Comparison of bovine serum albumin glycation by ribose and fructose in vitro and in vivo

Advanced glycation end products (AGEs) play a critical pathogenic role in the development of diabetic complications. Recent studies have shown that diabetes is associated with not only abnormal glucose metabolism but also abnormal ribose and fructose metabolism, although glucose is present at the highest concentration in humans. The glycation ability and contribution of ribose and fructose to diabetic complications remain unclear. Here, the glycation ability of ribose, fructose and glucose under a mimic physiological condition, in which the concentration of ribose or fructose was one-fiftieth that of glucose, was compared. Bovine serum albumin (BSA) was used as the working protein in our experiments. Ribose generated more AGEs and was markedly more cytotoxic to SH-SY5Y cells than fructose. The first-order rate constant of ribose glycation was found to be significantly greater than that of fructose glycation. LC-MS/MS analysis revealed 41 ribose-glycated Lys residues and 12 fructose-glycated residues. Except for the shared Lys residues, ribose reacted selectively with 17 Lys, while no selective Lys was found in fructose-glycated BSA. Protein conformational changes suggested that ribose glycation may induce BSA into amyloid-like monomers compared with fructose glycation. The levels of serum ribose were correlated positively with glycated serum protein (GSP) and diabetic duration in type 2 diabetes mellitus (T2DM), respectively. These results indicate that ribose has a greater glycation ability than fructose, while ribose largely contributes to the production of AGEs and provides a new insight to understand in the occurrence and development of diabetes complications.     

Site specificity of glycation and carboxymethylation of bovine serum albumin by fructose

Summary. We report an investigation of the site specificity, extent and nature of modification of bovine serum albumin (BSA) incubated with fructose or glucose at physiological temperature and pH. Sites of early glycation (Heyns rearrangement products (HRP) from fructose; fructoselysine (FL) from glucose) as well as advanced glycation (N^ε-(carboxymethyl)lysine; CML) were analyzed by liquid chromatography-mass spectrometry. The major site of modification by fructose, like glucose, is Lysine-524 and this results in, respectively, 31 and 76% loss of the corresponding unmodified tryptic peptide, Gln525-Lys533. In addition, total lysine, HRP, FL, CML and N^ε-(carboxyethyl)lysine in the incubations, was quantified. Almost all of the loss of lysine in the fructose-modified BSA was attributed to the formation of CML, with the yield of CML being up to 17-fold higher than glucose-modified BSA. A mechanism for the formation of CML from the HRP is proposed.     

Comparison of glycation of glutathione S-transferase by methylglyoxal, glucose or fructose

Glycation is a process closely related to the aging and pathogenesis of diabetic complications. In this process, reactive α-dicarbonyl compounds (e.g., methylglyoxal) cause protein modification accompanied with potential loss of their biological activity and persistence of damaged molecules in tissues. We suppose that glutathione S-transferases (GSTs), a group of cytosolic biotransformation enzymes, may be modified by glycation in vivo, which would provide a rationale of its use as a model protein for studying glycation reactions. Glycation of GST by methylglyoxal, fructose, and glucose in vitro was studied. The course of protein glycation was evaluated using the following criteria: enzyme activity, formation of advanced glycation end-products using fluorescence and western blotting, amine content, protein conformation, cross linking and aggregation, and changes in molecular charge of GST. The ongoing glycation by methylglyoxal 2 mM resulted in pronounced decrease in the GST activity. It also led to the loss of 14 primary amino groups, which was accompanied by changes in protein mobility during native polyacrylamide gel electrophoresis. Formation of cross links with molecular weight of 75 kDa was observed. Obtained results can contribute to understanding of changes, which proceed in metabolism of xenobiotics during diabetes mellitus and ageing.     

Kinetics of the glycation of bovine serum albumin by mannose and fucose in vitro

Glycation of bovine serum albumin was measured for mannose and fucose at 37 degrees C. Mannose as well as fucose demonstrated an initial rapid increase in rate of formation of total adducts followed by a slower secondary reaction. The equilibrium constant for Schiff base formation was almost two times larger for mannose than fucose, although the Schiff base formed by fucose rearranged 1.5 times faster than that for mannose. Both sugars showed parallel lines for the formation of total and acid stable products after three hours. Discussion integrates new mechanistic data with previously suggested mechanisms.     

Maillard induced complexes of bovine serum albumin - a dilute solution study

Association of bovine serum albumin (BSA) on heating in the presence and absence of 2% xylose has been studied using dynamic light scattering and sedimentation velocity. When 3% solutions of the protein alone are heated at 95°C association products are formed with molar masses of 2 × 10⁶g/mol, a value which is independent of the time of heating. These aggregates can be dissociated in solvents that disrupt non-covalent bonds. When the reducing sugar xylose is present there is a continuous change in the hydrodynamic properties with time. After 80 min a molar mass in excess of 7 × 10⁶g/mol is obtained. This increase in molar mass is attributed to additional non-disulphide linkages resulting from the Maillard reaction. Information about the gross conformation of the Maillard induced association products has been obtained from MHKS (Mark-Houwink-Kuhn-Sakarada) double logarithmic plots of D_20,w and s_20,w against molar mass. The values of the MHKS coefficients obtained are most consistent with a linear rod: i.e. the association is of an end-to-end type     

Oxidation of bovine serum albumin initiated by the Fenton reaction--effect of EDTA, tert-butylhydroperoxide and tetrahydrofuran

Oxidation of bovine serum albumin (BSA) was investigated using different oxidants: The water-soluble azo-initiator 2,2'azo-bis-(2-amidinopropane) hydrochloride (AAPH), a combination of FeCl₃ and ascorbate or the Fenton oxidant consisting of FeCl₂, H₂O₂ and EDTA. In addition, the effects of exogenous compounds such as tert-butyl hydroperoxide (tBuOOH) or solvents such as tetrahydrofuran (THF), often used in model systems, was evaluated. The extent of protein damage was studied by measuring protein carbonyl groups and protein hydroperoxides. The interaction between Fenton oxidant and EDTA, THF or tBuOOH was further characterized using spin trapping electron spin resonance (ESR) spectroscopy. The results showed that the extent of protein oxidation depended on the oxidant used. The Fenton oxidant was the most reactive of the initiators tested. However, in the absence of EDTA, the Fenton system produced protein carbonyl groups on BSA equivalent to that obtained with the other oxidants, however, significantly more protein hydroperoxide was produced. Surprisingly, it was also found that addition of tBuOOH or THF to BSA reduced protein damage when the oxidation was initiated with the Fenton oxidant. ESR investigation showed that EDTA played a key role in the generation of free radicals. It was also revealed that in an EDTA containing system both tBuOOH and THF were able to react with radicals without inducing protein damage in effect protecting BSA from oxidative damage.     

Characterization of bovine serum albumin glycated with glucose, galactose and lactose

The non-enzymatic reaction between reducing sugars and proteins, known as glycation, has received increased attention from nutritional and medical research. In addition, there is a large interest in obtaining glycoconjugates of pure well-characterized oligosaccharides for biological research. In this study, glycation of bovine serum albumin (BSA) by d-glucose, d-galactose and d-lactose under dry-heat at 60 degrees C for 30, 60, 120, 180 or 240 min was assessed and the glycated products studied in order to establish their biological recognition by lectins. BSA glycation was monitored using gel electrophoresis, determination of available amino groups and lectin binding assays. The BSA molecular mass increase and glycation sites were investigated by mass spectrometry and through digestion with trypsin and chymotrypsin. Depending on time and type of sugar, differences in BSA conjugation were achieved. Modified BSA revealed reduction of amino groups' availability and slower migration through SDS/PAGE. d-galactose was more reactive than d-glucose or d-lactose, leading to the coupling of 10, 3 and 1 sugar residues, respectively, after 120 minutes of reaction. BSA lysines (K) were the preferred modified amino acids; both K256 and K420 appeared the most available for conjugation. Only BSA-lactose showed biological recognition by specific lectins.     

Interaction of ergosterol with bovine serum albumin and human serum albumin by spectroscopic analysis

This study was designed to examine the interactions of ergosterol with bovine serum albumin (BSA) and human serum albumin (HSA) under physiological conditions with the drug concentrations in the range of 2.99-105.88?μM and the concentration of proteins was fixed at 5.0?μM. The analysis of emission spectra quenching at different temperatures revealed that the quenching mechanism of HSA/BSA by ergosterol was the static quenching. The number of binding sites n and the binding constants K were obtained at various temperatures. The distance r between ergosterol and HSA/BSA was evaluated according to F?ster non-radioactive energy transfer theory. The results of synchronous fluorescence, 3D fluorescence, FT-IR, CD and UV-Vis absorption spectra showed that the conformations of HSA/BSA altered in the presence of ergosterol. The thermodynamic parameters, free energy change (ΔG), enthalpy change (ΔH) and entropy change (ΔS) for BSA-ergosterol and HSA-ergosterol systems were calculated by the van't Hoff equation and discussed. Besides, with the aid of three site markers (for example, phenylbutazone, ibuprofen and digitoxin), we have reported that ergosterol primarily binds to the tryptophan residues of BSA/HSA within site I (subdomain II A).     

o-Quinones formed in plant extracts. Their reaction with bovine serum albumin

1. The reactions between chlorogenoquinone, the o-quinone formed during the oxidation of chlorogenic acid, and bovine serum albumin depend on the ratio of reactants. 2. When the serum albumin is in excess, oxygen is not absorbed and the products are colourless. This reaction probably involves the thiol group of bovine serum albumin; it does not occur with bovine serum albumin which has been treated with p-chloromercuribenzoate, iodoacetamide or Ellman's reagent. 3. When bovine serum albumin reacts with excess of chlorogenoquinone, oxygen is absorbed and the products are red. The red colour is probably formed by reaction of the lysine in-amino groups of bovine serum albumin, as it is prevented by treating the protein with formaldehyde, succinic anhydride or O-methylisourea. 4. Bovine serum albumin modified by a 1.5-fold (BSA-Q) and a fivefold (BSA-Q2) excess of chlorogenoquinone were separated by chromatography on DEAE-Sephadex A-50, and some of their properties observed. 5. Reaction of BSA-Q2 with fluorodinitrobenzene suggests that the terminal alpha-amino group, as well as lysine in-amino groups, are combined with chlorogenoquinone.     

Monitoring the formation of Maillard reaction products of glucosamine with fibrinogen and human serum albumin using capillary electrophoresis

Formation of Maillard reaction products (MRP) of glucosamine (GlcN) with fibrinogen and human serum albumin (HSA), under simulated physiological conditions, was detected by fluorescence (excitation/emission: 340/420 nm) and UV/Vis (max. 275 nm) spectroscopy. The application of polyacrylamide gel electrophoresis demonstrated the generation of high-molecular-weight fibrinogen and HSA MRP by GlcN. A simple and rapid capillary electrophoresis method was developed to separate MRP formed by the reaction of GlcN with proteins from GlcN autocondensation products.     

Maillard reaction of ribose 5-phosphate generates superoxide and glycation products for bovine heart cytochrome c reduction

Ribose 5-phosphate (R5P) is a sugar known to undergo the Maillard reaction (glycation) at a rapid rate. In a reaction with the lysines of bovine heart cytochrome c, R5P generates superoxide () that subsequently reduces ferri-cytochrome c to ferro-cytochrome c. The rate equation for the observed cytochrome c reduction is first order in respect to cytochrome c and half order in respect to R5P. The addition of amines to the cytochrome c-R5P system greatly increases the generation with rates of approximately 1.0 μM min⁻¹ being observed with millimolar levels of R5P and amine at 37 °C. Pre-incubation of R5P with the amine prior to cytochrome c addition further enhances the rate of cytochrome c reduction approximately twofold for every 30 min of incubation. While clearly accounting for a portion of the reduction of cytochrome c, is not the sole reductant of the system as the use of superoxide dismutase only partially limits cytochrome c reduction, and the contribution of proportionally decreases with longer amine-R5P incubation times. The remainder of the cytochrome c reduction is attributed to either the Amadori product or a cross-linked Schiff base created when a Maillard reaction-derived dicarbonyl compound(s) reacts with the amine. It is believed that these compounds directly transfer electrons to ferri-cytochrome c and subsequently become stable free-radical cations. ATP, a putative regulator of cytochrome c activity, does not inhibit electron transport from or the cross-linked Schiff base but does prevent R5P from reacting with surface lysines to generate superoxide. The spontaneous reaction between R5P and amines could serve as an alternative system for generating in solution.     

Interaction of allylisothiocyanate with bovine serum albumin

The interaction of allylisothiocyanate with bovine serum albumin was monitored by fluorescence titration. The interaction was weak with an apparent association constant of 2 × 10². The interaction was unaffected in the pH range of 5.0 to 8.3 and by NaCl. However, the addition of dioxane upto 4% increased the value of the association constant. N-Methyl bovine serum albumin and bovine serum albumin with sulphydryl groups blocked had the same affinity for allylisothiocyanate suggesting that amino and sulphydryl groups may not be involved in the interaction. Polyacrylamide gel electrophoresis and estimation of available lysine suggested that there were perhaps two types of groups involved in the interaction of allylisothiocyanate with bovine serum albumin. An erratum to this article is available at .     

Interactions of cannabinoids with bovine serum albumin

There is no shift of emission maximum (F₄₇₀nm) of bovine serum albumin (BSA)-l-anilino-8-naphthatene sulphonic acid (ANS) complex in the pesence of delta-9-tetrahydrocannabinol (delta-9-THC) alone and cannabidiol (CBD) or cannabinol (CBN) in the presence and absence of delta-9-THC. Delta-9-THC (1.66–13.33 M) and CBD at higher concentrations (13.33–20.0 M) produce a concentration-dependent significant quenching of fluorescence of BSA-ANS complex, but CBN (l.66–20.0 M) as well as CBD at lower concentrations (1.66–6.66 M) fails to produce any significant change in Iluorescence intensity under similar conditions. Furthermore, neither CBD nor CBN is able to affect the delta-9-THC-induced quenching of fluorescence intensity of BSA-ANS complex. These results indicate that the binding of cannabinoids to the ANS binding sites of BSA molecule are in the order detta-9-THC > CBr3 > CBN, and CBD or CBN does not have any influence on the binding of delta-9-THC to BSA molecules under these conditions.     

Interaction of wogonin with bovine serum albumin

The binding of wogonin with bovine serum albumin (BSA) was investigated at different temperatures by fluorescence, circular dichroism (CD) and Fourier transform infrared spectroscopy (FT-IR) at pH7.40. The association constants K were determined by Stern-Volmer equation based on the quenching of the fluorescence of BSA in the presence of wogonin, which were in agreement with the constants calculated by Scatchard plots. The thermodynamic parameters were calculated according to the Van't Hoff equation and the result indicated that DeltaH(0) and DeltaS(0) had a negative value (-12.02 kJ/mol) and a positive value (58.72 J/mol K), respectively. On the basis of the displacement experimental and the thermodynamic results, it is considered that wogonin binds to site I (subdomain IIA) of BSA mainly by hydrophobic interaction. The studied results by FT-IR and CD experiment indicated that the secondary structures of protein have been perturbed by the interaction of wogonin with BSA.     

Conjugate of bovine serum albumin with nicotine

The preparation and characterization of a nicotine-albumin conjugate are reported. The nicotine derivative, 6-(p-aminobenzamido)nicotine, which was coupled to bovine serum albumin(BSA), was synthesized by the following sequence; $\text{1}$-nicotine→6-aminonicotine→6-(p-nitrobenzamido)nicotine→6-(p-aminobenzamido)nicotine. Thirty molecules of conjugated nicotine per molecule of BSA were distinguished by the determination of 6-aminonicotine after hydrolysis. The distribution of the conjugated nicotine on BSA was examined by the stoichiometry of the composed amino acids before and after conjugation, and also by the electrophoretic behavior of the conjugated BSA on a polyacrylamide gel.     

Investigation of the antigen antibody reaction between anti-bovine serum albumin (a-BSA) and bovine serum albumin (BSA) using piezoresistive microcantilever based sensors

A new microsensor application based on piezoresistive microcantilever technology has been used to study the interaction of anti-bovine serum albumin (a-BSA) with bovine serum albumin (BSA). A thin layer of BSA attached to a glass slide was used as the active sensing layer for the detection of a-BSA in solution. This design produced a large, consistent cantilever deflection when exposed to the analyte. In this system, the cantilever deflection is measured as a simple resistance change in the piezoresistive channel within the cantilever. In a second set of experiments, 3:1 BSA:PEO protein/polymer blended substrates were used as the active sensing layer for the detection of a-BSA in an aerosol delivery. A distinct signature for the analyte, separate from the water vapor carrier, is obtained for this system.     

Induction of GM-CSF production of macrophages by advanced glycation end products of the Maillard reaction

We previously reported that AGEs can induce macrophage growth. In this paper, we examined whether advanced glycation end products (AGE) of protein induced GM-CSF production of macrophages. AGE of bovine serum albumin markedly stimulated not only the expression of GM-CSF mRNA, but also GM-CSF secretion in macrophage supernatant. Thus GM-CSF is suggested to be an endogenous signal for macrophage growth induction by AGEs.