Oxidation Of Human Serum Albumin Research Articles

Prunin from Poncirus trifoliata (L.) Rafin Inhibits Aldose Reductase and Glucose-Fructose-Mediated Protein Glycation and Oxidation of Human Serum Albumin.

Diabetes complications are associated with aldose reductase (AR) and advanced glycation end products (AGEs). Using bioassay-guided isolation by column chromatography, 10 flavonoids and one coumarin were isolated from Poncirus trifoliata Rafin and tested in vitro for an inhibitory effect against human recombinant AR (HRAR) and rat lens AR (RLAR). Prunin, narirutin, and naringin inhibited RLAR (IC50 0.48-2.84 μM) and HRAR (IC50 0.68-4.88 μM). Docking simulations predicted negative binding energies and interactions with the RLAR and HRAR binding pocket residues. Prunin (0.1 and 12.5 μM) prevented the formation of fluorescent AGEs and nonfluorescent Nε-(carboxymethyl) lysine (CML), as well as the fructose-glucose-mediated protein glycation and oxidation of human serum albumin (HSA). Prunin suppressed the formation of the β-cross-amyloid structure of HSA. These results indicate that prunin inhibits oxidation-dependent protein damage, AGE formation, and AR, which may help prevent diabetes complications.

Spontaneous and Ionizing Radiation-Induced Aggregation of Human Serum Albumin: Dityrosine as a Fluorescent Probe.

The use of spectroscopic techniques has shown that human serum albumin (HSA) undergoes reversible self-aggregation through protein–protein interactions. It ensures the subsequent overlapping of electron clouds along with the stiffening of the conformation of the interpenetrating network of amino acids of adjacent HSA molecules. The HSA oxidation process related to the transfer of one electron was investigated by pulse radiolysis and photochemical methods. It has been shown that the irradiation of HSA solutions under oxidative stress conditions results in the formation of stable protein aggregates. The HSA aggregates induced by ionizing radiation are characterized by specific fluorescence compared to the emission of non-irradiated solutions. We assume that HSA dimers are mainly responsible for the new emission. Dityrosine produced by the intermolecular recombination of protein tyrosine radicals as a result of radiolysis of an aqueous solution of the protein is the main cause of HSA aggregation by cross-linking. Analysis of the oxidation process of HSA confirmed that the reaction of mild oxidants (, , ) with albumin leads to the formation of covalent bonds between tyrosine residues. In the case of •OH radicals and partly, , species other than DT are formed. The light emission of this species is similar to the emission of self-associated HSA.

Photosensitized protein damage by water-soluble phthalocyanine zinc(II) and gallium(III) complexes through electron transfer and singlet oxygen production

The cationic phthalocyanines (ZnPyPc and GaPyPc) photosensitized the oxidation of human serum albumin (HSA). The scavenger experiment using sodium azide suggested the contribution of 1O2. However, the HSA photodamage was observed in the presence of sodium azide. Because the redox potential of these phthalocyanines were relatively small and the Gibbs energy of the electron transfer from tryptophan to singlet excited states of these phthalocyanines is negative, electron-transfer oxidation is possible in terms of thermodynamics. Fluorescence lifetime measurement supported the electron-transfer mechanism. This study showed that electron transfer–supported oxidation plays an important role in the protein photodamage by cationic phthalocyanines.

Electrochemically-functionalized CNT/ABTS nanozyme enabling sensitive and selective voltammetric detection of microalbuminuria

Developing a cost-effective and reliable sensing platform for the routine screening of microalbuminuria and early diagnosis of chronic kidney disease is of great importance. Herein, we report on the high specificity and electrocatalytic activity of CNT/ABTS(CV) nanozyme, derived from the electrochemical functionalization of the carbon nanotube/2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) nanocomposite CNT/ABTS), towards electrochemical oxidation of human serum albumin (HSA) in urine. The potential cycling of CNT/ABTS electrode resulted in the surface functionalization with imine and carbonyl functional groups, which enhances electrode's affinity towards the electroactive tyrosine and tryptophan amino acids and thus facilitates the adsorption and subsequent electrochemical oxidation of HSA. The developed CNT/ABTS(CV) nanozyme modified electrode showed promising sensing characteristics for the point-of-care diagnosis of microalbuminuria without any sample pretreatment and inclusion of additional label or redox probes during measurement, including a practical sensing range up to 1.50 μM, a low detection limit of 60 nM, good reusability (>5 cycles), good long-term stability (>28 days), and high accuracy (error <15%) for the detection of HSA in urine. This work opens a new avenue for the detection and quantification of proteins in physiological conditions.

Photosensitized Protein Damage by DiethyleneglycoxyP(V)tetrakis(p-n-butoxyphenyl)porphyrin Through Electron Transfer: Activity Control Through Self-aggregation and Dissociation.

DiethyleneglycoxyP(V)tetrakis(p-n-butoxyphenyl)porphyrin (EGP(V)TBPP) forms a self-aggregation in an aqueous solution, and the photoexcited state of this molecule was effectively deactivated. Association with human serum albumin (HSA), a water-soluble protein, causes dissociation of the self-aggregation, resulting in recovery of the photosensitizer activity of EGP(V)TBPP. Under visible light irradiation, EGP(V)TBPP photosensitized HSA oxidation. The photosensitized singlet oxygen-generating activity of EGP(V)TBPP was confirmed by near-infrared emission measurement. A singlet oxygen quencher, sodium azide, partially inhibited the HSA photodamage; however, the quenching effect was estimated to be 57%. Another 43% of the HSA photodamage could be explained by the electron transfer mechanism. The redox potential of EGP(V)TBPP and the calculated Gibbs energy of electron transfer from tryptophan to photoexcited EGP(V)TBPP demonstrated the possibility of HSA oxidation through electron extraction. Fluorescence lifetime measurements of EGP(V)TBPP verified the electron transfer from HSA. The photosensitizer activity of EGP(V)TBPP can be controlled through an association with biomolecules, such as protein, and the electron transfer-mediated biomolecule photooxidation plays an important role in photodynamic therapy under hypoxia.

Albumin in patients with liver disease shows an altered conformation

Human serum albumin (HSA) constitutes the primary transporter of fatty acids, bilirubin, and other plasma compounds. The binding, transport, and release of its cargos strongly depend on albumin conformation, which is affected by bound ligands induced by physiological and pathological conditions. HSA is both highly oxidized and heavily loaded with fatty acids and bilirubin in chronic liver disease. By employing small-angle X-ray scattering we show that HSA from the plasma of chronic liver disease patients undergoes a distinct opening compared to healthy donors. The extent of HSA opening correlates with clinically relevant variables, such as the model of end-stage liver disease score, bilirubin, and fatty acid levels. Although the mild oxidation of HSA in vitro does not alter overall structure, the alteration of patients’ HSA correlates with its redox state. This study connects clinical data with structural visualization of albumin dynamicity in solution and underlines the functional importance of albumin’s inherent flexibility.

Effects of Oxidation of Human Serum Albumin on the Binding of Aripiprazole.

Aripiprazole (ARP) is one of antipsychotics and binds to human serum albumin (HSA) with a high affinity. In this study, we investigated the binding characteristics of ARP to oxidized HSA as observed in chronic disease conditions. Oxidized HSAs were prepared using chloramine-T (CT-HSA) or metal-catalyzed oxidation system (MCO-HSA) in vitro, respectively. An increase in the carbonyl content was confirmed in oxidized HSAs. From the results of circular dichroism (CD) and tryptophan fluorescence spectra, no significant structural change of oxidized HSAs was observed. These results indicate that prepared HSAs are mildly oxidized and well reflects the status of HSA during chronic diseases. However, oxidized HSAs were observed to have a significant decrease in binding to ARP. The results of the induced CD spectrum suggested that ARP bound to oxidized HSAs with a similar orientation. These results suggest that oxidation of HSA during chronic disease state significantly affected the microenvironment of the binding site for ARP and binding capacity of HSA to ARP.

Oxidative Alteration of Trp-214 and Lys-199 in Human Serum Albumin Increases Binding Affinity with Phenylbutazone: A Combined Experimental and Computational Investigation.

Human serum albumin (HSA) is a target for reactive oxygen species (ROS), and alterations of its physiological functions caused by oxidation is a current issue. In this work, the amino-acid residues Trp-214 and Lys-199, which are located at site I of HSA, were experimentally and computationally oxidized, and the effect on the binding constant with phenylbutazone was measured. HSA was submitted to two mild oxidizing reagents, taurine monochloramine (Tau-NHCl) and taurine dibromamine (Tau-NBr2). The oxidation of Trp-214 provoked spectroscopic alterations in the protein which were consistent with the formation of N′-formylkynurenine. It was found that the oxidation of HSA by Tau-NBr2, but not by Tau-NHCl, provoked a significant increase in the association constant with phenylbutazone. The alterations of Trp-214 and Lys-199 were modeled and simulated by changing these residues using the putative oxidation products. Based on the Amber score function, the interaction energy was measured, and it showed that, while native HSA presented an interaction energy of −21.3 kJ/mol, HSA with Trp-214 altered to N′-formylkynurenine resulted in an energy of −28.4 kJ/mol, and HSA with Lys-199 altered to its carbonylated form resulted in an energy of −33.9 kJ/mol. In summary, these experimental and theoretical findings show that oxidative alterations of amino-acid residues at site I of HSA affect its binding efficacy.

Metal-catalyzed oxidation of human serum albumin does not alter the interactive binding to the two principal drug binding sites

It is well known that various physiological factors such as pH, endogenous substances or post-translational modifications can affect the conformational state of human serum albumin (HSA). In a previous study, we reported that both pH- and long chain fatty acid-induced conformational changes can alter the interactive binding of ligands to the two principal binding sites of HSA, namely, site I and site II. In the present study, the effect of metal-catalyzed oxidation (MCO) caused by ascorbate/oxygen/trace metals on HSA structure and the interactive binding between dansyl-L-asparagine (DNSA; a site I ligand) and ibuprofen (a site II ligand) at pH 6.5 was investigated. MCO was accompanied by a time-dependent increase in carbonyl content in HSA, suggesting that the HSA was being oxidized. In addition, The MCO of HSA was accompanied by a change in net charge to a more negative charge and a decrease in thermal stability. SDS-PAGE patterns and α-helical contents of the oxidized HSAs were similar to those of native HSA, indicating that the HSA had not been extensively structurally modified by MCO. MCO also caused a selective decrease in ibuprofen binding. In spite of the changes in the HSA structure and ligand that bind to site II, no change in the interactive binding between DNSA and ibuprofen was observed. These data indicated that amino acid residues in site II are preferentially oxidized by MCO, whereas the spatial relationship between sites I and II (e.g. the distance between sites), the flexibility or space of each binding site are not altered. The present findings provide insights into the structural characteristics of oxidized HSA, and drug binding and drug-drug interactions on oxidized HSA.

Relationship between Xanthine Oxidoreductase Redox and Oxidative Stress among Chronic Kidney Disease Patients.

Xanthine oxidase (XO), an isoform of xanthine oxidoreductase (XOR), is thought to increase the cardiovascular burden among chronic kidney disease (CKD) patients via oxidative radical production. Plasma XOR redox, which is characterized by the ratio of XO to total XOR, changes under different oxidative conditions associated with kidney dysfunction. However, the relationship between plasma XOR redox and oxidative stress (OS) is unclear. Thus, we aimed to clarify whether OS is related to XOR redox. We used the redox state of human serum albumin (HSA) as a marker to investigate the status of OS in CKD patients. HSA is composed of human mercaptoalbumin (HMA), which possesses not oxidized cysteine residues, reversibly oxidized human nonmercaptoalbumin-1 (HNA-1), and strongly oxidized human nonmercaptoalbumin-2 (HNA-2). The subjects included 13 nondialysis patients (7 males and 6 females) with varying degrees of kidney function. We found that ƒ(HMA) was negatively (R = −0.692, P = 0.0071) and ƒ(HNA-1) was positively (R = 0.703, P = 0.0058) correlated with plasma XO/XOR. ƒ(HNA-2) showed no correlation with XO/XOR (R = 0.146, P = 0.6412), indicating that plasma XOR redox is not related to the irreversible oxidation of HSA. In conclusion, plasma XOR redox is closely related to HSA redox, particularly reversible oxidation of HSA.

Ultrasound-assisted interaction between chlorin-e6 and human serum albumin: pH dependence, singlet oxygen production, and formulation effect

The interaction between chlorin e6 (Ce6) and human serum albumin (HSA) in the presence and absence of ultrasound have been investigated by ultraviolet-visible absorption spectroscopy and fluorescence spectroscopy. Ce6 is found to bind strongly to HSA at or near physiological pH conditions, but the strength of the binding is significantly weakened at lower pHs. The intrinsic fluorescence of HSA is incrementally quenched with increasing concentration of Ce6, and the quenching is enhanced after exposure to high-frequency ultrasound. Our experimental results suggest that Ce6-induced sonodynamic oxidation of HSA is mainly mediated by singlet oxygen. The formulation of Ce6 by high molecular weight polyvinylpyrrolidone (PVP) increased its stability in aqueous solutions and its quantum yield of singlet oxygen under ultrasound irradiation.

Oxidation of human serum albumin exhibits inter-individual variability after an ultra-marathon mountain race.

The aim of this study was to examine the oxidation of human serum albumin (HSA) caused by oxidative stress following exhaustive and demanding exercise, such as an ultra-marathon race. For this purpose, blood samples from 12 adult runners who underwent a 103 km mountain ultra-marathon race were collected before the race, and also at 24, 48 and 72 h post-race. HSA was partially purified using affinity chromatography and consequently subjected to western blot analysis in order to determine the levels of disulfide dimers indicating oxidation. For reasons of comparison, the results were correlated with those from a previous study, in which the same samples were analyzed using different oxidative stress markers. The results revealed a good correlation between albumin dimers and protein carbonyls at all time points, while there was also a significant correlation with static oxidation reduction potential at 24 h, and a negative correlation with capacity oxidation reduction potential at 24 and 48 h. In addition, an individual analysis of albumin dimers exhibited great inter-individual differences, indicating the variation of HSA oxidation between different athletes. Namely, in some athletes, HSA seemed to be the main oxidation target of serum proteins, while in other athletes, there was even a reduction of HSA. This inter-individual variability in the oxidation of HSA may suggest that different interventions (e.g., through diet) may be required in order to confront the effects on athletes following strenuous exercise. On the whole, this study suggests the importance of the assessment of albumin dimers as a predictive marker for exercise-induced oxidative stress.

A Robust Analytical Approach for the Identification of Specific Protein Carbonylation Sites: Metal-Catalyzed Oxidations of Human Serum Albumin

ABSTRACTThe formation of protein carbonyls in the metal-catalyzed oxidation of human serum albumin (HSA) is characterized using a new analytical approach that involves tagging the modification site with multiple hydrazide reagents. Protein carbonyl formation at lysine and arginine residues was catalyzed with copper and iron ions, and the resulting oxidation patterns in HSA are contrasted. A total of 18 modification sites were identified with iron-ion catalysis and 14 with copper-ion catalysis. However, with the more stringent requirement of identification with at least two tagging reagents, the number of validated modification sites drops to 10 for iron and nine for copper. Of the 14 total validated sites, there were only five in common for the two metal ions. The results illustrate the value of using multiple tagging agents and highlight the selective and specific nature of metal-catalyzed protein oxidations.

Photoexcited riboflavin induces oxidative damage to human serum albumin

Photoexcited riboflavin induced damage of human serum albumin (HSA), a water soluble protein, resulting in the diminishment of fluorescence from the tryptophan residue. Because riboflavin hardly photosensitized singlet oxygen generation and sodium azide, a singlet oxygen quencher, did not inhibit protein damage, electron transfer-mediated oxidation of HSA was speculated. Fluorescence lifetime of riboflavin was not affected by HSA, suggesting that the excited triplet state of riboflavin is responsible for protein damage through electron transfer. In addition, the preventive effect of xanthone derivatives, triplet quenchers, on photosensitized protein damage could be evaluated using this photosensitized reaction system of riboflavin and HSA.

Spectroscopic analysis of the impact of oxidative stress on the structure of human serum albumin (HSA) in terms of its binding properties

Oxygen metabolism has an important role in the pathogenesis of rheumatoid arthritis (RA). Reactive oxygen species (ROS) are produced in the course of cellular oxidative phosphorylation and by activated phagocytic cells during oxidative bursts, exceed the physiological buffering capacity and result in oxidative stress. ROS result in oxidation of serum albumin, which causes a number of structural changes in the spatial structure, may influence the binding and cause significant drug interactions, particularly in polytherapy. During the oxidation modification of amino acid residues, particularly cysteine and methionine may occur.The aim of the study was to investigate the influence of oxidative stress on human serum albumin (HSA) structure and evaluate of possible alterations in the binding of the drug to oxidized human serum albumin (oHSA). HSA was oxidized by a chloramine-T (CT). CT reacts rapidly with sulfhydryl groups and at pH 7.4 the reaction was monitored by spectroscopic techniques. Modification of free thiol group in the Cys residue in HSA was quantitatively determined by the use of Ellman’s reagent. Changes of albumin conformation were examined by comparison of modified (oHSA) and nonmodified human serum albumin (HSA) absorption spectra, emission spectra, red-edge shift (REES) and synchronous spectroscopy. Studies of absorption spectra indicated that changes in the value of absorbance associated with spectral changes in the region of 200–250nm involve structural alterations in peptide backbone conformation. Synchronous fluorescence spectroscopy technique confirmed changes of position of tryptophanyl and tyrosyl residues fluorescent band caused by CT. Moreover analysis of REES effect allowed to observe structural changes caused by CT in the region of the hydrophobic pocket containing the tryptophanyl residue.Effect of oxidative stress on binding of anti-rheumatic drugs, sulfasalazine (SSZ) and sulindac (SLD) in the high and low affinity binding sites was investigated by spectrofluorescence, ITC and 1H NMR spectroscopy, respectively. SSZ and SLD change the affinity of each other to the binding site in non- and modified human serum albumin. The presence of SLD causes the increase of association constant (Ka) of SSZ–oHSA system and the strength of binding and the stability of the complexes has been observed while in the presence of SSZ a displacement of SLD from the SLD–HSA has been recorded. The analysis of 1H NMR spectral parameters i.e. changes of chemical shifts of the drug indicate that the presence of SSZ and SLD have a mutual influence on changes in the affinity of human serum albumin binding site and this competition takes place not only due to the additional drug but also to the oxidation of HSA.

Elucidation of the mechanism responsible for the oxidation of serum albumin and its application in treating oxidative stress-related diseases

Oxidative damage results in protein modification and is observed in numerous diseases. Human serum albumin (HSA), the most abundant protein in the plasma, exerts important antioxidant activities to prevent oxidative damage. This paper focuses on the characterization of chemical changes in HSA that are induced by oxidative damage, their relevance to human pathology, and the most recent advances in clinical applications. The antioxidant properties of HSA are largely dependent on Cys-34 and its contribution to the maintenance of intravascular homeostasis, including protecting the vascular endothelium under conditions related to oxidative stress. Recent studies have also evaluated the susceptibility of other important amino acid residues to exposure to free radicals. The findings suggest that a redox change in HSA is related to the oxidation of several amino acid residues by different oxidants. On the other hand, the ratio of the oxidized form to the normal form of albumin (HMA/HNA), which is a function of the redox states of Cys-34, could serve as a useful marker for evaluating systemic redox states, which would be useful for the evaluation of disease progression and therapeutic efficacy. This review provides new insights into our current understanding of the mechanism of HSA oxidation, based on in vitro and in vivo studies.

Carnosic acid protects biomolecules from free radical-mediated oxidative damage in vitro

The protective effects of carnosic acid (CA), a naturally occurring catechol-type poly-phenolic diterpene, against free radical-induced oxidative damage to lipids, proteins, and DNA were evaluated in this study. The oxidation of human serum albumin and rat liver homogenate were induced by Cu2+/H2O2. Fe2+/VitC and 2,2′-azobis(2,4-dimethylvaleronitrile) were used to initiate linoleic acid (LA) peroxidation. Oxidative damage of DNA was carried out using 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH). The results showed that CA significantly inhibited the oxidative damages to proteins, LA and DNA caused by various types of free radicals. Moreover, in the Fe2+/VitC induced-LA peroxidation reaction system, the addition of CA also inhibited the cytotoxic effects of oxidized linoleic acid on RAW 264.7 cells. These results suggest that it might be possible to use CA to prevent or treat the oxidative stress-related disorders and diseases.

Peroxynitrite induced structural changes result in the generation of neo-epitopes on human serum albumin

Human serum albumin (HSA), the most abundant plasma protein, is quite vulnerable to oxidizing and nitrating agents. In this study, peroxynitrite induced nitration and oxidation of HSA was assessed by various physicochemical techniques. Cross-linking of HSA was evident on polyacrylamide gel electrophoresis. The carbonyl content was markedly elevated in peroxynitrite-modified HSA as compared to the native protein. Dityrosine and 3-nitrotyrosine were present only in peroxynitrite-modified HSA. The peroxynitrite-modified HSA induced high titre antibodies in experimental animals showing high specificity towards the immunogen. Spectroscopic studies showed structural alterations in the HSA molecule upon peroxynitrite treatment which result in the generation of neo-epitopes and enhanced immunogenicity. The possible role of damaged HSA in various diseases has been suggested.

Human Serum Albumin and Its Relation with Oxidative Stress

Human serum albumin, a negative acute phase reactant and marker of nutritive status, presents at high concentrations in plasma. Albumin has always been used in many clinical states especially to improve circulatory failure. It has been showed that albumin is involved in many bioactive functions such as regulation of plasma osmotic pressure, binding and transport of various endogenous or exogenous compounds, and finally extracellular antioxidant defenses. Molecules like transferrin, caeruloplasmin, haptoglobin, uric acid, bilirubin, alpha-tocopherol, glucose, and albumin constitute extracellular antioxidant defenses in blood plasma but albumin is the most potent one. Most of the antioxidant properties of albumin can be attributed to its unique biochemical structure. The protein possesses antioxidant properties such as binding copper tightly and iron weakly, scavenging free radicals, e.g., hypochlorous acid (HOCl) and Peroxynitrite (ONOOH) and providing thiol group (-SH). Whether it is chronic or acute, during many pathological conditions, biomarkers of oxidative protein damage increase and this observation continues with considerable oxidation of human serum albumin. There is an important necessity to specify its interactions with Reactive Oxygen Species. Generally, it may lower the availability of pro-oxidants and be preferentially oxidized to protect other macromolecules but all these findings make it necessary that researchers give a more detailed explanation of albumin and its relations with oxidative stress.

Redox-active Magnetic Resonance Imaging Contrast Agents: Studies with Thiol-bearing 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetracetic Acid Derivatives

The synthesis and structure-activity relationships of a homologous series of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid gadolinium(III) complexes bearing thiol-terminated alkyl side chains from three to nine carbons in length are reported. The observed binding with human serum albumin (HSA) of the compounds having C-3 through C-7 side chain lengths was inhibited by homocysteine in a manner consistent with single-site binding. The observed binding with HSA of the compounds having C-8 and C-9 side chain lengths was only partly inhibited by homocysteine, consistent with multisite binding. The binding affinity of the C-7 compound could be related to the HSA oxidation state. 2D 1H-1H NMR TOCSY provided evidence of covalent binding of the europium analog of the C-6 compound to HSA-Cys34. The longitudinal water-proton MRI relaxivities of the gadolinium complexes at 7 T increased upon binding to HSA. On the basis of these results, the C-6 and C-7 compounds were identified as promising redox-sensitive MRI contrast agents.