In this study the β-lactoglobulin fibrillation, in the presence or absence of lead ions, aflatoxin M1 and curcumin, was evaluated using ThT fluorescence, Circular dichroism spectroscopy and atomic force microscopy. To investigate the toxicity of the different form of β-Lg fibrils, in the presence or absence of above toxins and curcumin, we monitored changes in the level of reactive oxygen species and morphology of the differentiated neuron-like PC12 cells. The cell viability, cell body area, average neurite length, neurite width, number of primary neurites, percent of bipolar cells and node/primary neurite ratios were used to assess the growth and complexity of PC12 cells exposed to different form of β-Lg fibrils. Incubation of β-Lg with curcumin resulted in a significant decrease in ROS levels even in the presence of lead ions and aflatoxin M1. The β-Lg fibrils formed in the presence of lead ions and aflatoxin M1 attenuated the growth and complexity of PC12 cells compared with other form of β-Lg fibrils. However, the adverse effects of these toxins and protein fibrils were negated in the presence of curcumin. Furthermore, the antioxidant and inhibitory effects of curcumin protected PC12 cells against fibril neurotoxicity and enhanced their survival. Thus, curcumin may provide a protective effect toward β-Lg, and perhaps other protein, fibrils mediated neurotoxicity.
While many proteins are recognized to undergo folding via intermediate(s), the heterogeneity of equilibrium folding intermediate(s) along the folding pathway is less understood. In our present study, FTIR spectroscopy, far- and near-UV circular dichroism (CD), ANS and tryptophan fluorescence, near IR absorbance spectroscopy and dynamic light scattering (DLS) were used to study the structural and thermodynamic characteristics of the native (N), denatured (D) and intermediate state (X) of goat cytochorme c (cyt-c) induced by weak salt denaturants (LiBr, LiCl and LiClO4) at pH 6.0 and 25°C. The LiBr-induced denaturation of cyt-c measured by Soret absorption (Δε400) and CD ([θ]409), is a three-step process, N ↔ X ↔ D. It is observed that the X state obtained along the denaturation pathway of cyt-c possesses common structural and thermodynamic characteristics of the molten globule (MG) state. The MG state of cyt-c induced by LiBr is compared for its structural and thermodynamic parameters with those found in other solvent conditions such as LiCl, LiClO4 and acidic pH. Our observations suggest: (1) that the LiBr-induced MG state of cyt-c retains the native Met80-Fe(III) axial bond and Trp59-propionate interactions; (2) that LiBr-induced MG state of cyt-c is more compact retaining the hydrophobic interactions in comparison to the MG states induced by LiCl, LiClO4 and 0.5 M NaCl at pH 2.0; and (3) that there exists heterogeneity of equilibrium intermediates along the unfolding pathway of cyt-c as highly ordered (X1), classical (X2) and disordered (X3), i.e., D ↔ X3 ↔ X2 ↔ X1 ↔ N.
Glycation of serum albumin and its consequence products were considered as an important factor in drug distribution and diabetic complications, therefore finding the glycation inhibitors and their inhibitory mechanisms became a valuable field of study. In this work, bovine serum albumin (BSA) became a subject as a model protein for analyzing the inhibitory mechanism of flavonoids, known as natural BSA glycation inhibitors in the early stage of glycation.
Firstly, for theoretical study, the three-dimensional model of BSA structure was generated by homology modeling and refined through molecular dynamic simulation. Secondly, several validation methods (statistical assessment methods and also neural network methods) by simultaneous docking study were employed for insurance about accuracy of our simulation. Then docking studies were performed for visualizing the relation between flavonoids’ binding sites and BSA glycation sites besides, the correlation analyzes between calculated binding energy and reported experimental inhibitory IC50 values of the flavonoids set, was considered to explore their molecular inhibitory mechanism.
The quality assessment methods and simultaneous docking studies on interaction of quercetin (as the most studied flavonoids) with BSA and Human serum albumin (HAS), confirm the accuracy of simulation and the second stage of docking results which were in close agreement with experimental observations, suggest that the potential residues in flavonoids binding sites (which were place neighbor of tryptophan 212 within 5Ǻ) cannot be considered as one of glycation sites.
Based on the results, flavonoids don’t participate in inhibitory interference mechanism, and also, the differentiation between complexes of flavonoids with BSA and HSA could destroy the speculation of using them as an exchangeable model protein in study of serum albumin and flavonoids interactions.
Homology modeling; Molecular dynamics simulation; Correlation analyzes; Glycation sites; Flavonoids; BSA
Nanocrystalline mixed metal oxides (MMO) of various metal cations were synthesized and were used for coating a piece of copper wire as a new high sensitive solid phase micro extraction (SPME) fiber in extraction and determination of BTEX compounds from the headspace of aqueous samples prior to GC-FID analysis. Under optimum extraction conditions, the proposed fiber exhibited low detection limits, and quantification limits, good reproducibility, simple and fast preparation method, high fiber capacity and high thermal and mechanical durability. These are some of the most important advantages of the new fiber. The proposed fiber was used for human hemoglobin upon interaction with benzene. Binding isotherm, Scatchard and Klotz logarithmic plots were constructed using HS-SPME-GC data, accurately. The obtained binding isotherm analyzed using Hill method. The Hill parameters have been obtained by calculating saturation parameter from the ratio of measured chromatographic peak areas in the presence and absence of hemoglobin. In this interaction, Hill coefficient and Hill constant determined as (nH = 6.14 and log KH = 6.47) respectively. These results reveal the cooperativity of hemoglobin upon interaction with benzene.
Glycation is a general spontaneous process in proteins which has significant impact on their physical and functional properties. These changes in protein properties could be related to several pathological consequences such as cataract, arteriosclerosis and Alzheimer’s disease. Among the proteins, glycation of Human serum albumin (HSA) is of special interest. Human serum albumin is the most abundant protein in the plasma and because of its high sensitivity for glycation, undergoes structural and functional changes due to binding of reducing sugars in vitro. The glycation process occurs by plasma glucose in vivo which has great impacts on the three dimensional structure of protein. These changes are efficient and stable enough which makes the protein to be considered as a new special disease marker instead of HbA1C for diabetes. In some cases, glycated albumin was used as an alternative marker for glycemic control. Glycated albumin reacts with glucose ten times more rapidly than HbA1C and has shorter half-life which makes it more reliable for indicating glycemic states. In this review, glycation of Human Serum Albumin has been overviewed, starting from overall concepts of glycation, followed by some Examples of pathological consequences of protein glycation. The BSA aggregation was reviewed in terms of structural and biological impacts of glycation on the protein followed by reporting documents which indicate possibility of glycated albumin to be used as specific marker for diabetes. Finally, some of the studies related to the models of glycated albumin have been briefly described, with an emphasis on In vitro studies. It is interesting to note the relationship found between in vitro glycation experiments and the propensity of proteins to form amyloid structures, a point that could be further explored as to its significance in hyperglycemic states.
Glycation; Human serum albumin; Disease marker; Diabetes
Ficin (EC 126.96.36.199), a cysteine endoproteolytic protease in fig trees’ latex, has multiple isoforms. Until now, no data on autolysis of individual ficins (ficin isoforms) are available. Following purification, ficins’ autolysis was determined by HPLC chromatogram changes and ultrafiltrations at different temperatures and storage times. These results showed that the number of HPLC peaks in latex proteins purification of Ficus carica cv. Sabz varied from previous fig varieties or cultivars. Proteolytic activity of ficins was inhibited by specific cysteine protease inhibitors, confirming the participation of the cysteine residue in the active site. The zeta potential of the first two eluted peaks (I and II) was negative, while that of other peaks were positive. All ficins were susceptible to autolysis when stored at high temperatures. In contrast, only the last two ficins (B, C) were prone to autolysis at cold temperature after long storage period. The rate of degradation of the ficins was significantly increased with the increased storage time. The ficin (A) related to peak (III) had the highest and the lowest surface hydrophobic patches and ratio of autolytic to proteolytic activity, respectively.
Ficin; Autolysis; Hydrophobic patch; Cysteine protease; Fig latex
Varieties of hemoglobin (Hb) forms exist in fish, which are usually well adapted to the different ecological conditions or various habitats. In the current study, Hbs from two Sturgeon species of the Southern Caspian Sea Basin were purified and studied upon interaction with n-dodecyl trimethylammonium bromide (DTAB; as a cationic surfactant) by various methods including UV-visible absorption, dynamic light scattering (DLS), and ANS fluorescence spectrophotometry. The chemometric analysis of Hbs was investigated upon interaction with DTAB under titration, using UV-visible absorption spectra. The chemometric resolution techniques were used to determine the number of the components and mole fraction of the oxidized Hbs. These results provided the evidence for the existence of three different molecular components including native (N), intermediate (I) and denatured (D) in sturgeon Hbs. According to the distribution of intermediates, which were broadened in a range of DTAB concentration, the aggregation states, DLS experiments, and thermal stability (Tm obtained by differential scanning calorimetry (DSC)), the Acipenser stellatus Hb was more stable compared to Acipenser persicus Hb. These results demonstrate a significant relationship between the stability of fish Hbs and the habitat depth requirements.
Sturgeon hemoglobins; DTAB; Intermediates; Chemometric analysis; Aggregation; Thermal denaturation
Silica nanoparticle supported imidazolium ionic liquid [SNImIL] was synthesized and utilized as a biocompatible additive for studying the thermal reversibility of human carbonic anhydrase II (HCA II). For this purpose, we prepared additive by modification of nanoparticles through the grafting of ionic liquids on the surface of nanoparticles (SNImIL). The SNImIL were fully characterized by Fourier Transform Infrared spectroscopy, scanning electron microscopy and thermo gravimetric analysis. The characterization of HCA II was investigated by various techniques including UV–Vis and ANS fluorescence spectrophotometry, differential scanning calorimetry, and docking study. SNImIL induced disaggregation, enhanced protein stability and increased thermal reversibility of HCA II by up to 42% at pH 7.75.
Disaggregation; Human carbonic anhydrase II; Protein stability; Silica nanoparticle supported imidazolium ionic liquid; Thermal reversibility
Direct electron transfer of hemoglobin (Hb) was realized by immobilizing Hb on a carboxyl functionalized multi-walled carbon nanotubes (FMWCNTs) and gold nanoparticles (AuNPs) nanocomplex-modified glassy carbon electrode. The ultraviolet-visible absorption spectrometry (UV-Vis), transmission electron microscopy (TEM) and Fourier transform infrared (FTIR) methods were utilized for additional characterization of the AuNPs and FMWCNTs. The cyclic voltammogram of the modified electrode has a pair of well-defined quasi-reversible redox peaks with a formal potential of −0.270 ± 0.002 V (vs. Ag/AgCl) at a scan rate of 0.05 V/s. The heterogeneous electron transfer constant (ks) was evaluated to be 4.0 ± 0.2 s−1. The average surface concentration of electro-active Hb on the surface of the modified glassy carbon electrode was calculated to be 6.8 ± 0.3 × 10−10 mol cm−2. The cathodic peak current of the modified electrode increased linearly with increasing concentration of hydrogen peroxide (from 0.05 nM to 1 nM) with a detection limit of 0.05 ± 0.01 nM. The apparent Michaelis-Menten constant (Kmapp) was calculated to be 0.85 ± 0.1 nM. Thus, the modified electrode could be applied as a third generation biosensor with high sensitivity, long-term stability and low detection limit.
hemoglobin; direct electrochemistry; functionalized multi-walled carbon nanotubes; gold nanoparticles; nanocomplex
Botulinum neurotoxin-derived recombinant proteins have been recently recognized as potential botulism vaccines. Here considering botulinum neurotoxin type E (BoNT/E), we studied two of its binding domain-based recombinant proteins. A multivalent chimera protein which is composed of botulinum neurotoxin serotypes A, B and E binding subdomains, and a monovalent recombinant protein containing the 93 amino acid residues from C-terminal heavy chain of BoNT/E (rBoNT/E-HCC). We compared their efficiency in antibody production, structural differences, and BoNT/E-epitope location. Immunological studies indicated that the antibody yields against rBoNT/E-HCC was higher than chimera protein. Cross Enzyme-linked immunosorbent assays confirmed that the antibodies against the chimera protein recognized rBoNT/E-HCC more efficiently. However, both antibody groups were able to recognize other protein. These results suggested that BoNT/E-epitope in rBoNT/E-HCC is more exposed. Computational protein modeling and hydrophobicity predictions indicated a more exposed location for BoNT/E-epitope sequence (YYTHMRD) in rBoNT/E-HCC than the chimera protein. These results were in strong agreement with our immunological studies. Together our studies suggest that the lower antibody titer and lower antibody affinities for the chimera protein compared to rBoNT/E-HCC are influenced by the location of BoNT/E-epitope in the two recombinant proteins.
Botulinum neurotoxin type E; Cross Enzyme-linked immunosorbent assay; Computational modeling; recombinant vaccine-candidates
Prolyl hydroxylase domain 2 containing protein (PHD2) is a key protein in regulation of angiogenesis and metastasis. In normoxic condition, PHD2 triggers the degradation of hypoxia-inducible factor 1 (HIF-1α) that induces the expression of hypoxia response genes. Therefore the correct function of PHD2 would inhibit angiogenesis and consequent metastasis of tumor cells in normoxic condition. PHD2 mutations were reported in some common cancers. However, high levels of HIF-1α protein were observed even in normoxic metastatic tumors with normal expression of wild type PHD2. PHD2 malfunctions due to protein misfolding may be the underlying reason of metastasis and invasion in such cases. In this study, we scrutinize the unfolding pathways of the PHD2 catalytic domain’s possible species and demonstrate the properties of their unfolding states by computational approaches. Our study introduces the possibility of aggregation disaster for the prominent species of PHD2 during its partial unfolding. This may justify PHD2 inability to regulate HIF-1α level in some normoxic tumor types.
Formation of protein amyloid fibrils consists of a series of intermediates including oligomeric aggregates, proto-fibrillar structures, and finally mature fibrils. Recent studies show higher toxicity for oligomeric and proto-fibrillar intermediates of protein relative to their mature fibrils. Here the kinetic of the insulin amyloid fibrillation was evaluated using a variety of techniques including ThT fluorescence, Congo red absorbance, circular dichroism, and atomic force microscopy (AFM). The solution surface tension changes were attributed to hydrophobic changes in insulin structure and were detected by Du Noüy Ring method. Determination of the surface tension of insulin oligomeric, proto-fibrillar and fibrillar forms indicated that the hydrophobicity of solution is enhanced by the formation of the oligomeric forms of insulin compared to other forms. In order to investigate the toxicity of the different forms of insulin we monitored morphological alterations of the differentiated neuron-like PC12 cells following incubation with native, oligomeric aggregates, proto-fibrillar, and fibrillar forms of insulin. The cell body area, average neurite length, neurite width, number of primary neurites, and percent of bipolar cells and node/primary neurite ratios were used to assess the growth and complexity of PC12 cells exposed to different forms of insulin. We observed that the oligomeric form of insulin impaired the growth and complexity of PC12 cells compared to other forms. Together our data suggest that the lower surface tension of oligomers and their perturbation affects the morphology of PC12 cells, mainly due to their enhanced hydrophobicity and detergent-like structures.
Curcumin has many pharmaceutical applications, many of which arise from its potent antioxidant properties. The present research examined the antioxidant activities of curcumin in polar solvents by a comparative study using ESR, reduction of ferric iron in aqueous medium and intracellular ROS/toxicity assays. ESR data indicated that the steric hindrance among adjacent big size groups within a galvinoxyl molecule limited the curcumin to scavenge galvinoxyl radicals effectively, while curcumin showed a powerful capacity for scavenging intracellular smaller oxidative molecules such as H2O2, HO•, ROO•. Cell viability and ROS assays demonstrated that curcumin was able to penetrate into the polar medium inside the cells and to protect them against the highly toxic and lethal effects of cumene hydroperoxide. Curcumin also showed good electron-transfer capability, with greater activity than trolox in aqueous solution. Curcumin can readily transfer electron or easily donate H-atom from two phenolic sites to scavenge free radicals. The excellent electron transfer capability of curcumin is because of its unique structure and different functional groups, including a β-diketone and several π electrons that have the capacity to conjugate between two phenyl rings. Therfore, since curcumin is inherently a lipophilic compound, because of its superb intracellular ROS scavenging activity, it can be used as an effective antioxidant for ROS protection within the polar cytoplasm.
While various approaches exist to study protein localization, it is still a challenge to predict where proteins localize. Here, we consider a mechanistic viewpoint for membrane localization. Taking into account the steps for the folding pathway of α-helical membrane proteins and relating biophysical parameters to each of these steps, we create a score capable of predicting the propensity for membrane localization and call it FP3mem. This score is driven from the principal component analysis (PCA) of the biophysical parameters related to membrane localization. FP3mem allows us to rationalize the colocalization of a number of channel proteins with the Cav1.2 channel by their fewer propensities for membrane localization.
A new detection technique called Fast Fourier Transform Square-Wave Voltammetry (FFT SWV) is based on measurements of electrode admittance as a function of potential. The response of the detector (microelectrode), which is generated by a redox processes, is fast, which makes the method suitable for most applications involving flowing electrolytes. The carbon paste electrode was modified by nanostructures to improve sensitivity. Synthesized dysprosium nanowires provide a more effective nanotube-like surface [1-4] so they are good candidates for use as a modifier for electrochemical reactions. The redox properties of diclofenac were used for its determination in human serum and urine samples. The support electrolyte that provided a more defined and intense peak current for diclofenac determination was a 0.05 mol L−1 acetate buffer pH = 4.0. The drug presented an irreversible oxidation peak at 850 mV vs. Ag/AgCl on a modified nanowire carbon paste electrode which produced high current and reduced the oxidation potential by about 100 mV. Furthermore, the signal-to-noise ratio was significantly increased by application of a discrete Fast Fourier Transform (FFT) method, background subtraction and two-dimensional integration of the electrode response over a selected potential range and time window. To obtain the much sensivity the effective parameters such as frequency, amplitude and pH was optimized. As a result, CDL of 2.0 × 10−9 M and an LOQ of 5.0 × 10−9 M were found for the determination for diclofenac. A good recovery was obtained for assay spiked urine samples and a good quantification of diclofenac was achieved in a commercial formulation.
Fast Fourier Transformation; square wave voltammetry; carbon paste electrode dysprosium nanowire; diclofenac; oxidation-reduction
A bromide–modified silver electrode is reported, in the present study, to catalyze the redox reactions of metalloproteins. This study describes that the bromide ions show very good redox behavior with silver electrode. The cathodic and anodic peak potentials were related to the concentration of bromide ions involved in making bromide-modified silver electrode. The electrode reaction in the bromine solution was a diffusion-controlled process. Positive potential shift of the bromide ions was seen when different proteins were added to the solution using a silver electrode. New cathodic and anodic peaks were observed at different potential ranges for myoglobin, cytochrome c and catalase. A linearly increasing cathodic peak current of bromide ions was seen when the concentration of superoxide dismutase was increased in the test solution. However, no change for albumin was observed when its concentration was increased in the test solution. Present data proves our methodology as an easy-to-use analysis for comparing the redox potentials of different metalloproteins and differentiating the metallo-from non-metalloproteins. In this study, we introduced an interesting method for bio-electrochemical analyses.
Bromide ions; Mediator; Silver electrode; metalloprotein; Bio-electrochemistry