Reaction Of Bovine Serum Albumin Research Articles

Photo-Crosslinkable Polymeric Coatings Providing Chemically Versatile Reactive Surfaces on Various Substrates

We demonstrate a chemical route to attain simple, clean, scalable, photopatternable, chemically adjustable, and versatile systems to fabricate reactive organic thin films. Their surfaces exhibit effective biocompatibility, controlled reactivity, and processability on various surface types, i.e., photo-crosslinkable polymeric thin films with various functionalities, further applicable for the definition of surface activity for interfacing biological objects. The copolymers were synthesized with three monomers: a poly(ethylene glycol)-containing monomer, a monomer releasing a primary amine upon exposure to UV light, and a monomer bearing cyclic dithiocarbonate or glycidyl groups that are highly reactive with the amine. The resulting copolymers were processed with polar solvents such as water or alcohol for coating and were readily crosslinked under UV light illumination to form a highly stable molecular network, beneficial for defining selective reactive surfaces in a desired region on a variety of organic and inorganic substrates. The designed system was chemically versatile in immobilizing biologically relevant molecules on the surfaces by (i) post-crosslinking modification through the reaction of bovine serum albumin on the remaining surface reactive group with a primary amine in the protein and (ii) pre-crosslinking formulation with reactive gelatin that has the functionality to crosslink in the composite thin film. We further showed that the resulting coating on a glass or polystyrene substrate provided excellent biocompatibility and growth of C2C12 murine myocytes.

Antinutrient and Sugar Type Effect on Structure and Maillard Reaction of Bovine Serum Albumin Submitted to Mild Heating

Antinutrient and Sugar Type Effect on Structure and Maillard Reaction of Bovine Serum Albumin Submitted to Mild Heating

Controlled Conversion of Proteins into High-Molecular-Weight Peptides without Additives with High-Temperature Water and Fast Heating Rates

Reaction of bovine serum albumin (BSA) protein in high-temperature (200−260 °C) water (HTW) with fast heating rates (ca. 135−180 K·s–1) without acid or base additives gives high-molecular-weight (1500–8300 Da) peptides with minimal formation of amino acids and ammonia. The decrease in the number-average molecular weight of peptides after HTW treatment of BSA could be described by a kinetic model based on random scission mechanism of the polymer chain. Reaction of BSA in HTW under identical conditions with slow heating rates (ca. 0.25 K·s–1) gives peptides of low molecular weight with formation of amino acids and ammonia for which the kinetics could not be described by a random scission mechanism. The activation energy determined for the conversion of BSA into high-molecular-weight peptides with fast heating rates in high-temperature water was 16.4 kJ·mol–1. Reaction of proteins in high-temperature water with fast heating rates inhibits initial aggregation that occurs during slow heating rates and allows controlled conversion of the denatured polymer chain into high-molecular-weight peptides.

Evaluation of the binding interaction between bovine serum albumin and dimethyl fumarate, an anti-inflammatory drug by multispectroscopic methods

The information of the quenching reaction of bovine serum albumin with dimethyl fumarate is obtained by multi-spectroscopic methods. The number of binding sites, n and binding constants, KA were determined at different temperatures. The effect of increasing temperature on Stern–Volmer quenching constants (KD) indicates that a dynamic quenching mechanism is involved in the interaction. The analysis of thermodynamic quantities namely, ∆H° and ∆S° suggested hydrophobic forces playing a major role in the interaction between dimethyl fumarate and bovine serum albumin. The binding site of dimethyl fumarate on bovine serum albumin was determined by displacement studies, using the site probes viz., warfarin, ibuprofen and digitoxin. The determination of magnitude of the distance of approach for molecular interactions between dimethyl fumarate and bovine serum albumin is calculated according to the theory of Förster energy transfer. The CD, 3D fluorescence spectra, synchronous fluorescence measurements and FT-IR spectral results were indicative of the change in secondary structure of the protein. The influence of some of the metal ions on the binding interaction was also studied.

Advanced glycation end products promote differentiation of CD4+ T helper cells toward pro-inflammatory response

This study investigated the effect of advanced glycation end products (AGEs) on differentiation of naïve CD4(+) T cells and the role of the receptor of AGEs (RAGE) and peroxisome proliferator-activated receptors (PPARs) activity in the process in order to gain insight into the mechanism of immunological disorders in diabetes. AGEs were prepared by the reaction of bovine serum albumin (BSA) with glucose. Human naïve CD4(+) T cells, enriched from blood of healthy adult volunteers with negative selection assay, were cultured in vitro and treated with various agents including AGEs, BSA, high glucose, PGJ2 and PD68235 for indicated time. In short hairpin (sh) RNA knock-down experiment, naïve CD4(+) T cells were transduced with media containing shRNA-lentivirus generated from lentiviral packaging cell line, Lent-X(TM) 293 T cells. Surface and intracellular cytokine stainings were used for examination of CD4(+) T cell phenotypes, and real-time PCR and Western blotting for detection of transcription factor mRNA and protein expression, respectively. The suppressive function of regulatory T (Treg) cells was determined by a [(3)H]-thymidine incorporation assay. The results showed that AGEs induced higher pro-inflammatory Th1/Th17 cells differentiated from naïve CD4(+) T cells than the controls, whereas did not affect anti-inflammatory Treg cells. However, AGEs eliminated suppressive function of Treg cells. In addition, AGEs increased RAGE mRNA expression in naïve CD4(+) T cells, and RAGE knock-down by shRNA eliminated the effect of AGEs on the differentiation of CD4(+) T cells and the reduction of suppressive function of Treg cells. Furthermore, AGEs inhibited the mRNA expression of PPARγ, not PPARα PPARγ agonist, PGJ2, inhibited the effect of AGEs on naïve CD4(+) T cell differentiation and reversed the AGE-reduced suppressive function of Treg cells; on the other hand, PPARγ antagonist, PD68235, attenuated the blocking effect of RAGE shRNA on the role of AGEs. It was concluded that AGEs may promote CD4(+) T cells development toward pro-inflammatory state, which is associated with increased RAGE mRNA expression and reduced PPARγ activity.

Hydogen peroxide-dependent photocytotoxicity by phloxine B, a xanthene-type food colorant

BackgroundPhloxine B (PhB; 2′,4′,5′,7′-tetrabromo-4,5,6,7-tetrachloro-fluorescein), an artificial xanthene colorant, has been used as a red coloring agent in drugs and cosmetics as well as foods in some countries. However, little effort has been devoted to the study of this colorant as a potentially useful medicinal agent. MethodsWe investigated the daily light-induced photocytotoxicity of PhB in two human leukemia cells, HL-60 and Jurkat, and its underlying mechanisms by in vitro experiments using antioxidants. Reuslts and conclusionsPhB inhibited cell proliferation more preferentially to HL-60 cells than to Jurkat cells. Co-treatment of catalase completely blocked the photocytotoxicity by PhB in HL-60 cells, whereas the effect of histidine was only partial, suggesting that hydrogen peroxide (H2O2), rather than singlet oxygen, might be a prerequisite for the PhB-induced HL-60 cell death. Actually, PhB produced a significant amount of H2O2 in the media as well as in the cells in concentration- and light-dependent manners. Furthermore, methionine, a hypochlorous acid (HOCl) scavenger, also significantly attenuated the cytotoxicity in HL-60 cells, but not in Jurkat cells, indicating the involvement of myeloperoxidase (MPO)-dependent hypohalous acid formation during the photocytotoxicity. In vitro experiments revealed that halogenated tyrosine was generated from the reaction of bovine serum albumin with PhB and HL-60 cell lysate. The present findings suggested that PhB induced a differential photodynamic action in the MPO-containing leukemia cells through an H2O2-dependent mechanism. General significanceOur findings provide new insights into the molecular mechanisms underlying the PhB-induced apoptosis and also evaluated PhB as a promising PDT agent.

A novel proteinaceous photolinker for simultaneous binding to an inert surface and a biomolecule

A simple and versatile method is developed for covalently binding a protein ligand onto a matrix irrespective of functional groups either on the ligand or the matrix. Prerequisite of the method is a novel proteinaceous photolinker having multiple light-activable functional groups. We have made photoreactive-BSA – a proteinaceous photolinker by the reaction of bovine serum albumin (BSA) with excess of 1-fluoro-2-nitro-4-azidobenzene (FNAB). When an enzyme is placed on an inert polystyrene matrix in presence of photoreactive-BSA and exposed to light the later forms highly reactive nitrenes some of which bind to the matrix and the rest to the ligand resulting simultaneous formation of covalent bonds with the matrix and the enzyme. The method is further exemplified by performing ELISA by covalent binding of antigen or antibody on a polystyrene microtiter plate in just 30 min using photoreactive-BSA. ELISA carried out in less than 3 h using photoreactive-BSA showed comparable results with that of conventional ELISA carried out in 18 h. Thus the method is potentially useful for rapid ELISA or covalent immobilization of ligands onto an inert surface without prior activation.

Exploiting Soft and Hard X-Ray Absorption Spectroscopy to Characterize Metallodrug/Protein Interactions: the Binding of [trans-RuCl4(Im)(dimethylsulfoxide)][ImH] (Im = imidazole) to Bovine Serum Albumin

The reaction of bovine serum albumin (BSA) with [ trans-RuCl 4(Im)(dimethylsulfoxide)][ImH] (Im = imidazole) (NAMI-A), an experimental ruthenium(III) anticancer drug, and the formation of the respective NAMI-A/BSA adduct were investigated by X-ray absorption spectroscopy (XAS) at the sulfur and chlorine K-edges and at the ruthenium K- and L 3-edges. Ruthenium K and L 3-edge spectra proved unambiguously that the ruthenium center remains in the oxidation state +3 after protein binding. Comparative analysis of the chlorine K-edge XAS spectra of NAMI-A and NAMI-A/BSA, revealed that the chlorine environment is greatly perturbed upon protein binding. Only modest changes were observed in the sulfur K-edge spectra that are dominated by several protein sulfur groups. Overall, valuable information on the nature of this metallodrug/protein adduct and on the mechanism of its formation was gained; XAS spectroscopy turns out to be a very suitable method for the characterization of this kind of systems.

Reaction of BSA, CT-DNA, Adenine and Guanine with [Pt(RaaiR′)Cl2] and Characterization of the Products [Pt(RaaiR′)(NB)(H2O)](PF6)2] [RaaiR′=1-alkyl-2-(arylazo)imidazole

The reaction of [Pt(RaaiR′)(solvent)2]2+ with nucleobases (NB), adenine and guanine was studied and the products [Pt(RaaiR′)(NB)(H2O)](PF6)2RaaiR′ = 1-alkyl-2-(arylazo)imidazole, R′ = Me (1), Et (2); R = H (a),OMe (b), NO2(c)] characterized by i.r, u.v.–vis. and 1H-n.m.r spectroscopy. The solution spectra exhibit metal-to-ligand charge-transfer transitions (MLCT) and intra-ligand charge-transfer transitions. The position and symmetry of the bands depend on the nucleobase and arylazoimidazole. The coordination of the ligand is supported by 1H-n.m.r. spectral data. The redox property has been examined by cyclic voltammetric technique and shows the involvement of the azo group of the chelated ligand, RaaiR′ in the reduction process along with the irreversible reduction of the coordinated nucleobase. Binding with Bovine Serum Albumin (BSA) and Calf Thymus DNA was performed spectrophotometrically in the physiological buffer medium and spectral profile was recorded in 24 h.

Non-enzymatic posttranslational modifications of bovine serum albumin by oxo-compounds investigated by chromatographic and electrophoretic methods

Non-enzymatic posttranslational modifications of bovine serum albumin (BSA) by oxo-compounds, particularly glucose, ribose, glyoxal and glutardialdehyde, have been investigated using a set of modern chromatographic and electrophoretic separation methods. High-performance liquid chromatography (HPLC) alternatively with UV spectrophotometric (diode array) or mass spectrometric (MS) detection, polyacrylamide gel electrophoresis (PAGE) with Coomassie brilliant blue staining detection, and capillary zone electrophoresis (CZE) with UV spectrophotometric detection have been employed for the investigation of the chemical and structural changes of BSA caused by its reaction with the above oxo-compounds exhibiting different degree of reactivity. The extent of modifications was found to be dependent on the nature of the oxo-compound used and progressed in the glucose < ribose < glyoxal < glutaraldehyde order. With the aid of HPLC/UV/MS and CZE/UV tryptic peptide mapping and amino acid analysis of both unmodified and modified BSA it was revealed that the mildest modification resulted from the reaction of BSA with glucose, in this case presumably only monofunctional derivatives have arisen, whereas the most intensive modifications were found after BSA reaction with glutardialdehyde, which resulted in high degree of both inter- and intra-molecular cross-linking, due to which no unmodified peptides were detected after tryptic cleavage of BSA modified by this agent.

Observation of Protein-derived (BSA) Oxygen-centered Radicals by EPR Spin-trapping Techniques

Electron paramagnetic resonance (EPR) spin-trapping experiments, employing the novel spin-trap DEPMPO, provide evidence for the formation of protein-peroxyl radicals from the reaction of bovine serum albumin (BSA) or lysozyme with HO · in the presence of O 2 . Spin-trapping leads to the detection of anisotropic spectra of partially immobilized protein-peroxyl spin-adducts; positive identification is based on a novel spectrum simulation approach (through which broadened anisotropic spectra are simulated and compared with experiment) and by comparison of results with those obtained when MeO 2 · is trapped and the adduct frozen in a solid matrix.

Generation of Acetate and Production of Ethyl-Lysine in the Reaction of Acetaldehyde Plus Serum Albumin

We report that incubation of acetaldehyde with bovine serum albumin results in the generation of acetate in a reaction that is directly proportional to the levels of albumin and exponentially dependent on the concentration of acetaldehyde. Both reactants need to be present for acetate to be formed. The oxidation of acetaldehyde into acetate requires that a reduced product also be generated in the reaction. It was hypothesized that, at high concentrations, acetaldehyde itself may reduce the Schiff bases formed in the reaction of a second molecule of acetaldehyde with amino groups in the protein, resulting in the generation of ethyl-lysine moieties. Incubation of acetaldehyde (240 mM) with bovine serum albumin was found to generate ethyl-lysine moieties as determined by a specific monoclonal antibody. Immunization of rabbits with products of the reaction of bovine serum albumin with acetaldehyde led to the generation of antibodies that reacted to reduced adducts formed in the reaction of acetaldehyde and proteins in the presence of sodium cyanoborohydride. However, the generation of acetate from acetaldehyde plus albumin was 60-fold greater than could be explained by the reduction of Schiff bases, as determined by the maximal incorporation of [ 14C]-acetaldehyde into an acid-precipitable protein fraction. Thus, other mechanisms to generate acetate also occur. The present findings provide an explanation for earlier reports that acetaldehyde adducts formed under “nonreducing” conditions generate antibodies that recognize reduced acetaldehyde protein adducts. However, the mechanism by which the bulk of acetate is generated in the reaction of acetaldehyde and bovine serum albumin remains to be elucidated.

Capabilities of polymer-modified monodisperse colloidal silica particles as biomaterial carrier

Polymer modification of monodispersed colloidal silica (0.5 µm) with poly(maleic anhydride-co-styrene) (P(MA-ST)) and poly (maleic anhydride-co-methyl methacrylate) (P(MA-MMA)) and application of the composite particle; to biomaterial carriers were investigated. The reaction of bovine serum albumin(BSA)-immobilized P(MA-MMA)/SiO2 with the anti-BSA antibody showed higher sensitivity in immunological agglutination test than BSA-P(MA-ST)/SiO2, though immobilization efficiency of BSA on P(MA-MMA)/SiO2 was lower than that on P(MA-ST)/SiO2. Alkaline phos-phatase and glucose oxidase immobilized on the composite particles exhibited extremely low activities, but a-chymotrypsin immobilized on P(MA-MMA)/SiO2 and its derivative particles showed the relative activity of 12.5% and 16.1% to the native enzyme, respectively. Grafting of a hydrophilic polymer of poly(acrylic acid) to P(MA-ST)/SiO2 let to an increase of the immobilized a-chymotrypsin activity to give the maximum relative activity of 55.5%.

Changes induced in bovine serum albumin following interactions with the lipid peroxidation product E-2-octenal

Bovine serum albumin (BSA) undergoes a number of deteriorative changes when exposed to E-2-octenal. Reaction of BSA with E-2-octenal produced fluorescent BSA with an excitation maximum at 350 nm and emission maximum at 440 nm and promoted polymerization. Amino acid analysis of the modified BSA showed that the E-2-octenal treatment leads to the selective loss of lysine residues and the formation of new amino acid derivatives. The same products were detected in acid hydrolysates of poly- l-lysine and N 2- (carbobenzyloxy)- l-lysine after their reactions with E-2-octenal. The reaction of N 2- (carbobenzyloxy)- l-lysine with E-2-octenal led to the production of 1-[N 2- (carbobenzyloxy)- l-lysyl ]-2- (1′- carboxymethyl)-4- pentylpyridinium betaine, 1-[N 2- (carbobenzyloxy)- l-lysyl ]-2- (3′- carboxy-2′-E- propen-1′- yl)-4- pentylpyridinium betaine and bis[1-[N' 2- (carbobenzyloxy)- l-lysyl ]-2- (3′- carboxy-2′- propen-1′,2′- diyl)-4- pentylpyridinium betaine] (isomeric mixture). Upon acid hydrolysis, these quaternary pyridinium salts led to new amino acid derivatives, presumably 1-( l-lysyl )-2-(1′- carboxymethyl)-4- pentylpyridinium betaine, 1-( l-lysyl )-2-(3′- carboxy-2′-E- propen-1′- yl) - 4-pentylpyridinium betaine and bis[1-( l-lysyl )-2-(3′- carboxy-2′- propen-1′,2′- diyl) - 4-pentylpyridinium betaine] (isomeric mixture) that were indistinguishable from those obtained from BSA, poly- l-lysine and N 2- (carbobenzyloxy)- l-lysine after similar treatment. The reaction of lysine residues with E-2-octenal provides the basis for methods by which the contributions of E-2-octenal in the modifications of proteins can be determined.

Modification of Histidine Residues in Bovine Serum Albumin by Reaction with (E)-2-Octenal

Reaction of bovine serum albumin (BSA) with the lipid peroxidation product (E)-2-octenal leads to the generation of (E)-2-octenal-BSA adducts. Amino acid analysis of the modified protein (after reduction with NaBH4) showed that histidine residues were major targets of the reaction and showed the formation of new amino acid derivatives. The same products were detected in acid hydrolysates of poly(L-histidine) and N-(carbobenzoxy)-L-histidine after their reactions with (E)-2-octenal and NaBH4. The reaction of N-(carbobenzoxy)-L-histidine with (E)-2-octenal led to the production of isomeric forms of N-(carbobenzoxy)-1(3)-[1' (formylmethyl)heyl]-L-histidine dihydrate. Upon acid hydrolysis, these compounds yielded stoichiometric amounts of histidine. However, after reduction with NaBH4, acid hydrolysis led to a mixture of amino acid derivatives [presumably, isomeric forms of 1(3)-[1'-(hydroyethyl)hexyl]-L-histidine] that were indistinguishable from those obtained from BSA, poly(L-histidine), and N-(carbobenzoxy)-L-histidine after similar treatment. Although other possibilities are not excluded, it is suggested that the modification of histidine residues in BSA by (E)-2-octenal involves a Michael-type addition of the imidazole nitrogen atom of histidine to the alpha,beta-unsaturated bond of (E)-2-octenal. The reaction of histidine residues with (E)-2-octenal provides the basis for methods by which the contributions of (E)-2-octenal in the modification of proteins can be determined.

Transformation of albumin into melanin by hydroxyl radicals

Bovine serum albumin (BSA) was exposed to hydroxyl radicals generated by the Fenton reaction. The reaction of the BSA solution with hydroxyl radicals resulted in a colour change from clear transparent to dark brown. The reaction was followed spectrophometrically. It was observed that during the reaction of BSA with hydroxyl radicals, a melanin-like absorption spectrum developed. The reaction with ferricyanide and the dark brown BSA solution resulted in the same dark blue-green colour as is typical for melanin. The results suggest that, by the reaction of BSA with hydroxyl radicals, melanin was formed.

Acrylodan can label amino as well as sulfhydryl groups: Results with low-density lipoprotein, lipoprotein[a], and lipid-free proteins

Human plasma lipoprotein[a] and autologous low-density lipoprotein were reacted with the fluorescent probe 6-acryloyl-2-(dimethylamino)naphthalene (acrylodan) previously reported to be specific for sulfhydryl groups. Reaction kinetics were biphasic in both cases. The reaction of bovine serum albumin with acrylodan was also biphasic. Monophasic kinetics were observed when protein free sulfhydryl groups were blocked by carboxamidomethylation prior to acrylodan reaction. A significant increase in total fluorescence was observed in the reaction of acrylodan with proteins containing no free sulfhydryl groups and with polylysine. The rates of these reactions were highly sensitive to pH. Fluorescence changes due to dissolution of probe into hydrophobic protein or lipid domains were minimal as was reaction of probe with phospholipid head groups. When isolated from acrylodan-labeled Lp[a], apo[a], which contains no free sulfhydryl groups, contained covalently bound acrylodan. These results suggest that acrylodan can modify the lysine residues of lipid-free proteins and may modify not only the free sulfhydryl groups of low-density lipoprotein and lipoprotein[a] but also reactive amino groups. We conclude that under these conditions, the use of this probe to quantify free sulfhydryl groups in these lipoproteins is infeasible.

Effects of 3-deoxyglucosone on the Maillard reaction

We investigated the effect of exogenously applied 3-deoxyglucosone, a major carbonyl intermediate, on the Maillard reaction. The fluorescence intensity of the product of the reaction of bovine serum albumin with 3-deoxyglucosone was higher than that with an equivalent amount of glucose. Similarly the rate of polymerization of lysozyme in the presence of 3-deoxyglucosone was also greater than with glucose, and collagen incubated with 3-deoxyglucosone was less digestible than collagen incubated with glucose. By contrast, aminoguanidine inhibited an increase in fluorescence of the Maillard compounds and the polymerization of protein, both of which were stimulated by 3-deoxyglucosone. These results suggest that 3-deoxyglucosone accelerates the advanced stage of the Maillard reaction and that aminoguanidine acts on 3-deoxyglucosone to inhibit its action in the advanced stage of the Maillard reaction.

Interaction of cinnamaldehyde (a sensitizer in fragrance) with protein.

Formation of a cinnamaldehyde-protein conjugate in the skin by reaction of cinnamaldehyde with nucleophilic groups in proteins is considered to be responsible for the observed immunogenicity. The kinetics of the reaction of bovine serum albumin with cinnamaldehyde in aqueous solution at 30 degrees C have been investigated, and the rate of formation of conjugate over the pH range 6.4-10.5 showed a first-order dependence on cinnamaldehyde concentration. The cinnamaldehyde binding sites on the protein appeared mostly or exclusively to be the thiol groups of cysteine residues.

Maillard-reaktion von rinderserumalbumin mit glucose hochleistung-flüssigkeitschromatographischer nachweis des 2-formyl-5-(hydroxymethyl)pyrrol-1-norleucins nach alkalischer hydrolyse

Maillard reaction of bovine serum albumin with glucose. Determination of 2- formyl-5-(hydroxymethyl)pyrrole-1-norleucine by high-performance liquid chromatography after alkaline hydrolysis Reactions between glucose and bovine serum albumin proceed predominantly at the side chain amino groups of lysine residues. Among other products, proteinbound 2-formyl-5-(hydroxymethyl)pyrrole-1-norleucine is formed. After alkaline hydrolysis and fractionation of the protein hydrolysate on RP-18 material this substance can be separated by high-performance liquid chromatography. The identity of the norcleucine derivative with a synthesized compound can be determined with fast atom bombardment-mass spectrometry spectral data. A colour reaction with thiobarbituric acid is also suitable for detection.