near 1 base pair of DNA. The DNA-ethidium bromide (EB) complex, in the presence of Me₂SnCl₂, caused the quenching of the fluorescence emission. The Scatchard plots illustrated a non-intercalating manner for such quenching. The DNA-EB complex results indicated that the binding of Me₂SnCl₂ is with the phosphate groups of DNA at low ligand concentrations (<9 mM). This was confirmed with the IR spectrophotometric spectra. However, the binding at higher ligand concentrations (>9 mM) was with the base groups of DNA. Therefore, these results suggest that the Me₂SnCl₂ binding to DNA at low concentrations occurs through an outside interaction by an exothermic process. However, the partial unfolding of the DNA caused at higher concentrations of Me₂SnCl₂ is through an endothermic process involving interactions with the base groups.     

Stabilizing and suicide-peroxide protecting effect of Ni2+ on horseradish peroxidase

The present research discusses the structure stabilizing and protecting effects of Ni²⁺ against suicide-peroxide inactivation of horseradish peroxidase (HRP). Suicide inactivation of HRP by hydrogen peroxide (3 mM) was monitored by measuring change in the absorbance of the colored product (tetraguaiacol) of the catalytic reaction cycle at 470 nm. Progress curves of the catalytic reaction cycle were obtained at 27 °C, phosphate buffer (5 mM), pH 7.0. The corresponding kinetic parameters (e.g., initial enzyme activity (α_o) and the apparent rate constant (k_i) of suicide inactivation of HRP by peroxide) were evaluated using a kinetic equation derived in this study. Comparative activatory and inhibitory effects of Ni²⁺ on the kinetics of suicide-peroxide inactivation of HRP are discussed.     

Activity and structural changes of mushroom tyrosinase induced by n-alkyl sulfates

Catecholase activity and structural changes of mushroom tyrosinase (MT) were studied in the presence of some n-alkyl sulfate derivatives. Experiments showed that MT reached its optimal activity in the presence of 1.5, 0.6, and 0.2 mM of sodium n-octyl sulfate (SOS), sodium n-dodecyl sulfate (SDS) and sodium n-tetradecyl sulfate (STS), respectively. Native and incubated MT with the n-alkyl sulfates were also investigated from structural point of view by far-UV circular dichroism (CD) and intrinsic fluorescence spectroscopy. At the above mentioned concentrations of SOS, SDS, and STS no change in the secondary structure of MT was observed. However, changes in the tertiary structure of the enzyme due to the presence of n-alkyl sulfates were obvious. Results of this research indicate that n-alkyl sulfate with longer chain induces greater conformational changes in MT, hence, can activate it at lower concentrations.     

Unfolding mechanism of PHD2 as a vital protein: all-atom simulation approach

Prolyl hydroxylase domain 2 containing protein (PHD2) is a central protein in regulation of cellular response to hypoxia. This protein controls the responses of cell to oxygen level via the regulation of hypoxia inducible factor (HIF) stability. HIF induces the expression of many genes, especially ones orchestrate angiogenesis. There are some reports that mentioned in some tumor types the level of HIF is high in spite of the presence of wild-type PHD2 and normoxic environment. Therefore, the possibility of PHD2 misfolding in some cancer cells arises. Studying such important protein unfolding pathway is insightful for possible therapeutic approaches. In this study, the unfolding pathway of PHD2 illustrates utilizing molecular dynamics simulation of protein thermal denaturation. Based on current study results, we represent the possible mechanisms of PHD2 unfolding in detail. The possible intermediates of PHD2 thermal unfolding are characterized, and the most venomous state of its unfolding pathway is introduced.     

Synthesis and characterization of a newly designed di-copper(II)-based complex and study of its artificial enzyme catalytic activity

A di-copper(II) complex of the formula [(dien)Cu(μ-1,6-DAH)Cu(dien)(NO₃)₂](NO₃)₂, where μ-1,6-DAH = 1,6-diaminohexane, has been synthesized and characterized by X-ray crystallography, X-ray powder diffraction, thermal gravimetric (TG) and differential thermal analyses, cyclic voltammetry, infrared, ultraviolet visible spectroscopies and elemental analysis methods. It was crystallized in a monoclinic system, space group P2₁/n, with a = 8.0297(8) Å, b = 12.4937(14) Å, c = 15.3786(15) Å, β = 102.739(8) Å and z = 2. Each copper(II) has a square-based pyramidal coordination geometry with four N atoms building the basal plane (three from dien and one from μ-1,6-DAH). TGA study of the complex revealed the compound to be stable up to 245 °C. Electrochemical behavior of complex and enzyme-like catalytic activity of this complex, as a potential functional model for the active site of tyrosinase, was studied extensively. Kinetic studies show that the complex has the maximum enzymatic activity at pH 8, temperature of 40 °C and ionic strength of 50 mM.     

Electrocatalytic oxidation of the antiviral drug acyclovir on a copper nanoparticles-modified carbon paste electrode

The electrocatalytic oxidation of acyclovir (Zovirax) on two different copper-based electrodes: copper microparticles- and copper nanoparticles-modified carbon paste electrodes (denoted as micro-CPE and nano-CPE, respectively) was voltammetrically investigated. In the voltammogram recorded using micro-CPE, a single anodic oxidation peak appeared, while nano-CPE resulted in two overlapped anodic peaks. The anodic currents were related to the electrocatalytic oxidation of acyclovir via the electrogenerated active species of Cu(III) with an EC’ mechanism. Acyclovir was oxidized with higher rates at low potentials on nano-CPE compared to micro-CPE. This was related to the nanosize effect of copper nanoparticles. The constants of the electrocatalytic oxidation process and the diffusion coefficient of acyclovir were reported. A sensitive and time-saving determination procedure was developed for the analysis of acyclovir and the corresponding analytical parameters were reported. The proposed amperometric method was applied to the analysis of commercial pharmaceutical products (tablets and topical cream) and the results were in good agreement with the declared values.