Intensification of serum albumin amyloidogenesis by a glycation-peroxidation loop (GPL).

The interaction of reducing sugars with proteins leads to the formation of advanced glycation end products (AGE) and reactive oxidative species (ROS). ROS peroxidise free or membrane included unsaturated fatty acids, leading to generate reactive aldehydes as advanced lipid peroxidation end products (ALE). Aldehydes from lipid peroxidation (LPO) react with proteins to cause alteration of protein structure to exacerbate complication of diseases. Here we studied serum albumin glycation in the presence and absence of liposomes as a bio-membrane model to investigate protein structural changes using various techniques including intrinsic and extrinsic fluorescence spectroscopies and electron microscopy analysis. Accordingly, serum albumin glycation and fibrillation were accelerated and intensified in the presence of liposomes through a hypothesized glycation-peroxidation loop (GPL). Together, our results shed light on the necessity of reconsidering diabetic protein glycation to make it close to physiological conditions mimicry, more importantly, proteins structural change due to diabetic glycation is intensified in the proximity of cell membranes which probably potentiates programmed cell death distinct from apoptosis.

The ambivalent effect of Fe3O4 nanoparticles on the urea-induced unfolding and dilution-based refolding of lysozyme.

Due to the numerous biological applications of magnetite (Fe3O4) nanoparticles (MNPs), it is essential to identify the influence of these nanoparticles on basic biological processes. Therefore, in this research, the effect of MNPs on the structure and activity of hen egg white lysozyme (HEWL) (EC 3.2.1.1) as a model protein was examined using tryptophan intrinsic fluorescence, UV/Vis, and circular dichroism spectroscopy. Moreover, enzyme activities were analyzed by a turbidometric approach in the presence of MNPs at concentrations providing MNPs/HEWL ratios in the range of 0.04-1.25. As-synthesized MNPS were characterized by Fourier transform infrared spectroscopy, x-ray diffraction, scanning electron microscopy, transmission electron microscopy, vibrating sample magnetometry and the zeta potential of MNPs was measured to be -29 mV. The goal of this work was investigating the ordering or disordering effect of MNPs on protein structure at ratios lower or higher than 0.918 as concentration ratio of threshold (CRT), respectively, in order to answer the question: 'How can the denaturation and refolding of a model protein (HEWL) be affected by MNPs?' As has been reported recently, the protein folding, helicity, and half-life were improved at CRT to make the protein more disordered upon interaction with MNPs. The disordering effect of urea at >CRT and even at

Modeling, simulation, and employing dilution-dialysis microfluidic chip (DDMC) for heightening proteins refolding efficiency.

Miniaturized systems based on the principles of microfluidics are widely used in various fields, such as biochemical and biomedical applications. Systematic design processes are demanded the proper use of these microfluidic devices based on mathematical simulations. Aggregated proteins (e.g., inclusion bodies) in solution with chaotropic agents (such as urea) at high concentration in combination with reducing agents are denatured. Refolding methods to achieve the native proteins from inclusion bodies of recombinant protein relying on denaturant dilution or dialysis approaches for suppressing protein aggregation is very important in the industrial field. In this paper, a modeling approach is introduced and employed that enables a compact and cost-effective method for on-chip refolding process. The innovative aspect of the presented refolding method is incorporation dialysis and dilution. Dilution-dialysis microfluidic chip (DDMC) increases productivity folding of proteins with the gradual reduction of the amount of urea. It has shown the potential of DDMC for performing refolding of protein trials. The principles of the microfluidic device detailed in this paper are to produce protein on the dilution with slow mixing through diffusion of a denatured protein solution and stepwise dialysis of a refolding buffer flowing together and the flow regime is creeping flow. The operation of DDMC was modeled in two dimensions. This system simulated by COMSOL Multiphysics Modeling Software. The simulation results for a microfluidic refolding chip showed that DDMC was deemed to be perfectly suitable for control decreasing urea in the fluid model. The DDMC was validated through an experimental study. According to the results, refolding efficiency of denaturant Hen egg white lysozyme (HEWL) (EC 3.2.1.17) used as a model protein was improved. Regard to the remaining activity test, it was increased from 42.6 in simple dilution to 93.7 using DDMC.

Antiamyloidogenic Effects of Ellagic Acid on Human Serum Albumin Fibril Formation Induced by Potassium Sorbate and Glucose.

Oxidative stress has the main role in protein conformational changes and consequent direct involvement in different kind of diseases. Potassium sorbate as a widespread industrial preservative and glucose are two important oxidants that can be involved in oxidative stress. In this study the effect of ellagic acid as a phenolic antioxidant on amyloid fibril formation of human serum albumin upon incubation of potassium sorbate and glucose was studied using thioflavin T assay, surface tension, atomic force microscopy, Amadori product, and carbonyl content assays. The thioflavin T assay and atomic force microscopy micrographs demonstrated the antiamyloidogenic effect of ellagic acid on the human serum albumin fibril formation. This antioxidant also had the repair effect on surface tension of the modified human serum albumin (amyloid intermediates), which was destructed, caused by potassium sorbate and glucose. This mechanism takes place because of potent carbonyl stress suppression effect of ellagic acid, which was strengthening by potassium sorbate in the presence and absence of glucose.

Autolysis control and structural changes of purified ficin from Iranian fig latex with synthetic inhibitors.

The fig's ficin is a cysteine endoproteolytic enzyme, which plays fundamental roles in many plant physiological processes, and has many applications in different industries such as pharmaceutical and food. In this work, we report the inhibition and activation of autolysis and structural changes associated with reaction of ficin with iodoacetamide and tetrathionate using high-performance liquid chromatography (HPLC), ultra filtration membrane, and dynamic light scattering (DLS) methods. The ficin structural changes were also determined using UV-absorption, circular dichroism (CD), fluorescence spectroscopy, and differential scanning calorimetry (DSC) techniques. These techniques demonstrated that iodoacetamide completely inhibited ficin autolysis, which was irreversible. However, tetrathionate partially and reversibility inhibited its autolysis. The ficin structural changes with two synthetic inhibitors were associated with secondary structural changes related to decreased alpha-helix and increased beta sheet and random coil conformations, contributing to its aggregation.

Effect of covalent attachment of neomycin on conformational and aggregation properties of catalase.

The carboxylic groups of glutamic acid and aspartic acid residues of catalase (CAT) were chemically modified using the treatment of the enzyme with 1-ethyl-3-(3'-dimethylamino) carbodiimide hydrochloride (EDC) and neomycin. The effect of covalent attachment of neomycin on the enzymatic activity, conformational and aggregation properties of CAT was investigated. The modification of CAT with different concentrations of neomycin showed two different types of behavior, depending up on the concentration range of neomycin. In the concentration range from 0.0 to 5.2 mM, neomycin-modified CAT, compared to the native enzyme exhibited higher a-helix content, reduced surface hydrophobicity, little enhancement in CAT activity and a better protection against thermal aggregation, whereas at concentrations greater than 5.2 mM, the modified enzyme exhibited a significant decrease in CAT activity and an increase in random coil content which may result in disorder in the protein structure and increase in thermal aggregation. This modification is a rapid and simple approach to investigate the role of aspartate and glutamate residues in the structure, function and folding of CAT.

Electromembrane Extraction of Organic Acid Compounds in Biological Samples Followed by High-Performance Liquid Chromatography.

Electromembrane extraction (EME) coupled with high-performance liquid chromatography was developed for determination of organic compounds including citric, tartaric and oxalic acid in biological samples. Organic compounds moved from aqueous samples, through a thin layer of 1-octanol immobilized in the pores of a porous hand-made polypropylene tube, and into a basic aqueous acceptor solution present inside the lumen of the tube. This new set-up for EME has a future potential such as simple, cheap and fast sample preparation technique for extraction of organic compounds in various complicated matrices. The pH of acceptor phase, extraction time, voltage, ionic strength, temperature and stirring speed were studied and optimized. Optimum conditions were: the pH of acceptor phase, 7; extraction time, 30 min; voltage, 30 V and stirring speed, 500 rpm. At the optimum conditions, the preconcentration factors of 175-200, the limits of detection of 1.9-3.1 µg L(-1) were obtained for the analytes. The developed procedure was then applied to the extraction and determination of organic acid compounds from biological samples.

Application of merged spectroscopic data combined with chemometric analysis for resolution of hemoglobin intermediates during chemical unfolding.

Using tetradecyltrimethylammonium bromide (TTAB) as a surfactant denaturant, and augmentation of different spectroscopic data, helped to detect the intermediates of hemoglobin (Hb) during unfolding process. UV-vis, fluorescence, and circular dichroism spectroscopy were used simultaneously to monitor different aspects of hemoglobin species from the tertiary or secondary structure points of view. Application of the multivariate curve resolution-alternating least square (MCR-ALS), using the initial estimates of spectral profiles and appropriate constraints on different parts of augmented spectroscopic data, showed good efficiency for characterization of intermediates during Hb unfolding. These results indicated the existence of five protein species, including three intermediate-like compounds in this process. The unfolding pathway in the presence of TTAB included conversion of oxyhemoglobin into deoxyhemoglobin, and then ferrylhemoglobin, ferrihemoglobin or aquamethemoglobin, which finally transformed into hemichrome. This is the first application of chemometric analysis on the merged spectroscopic data related to chemical denaturation of a protein. These types of analysis in multisubunit proteins not only increase the domain of information, but also can reduce the ambiguities of the obtained results.

Differential propensity of citrate- and polyethylene glycol-coated silver nanoparticles to bovine hemoglobin.

Propensity of two different silver nanoparticles (Ag-NPs) to bovine hemoglobin (BHb) was investigated by means of spectroscopic methods. We have combined spectrophotometric and calorimetric methods to show that there is no significant interaction between citrate-coated Ag-NPs and BHb at physiological pH and 20°C. However, our previous results show that polyethylene glycol-coated Ag-NPs strongly bind to Hb and effect on the secondary and tertiary structures of BHb. Thus, a suitable surface coating and modification of surface charge would increase the NPs safety and reduce adverse biological responses.

Hemoglobin fructation promotes heme degradation through the generation of endogenous reactive oxygen species.

Protein glycation is a cascade of nonenzymatic reactions between reducing sugars and amino groups of proteins. It is referred to as fructation when the reducing monosaccharide is fructose. Some potential mechanisms have been suggested for the generation of reactive oxygen species (ROS) by protein glycation reactions in the presence of glucose. In this state, glucose autoxidation, ketoamine, and oxidative advance glycation end products (AGEs) formation are considered as major sources of ROS and perhaps heme degradation during hemoglobin glycation. However, whether fructose mediated glycation produces ROS and heme degradation is unknown. Here we report that ROS (H2O2) production occurred during hemoglobin fructation in vitro using chemiluminescence methods. The enhanced heme exposure and degradation were determined using UV-Vis and fluorescence spectrophotometry. Following accumulation of ROS, heme degradation products were accumulated reaching a plateau along with the detected ROS. Thus, fructose may make a significant contribution to the production of ROS, glycation of proteins, and heme degradation during diabetes.

Heme degradation upon production of endogenous hydrogen peroxide via interaction of hemoglobin with sodium dodecyl sulfate.

In this study the hemoglobin heme degradation upon interaction with sodium dodecyl sulfate (SDS) was investigated using UV-vis and fluorescence spectroscopy, multivariate curve resolution analysis, and chemiluminescence method. Our results showed that heme degradation occurred during interaction of hemoglobin with SDS producing three fluorescent components. We showed that the hydrogen peroxide, produced during this interaction, caused heme degradation. In addition, the endogenous hydrogen peroxide was more effective in hemoglobin heme degradation compared to exogenously added hydrogen peroxide. The endogenous form of hydrogen peroxide altered oxyHb to aquamethemoglobin and hemichrome at low concentration. In contrast, the exogenous hydrogen peroxide lacked this ability under same conditions.

Inhibition of fluorescent advanced glycation end products (AGEs) of human serum albumin upon incubation with 3-ß-hydroxybutyrate.

Advanced glycation end products (AGEs), which are the final products of glycation, have a major role in diabetic complication and neurodegenerative disorders. The 3-ß-hydroxybutyrate (3BHB), a ketone body which is produced by the liver, can be detected in increased concentrations in individuals post fasting and prolonged exercises and in diabetic (type I) patients. In this study, the inhibitory effect of 3BHB on AGEs formation by glucose from the human serum albumin (HSA) was studied at physiological conditions after 35 days of incubation, using physical techniques such as circular dichroism and fluorescence spectroscopy, as well as differential scanning calorimetry (DSC). The fluorescence intensity measurements of glycated HSA by glucose (GHSA) in the presence of 3BHB indicate a decrease in AGEs formation. The DSC deconvolution profile results also confirm the protective role of 3BHB on incubated with glucose by preventing the enthalpy reduction of the HSA tail segment, compared with the deconvolution profile seen for incubated with glucose alone. The concentration of 3BHB used in this study is in accordance with the concentration detected in the body of individuals post fasting and prolonged exercises.

Energetic domains and conformational analysis of human serum albumin upon co-incubation with sodium benzoate and glucose.

Sodium benzoate (SB), a powerful inhibitor of microbial growth, is one of the most commonly used food preservative. Here, we determined the effects of SB on human serum albumin (HSA) structure in the presence or absence of glucose after 35 days of incubation under physiological conditions. The biochemical, biophysical, and molecular approaches including free amine content assay (TNBSA assay), fluorescence, and circular dichroism spectroscopy (CD), differential scanning calorimetry (DSC), and molecular docking and LIGPLOT studies were utilized for structural studies. The TNBSA results indicated that SB has the ability to bind Lys residues in HSA through covalent bonds. The docking and LIGPLOT studies also determined another specific site via hydrophobic interactions. The CD results showed more structural helicity for HSA incubated with SB, while HSA incubated with glucose had the least, and HSA incubated with glucose + SB had medium helicity. Fluorescence spectrophotometry results demonstrated partial unfolding of HSA incubated with SB in the presence or absence of glucose, while maximum partial unfolding was observed in HSA incubated with glucose. These results were confirmed by DSC and its deconvoluted thermograms. The DSC results also showed significant changes in HSA energetic structural domains due to HSA incubation with SB in the presence or absence of glucose. Together, our studies showed the formation of three different intermediates and indicate that biomolecular investigation are effective in providing new insight into safety determinations especially in health-related conditions including diabetes.

Potassium sorbate as an AGE activator for human serum albumin in the presence and absence of glucose.

Advanced glycation end products (AGEs) are the predominant intermediates of glycation process, and mediate oxidative stress and complications of diabetes. Potassium sorbate (PS) as a widespread preservative is an oxidative agent and used in different dairy and drug products, which can readily enter biological matrices. Here we studied the PS interference with glycation of human serum albumin (HSA) in the presence of glucose (Glc) using various techniques. These included TNBSA assay, circular dichroism, fluorescence spectroscopy, differential scanning calorimetry (DSC), Th T assay, and atomic force microscopy. Our results indicated that HSA glycation was accelerated in the presence of PS. Furthermore, PS produced AGEs in the absence of glucose. Secondary and tertiary structural changes were also observed in HSA incubated with glucose in the presence or absence of PS through beta-sheet inducing effects. Th T assay demonstrated the role of PS in HSA fibril formation in the presence or absence of glucose. Atomic force microscopy determined different amyloid fibril formation in HSA incubated with PS in the presence or absence of glucose. Together our results indicated that PS has a stimulatory effect on glycation and fibrillation of HSA in the presence or absence of glucose, and could exacerbate complication of diabetes.

Investigation of thermal reversibility and stability of glycated human serum albumin.

Protein glycation, the process by which carbohydrates attach to proteins upon covalent binding, can alter protein thermal reversibility and stability. Protein stability and reversibility have important role in protein behavior and function. Also they are benefit properties for drug produce and protein industrial applications. In this research the thermal reversibility and stability changes in human serum albumin (HSA) were studied upon incubation with glucose (GHSA) under physiological conditions for 21 and 35 days. The thermal reversibility and stability changes in GHSA were evaluated using circular dichroism (CD), UV-vis spectroscopy, fluorescence spectroscopy and differential scanning calorimetry (DSC). Our results showed that the glycation of HSA increased its thermal reversibility and stability, but decreased its conformational entropy compared to fresh native HSA and untreated HSA. Free lysine content assay (TNBSA test) indicated glucose can bind to protein covalently. These alterations were mainly attributed to the formation of crosslink between the lysine residues of HSA upon incubation with glucose.

Thermodynamics of a molten globule state of human serum albumin by 3-ß-hydroxybutyrate as a ketone body.

The molten globule (MG) state is an intermediate which is considered as the third thermodynamic state of protein molecules. In this work the effect of incubating human serum albumin (HSA) at physiological condition in the presence of 3-ß-hydroxybutyrate for 7, 14, 21 and 35 days were studied by different techniques such as UV/vis, fluorescence and circular dichroism (CD) spectroscopy, differential scanning calorimetry (DSC) and dynamic light scattering (DLS). In this paper, we introduce the MG state for HSA upon 21 days incubation with 3-ß-hydroxybutyrate as a ketone body at physiological condition. The results from the HSA sample incubated for 21 days shows a similar secondary structure by CD, more surface hydrophobicity and a little change on tertiary structure by fluorescence, and a larger size by DLS as compared to the native sample or other incubated samples. These results were also confirmed by calculated parameters and DSC deconvoluted thermograms.

Synergic study of α-glucosidase inhibitory action of aloin and its antioxidant activity with and without camel ß-casein and its peptides.

Regular consumption of natural antioxidants reduces the risk of developing diseases. Aloin is one of the main active phenolic components of Aloe vera. The main disadvantage of aloin is its concentration limit of use that causes cell damage. One of the aims of this study was to investigate the antioxidant activity of aloin in the presence and absence of camel ß-casein ( ß-CN) and its peptide fractions. The mixture of aloin, ß-CN and peptides showed a very high antioxidant activity in a synergistic manner as compared to each component alone. The alpha ( α)-glucosidase inhibitory activity of aloin was also investigated in the presence and absence of ß-CN and its peptides. Aloin alone is a potent inhibitor of α-glucosidase. The α-glucosidase inhibitory activity of aloin is reduced in the presence of ß-CN or its peptides. The combination of aloin and ß-CN or its peptides makes a high antioxidant functional ingredient.

Effect of compatible and noncompatible osmolytes on the enzymatic activity and thermal stability of bovine liver catalase.

Catalase is an important antioxidant enzyme that catalyzes the disproportionation of H2O2 into harmless water and molecular oxygen. Due to various applications of the enzyme in different sectors of industry as well as medicine, the enhancement of stability of the enzyme is important. Effect of various classes of compatible as well as noncompatible osmolytes on the enzymatic activity, disaggregation, and thermal stability of bovine liver catalase have been investigated. Compatible osmolytes, proline, xylitol, and valine destabilize the denatured form of the enzyme and, therefore, increase its disaggregation and thermal stability. The increase in the thermal stability is accompanied with a slight increase of activity in comparison to the native enzyme at 25 °C. On the other hand, histidine, a noncompatible osmolyte stabilizes the denatured form of the protein and hence causes an overall decrease in the thermal stability and enzymatic activity of the enzyme. Chemometric results have confirmed the experimental results and have provided insight into the distribution and number of mole fraction components for the intermediates. The increase in melting temperature (Tm) and enzymatic rate could be further amplified by the intrinsic effect of temperature enhancement on the enzymatic activity for the industrial purposes.

Thermal inactivation and conformational lock of bovine carbonic anhydrase.

The kinetics of thermal inactivation of bovine carbonic anhydrase (BCA) was studied in a 50 mM Tris-HCl buffer, pH 7.8 using p-nitrophenyl acetate as substrate in absorbance of 400 nm by UV-VIS spectrophotometry. The number of conformational locks and inter-subunit amino acid residues of BCA were obtained by thermal inactivation analysis. The cleavage bonds between dimers of BCA during thermal dissociation and type of interactions between specific amino acid residues were also detected. The thermal inactivation curves were plotted in temperatures ranging between 40-70°C. It was shown several phases for inactivation of BCA at 65°C. Analyses of the curves were done by the conformational lock theory. The subunits are dissociated and several intermediates appear during inactivation through increasing the temperature in comparison with native state. Dynamic light scattering measurements was done to study the changes in hydrodynamic radius during thermal inactivation. Three distinct zones were shown in DLS data. Biochemical computation using ligplot is performed to find the inter-subunit amino acid residues for BCA.

Acidic residue modifications restore chaperone activity of ß-casein interacting with lysozyme.

An important factor in medicine and related industries is the use of chaperones to reduce protein aggregation. Here we show that chaperone ability is induced in ß-casein by modification of its acidic residues using Woodward's Reagent K (WRK). Lysozyme at pH 7.2 was used as a target protein to study ß-casein chaperone activities. The mechanism for chaperone activity of the modified ß-casein was determined using UV-vis absorbencies, fluorescence spectroscopy, differential scanning calorimetry and theoretical calculations. Our results indicated that the ß-casein destabilizes the lysozyme and increases its aggregation rate. However, WRK-ring sulfonate anion modifications enhanced the hydrophobicity of ß-casein resulting in its altered net negative charge upon interactions with lysozyme. The reversible stability of lysozyme increased in the presence of WRK-modified ß-casein, and hence its aggregation rate decreased. These results demonstrate the enhanced chaperone activity of modified ß-casein and its protective effects on lysozyme refolding.