Reaction Of Human Serum Albumin Research Articles

2 - The microenvironment of the sulfenic acid of human serum albumin: In search of clues for explaining its spontaneous decay

Human serum albumin (HSA) is the most abundant protein in plasma. It consists of a single, non-glycosylated polypeptide chain of 585 residues. Only one free cysteine, namely Cys34, is present in HSA structure. With a concentration of ∼500 μM, Cys34 constitutes more than 80 % of the plasma free thiol pool. HSA has many functions, such as transporting small molecules, maintaining the oncotic pressure and scavenging oxidant/electrophilic species. The latter is associated with Cys34 thiol, hereto identified as HSA-SH. Reaction of HSA with a two-electron oxidant (e.g. H2O2, HOONO, HOCl) leads to formation of a sulfenic acid, HSA−SOH, with a relatively long half-life [1]. HSA−SOH can follow three possible destinations: (a) reaction with a free thiol (e.g. free Cys) to give a mixed disulfide (HSA-SS-R); (b) reaction with a two-electron oxidant to yield a sulfinic acid (HSA−SO2H); and (c) spontaneously decay to an uncharacterized product, named HSA−SX, with a rate constant of ca. 10−3 s−1 (37 °C, pH 7.4).[2]

Fluid mixing using AC electrothermal flow on meandering electrodes in a microchannel

The mixing of fluids using AC electrothermal flow (AC-ETF) is presented. A pair of coplanar electrodes with a sinusoidal interelectrode gap was used to enhance the mixing in a microchannel. To demonstrate the performance of the mixer, conventional dilution experiments were conducted using Texas Red-labeled dextran. The dependence of mixing on the salt concentration (10(-3) ∼ 10(-1) mol dm(-3) ) of the solutions and frequency (100 kHz ∼ 5 MHz) of the applied voltage were investigated. AC-ETF was responsible for the mixing at salt concentrations >10(-2) mol dm(-3) , whereas the effect of AC-EOF was suggested to play a role at concentrations <10(-2) mol dm(-3) in the low-frequency region. The fluorogenic reaction of human serum albumin (HSA) with SYPRO Red in the mixer was also examined, and results showed that enrichment of fluorescence intensity and an almost uniform distribution of stained HSA were achieved. The present mixer can be employed as a powerful tool to facilitate efficient chemical and biomedical analysis on microfluidic devices.

Proteomics Study on Cross-Linking Reaction of Formaldehyde and HSA

Possible clinical diagnosis biomarkers of diabetes were proposed using proteomics and bioinformatics methods through the preliminary study on the formaldehyde-human serum albumin crosslinkage. Using LC/MS techniques, the experiment on in vivo cross-linking reaction of formaldehyde with pure protein or plasma protein revealed that the cross-linking reaction of human serum albumin and forrmaldehyde would occur and the concentrations of some normal peptides would change. A group of significant peptides of human serum albumin were selected. As the results of in vivo cross-linking reaction, Non-enzymatic glycosylation modification of HSA were observed in diabetic. The same change of the concentrations of some peptides happened. The above-mentioned results were demonstrated with the hematoplasmas of normal people and diabetics.

Evidence of changes in hydrophilic/hydrophobic balance and in chemical activity of HSA induced by thermal treatments

Abstract Samples of human serum albumin (HSA) obtained as a result of heat denaturation followed by refolding controlled by a cooling of the protein solution were studied by several methods: chromatographic measurements, kinetic of the reaction with a water soluble free radical and by electron paramagnetic resonance (EPR) spectroscopy. In this context the interaction of this protein with β-cyclodextrin (β-CD) and sodium dodecyl sulfate (SDS) was also investigated. Reversed phase thin layer chromatography (RP-TLC) showed changes in lipophylicity of HSA, which are related with the existence of different ensembles of conformers. The UV-Vis absorption spectra had shown the broadening of absorption band of the protein and a hyperchrom effect in the presence of SDS; β-CD reduces the effect of SDS on protein UV-Vis spectra. Kinetic measurements related to the reaction of HSA with a water soluble DPPH type free radical provided evidence that reactivity of the HSA denaturated conformers is higher compared with the natural conformer. The affinity of SDS to the albumins surface and the effect of β-CD on the SDS/protein aggregates were also evident by changes in the EPR spectra of the spin probe CAT16.

Homocystamides promote free-radical and oxidative damage to proteins.

Elevated levels of homocysteine are associated with several major diseases. However, it is not clear whether homocysteine is a marker or a causative agent. The majority (ca. 80%) of the homocysteine present in humans is protein bound. The study of the posttranslational modification of proteins by homocysteine and its cyclic congener, homocysteine thiolactone, is emerging as an area of great current interest for unraveling the ongoing "mediator/marker controversy" [Jacobsen DW (2009) Clin Chem 55:1-2]. Interestingly, many of the pathologies associated with homocysteine are also linked to oxidative stress. In the current study, chemical evidence for a causal relationship between homocysteine-bound proteins and oxidative damage is presented. For example, a reproducible increase in protein carbonyl functionality occurs as a consequence of the reaction of human serum albumin with homocysteine thiolactone. This occurs at physiological temperature upon exposure to air without any added oxidants or free-radical initiators. Alpha-amino acid carbon-centered radicals, well-known precursors of protein carbonyls, are shown to form via a hydrogen atom transfer process involving thiolactone-derived homocystamides. Model peptides in buffer as well as native proteins in human blood plasma additionally exhibit properties in keeping with the homocystamide-facilitated hydrogen atom transfer and resultant carbon-centered radicals.

Direct observation of covalent adducts with Cys34 of human serum albumin using mass spectrometry

The interactions of the unpaired thiol residue (Cys34) of human serum albumin (HSA) with low-molecular-weight thiols and an Au(I)-based antiarthritic drug have been examined using electrospray ionization mass spectrometry. Early measurements of the amount of HSA containing Cys34 as the free thiol suggested that up to 30% of circulating HSA bound cysteine as a mixed disulfide. It has also been suggested that reaction of HSA with cysteine, occurs only on handling and storage of plasma. In our experiments, there were three components of HSA in freshly collected plasma from normal volunteers, HSA, HSA + cysteine, and HSA + glucose in the ratio ∼50:25:25. We addressed this controversy by using iodoacetamide to block the free thiol of HSA in fresh plasma, preventing its reaction with plasma cysteine. When iodoacetamide was injected into a vacutaner tube as blood was collected, the HSA was modified by iodoacetamide, with 20–30% present as the mixed disulfide with cysteine (HSA + cys). These data provide strong evidence that 20–30% of HSA in normal plasma contains one bound cysteine. Reaction of HSA with [Au(S 2O 3) 2] 3− resulted in formation of the adducts HSA + Au(S 2O 3) and HSA + Au. Reaction of HSA with iodoacetamide prior to treatment with [Au(S 2O 3) 2] 3− blocked the formation of gold adducts.

Identification of subdomain IB in human serum albumin as a major binding site for polycyclic aromatic hydrocarbon epoxides.

Covalent adducts between serum albumin and low molecular weight organic electrophiles are formed with a high degree of regioselectivity mostly for nucleophilic amino acid residues located in subdomains IIA and IIIA. Previous studies have indicated that diol epoxide metabolites of polycyclic aromatic hydrocarbons (PAH) may target residues in a different subdomain. The regioselectivity of PAH epoxide and diol epoxide binding was examined in this study by reaction of human serum albumin in vitro with the racemic trans,anti-isomers of 7,8-dihydrobenzo[a]pyrene-7,8-diol 9,10-epoxide (1), 2,3-dihydrofluoranthene-2,3-diol 1,10b-epoxide (2), 1,2-dihydrochrysene-1,2-diol 3,4-epoxide (5), 6-methyl-1,2-dihydrochrysene-1,2-diol 3,4-epoxide (6), 5-methyl-1,2-dihydrochrysene-1,2-diol 3,4-epoxide (7), 3,4-dihydrobenzo[c]phenanthrene-3,4-diol 1,2-epoxide (8), 11,12-dihydrobenzo[g]chrysene-11,12-diol 13,14-epoxide (9), and 11,12-dihydrodibenzo[a,l]pyrene-11,12-diol 13,14-epoxide (10) and the racemic epoxides cyclopenta[cd]pyrene 3,4-epoxide (3) and benzo[a]pyrene 4,5-epoxide (4) followed by determination of the linkage site. Adducted albumin was digested enzymatically, and digests were chromatographed by reversed-phase HPLC to purify peptide adducts, which were analyzed by electrospray ionization collision-induced dissociation (CID) tandem mass spectrometry. Product ion spectra revealed that adducts fragmented predominantly by cleavage of the peptide-PAH bond with retention of charge by the peptide as well as by the hydrocarbon. Peptide sequences were determined by MS/MS analysis of the peptide ions formed by in-source CID to cleave the adduct bond. Longer peptide sequences established site selectivity by virtue of their uniqueness, while shorter sequences revealed the reactant amino acid within the site. Epoxide 4 and diol epoxides 1, 2, 5, and 6 reacted predominantly with His146; epoxide 3 and diol epoxides 7-9 reacted predominantly with Lys137. Both residues are situated in subdomain IB. The binding site for 10 could not be determined uniquely, but one of the several possibilities was Lys159, which is also located in subdomain IB. The results, taken together with previous findings, demonstrate that the reaction of polycyclic aromatic hydrocarbon epoxides with human serum albumin is highly selective for a small number of residues in subdomain IB.

The application of adsorptive stripping voltammetry for a study of the reaction of human serum albumin with anti-human serum albumin

Adsorptive stripping voltammetry has been used to study the interaction of human serum albumin with anti-human serum albumin. The adsorptive stripping voltammetric behaviour of both proteins has been optimised with respect to accumulation potential, accumulation time and scan rate. It has been demonstrated that adsorptive stripping voltammetry can be a useful technique for the study of the interaction of these two proteins directly in solution.

Reaction of human serum albumin with aldoses

The reaction of human serum albumin (HSA) with aldoses (C 3-C 6) and acetaldehyde has been studied. U.v. and fluorescent spectra of the HSA-glyceraldehyde and HSA-GlcN adducts reveal yellow chromophores absorbing at 300–350 nm and emitting at 435 nm. However, even limited reaction of HSA with acetaldehyde induced perturbation in the Trp microenvironment. C.d. spectra of the adducts show an average 20% decrement in mean residual ellipticity [θ], which is independent of the extent of the reaction and the aldose used. It is concluded that most of the reactions with aldoses occur at the surface of the HSA molecule. With the exception of the GlcN adduct, the HSA adducts rearrange to produce pyrrole rings on the protein surface. I.e.f. analysis shows that the pI values of the modified HSA are almost linearly correlated with the chain length of the reacting aldose: from pI 4.2 for HSA-glyceraldehyde up to pI 5.0 for HSA-GlcN.

Anthraniloyl-tyrosine 411 as a spectroscopic probe of fatty acid binding to human serum albumin.

Reaction of human serum albumin with p-nitrophenylanthranilate results in transesterification of the anthraniloyl group to tyrosine 411. Titration of anthraniloyl-Tyr-411-albumin with long chain or short chain fatty acids produces marked changes in the absorption and fluorescence spectra of the anthraniloyl moiety as fatty acids bind in the channel near it. It appears that the anthraniloyl group is a very sensitive probe that can follow binding of small molecules at the 3-AB subdomain of human serum albumin.

Kinetics of the Rapid Modification of Human Serum Albumin with Trinitrobenzenesulfonate and Localization of Its Site

□ The rapid reaction of human serum albumin with trinitrobenzenesulfonate (I) and the location of the reactive site were investigated to characterize the chemical modification of albumin by I. The modification proceeds through trinitrophenylation of a lysine residue of albumin and monoaddition of the byproduct, sulfite ion, to the trinitrophenylalbumin, as reported previously. The individual kinetic parameters for both reactions were determined at various pH values and 25°. The ϵ-amino group of the lysine residue which has a pKa value of ~8.9 was the reactive group involved in the trinitrophenylation. The dissociation constant of the sulfite monoadduct was about 10-fold smaller than that of the monoadduct of the model compound trinitrophenyl α-acetyllysine. The modification of albumin by I reduced the fluorescence intensity of the tryptophan-214 residue in the albumin amino acid sequence. Acetylation of the lysine-199 residue with aspirin and 5-nitroaspirin decreased the trinitrophenylation rate of albumin with I. These results on the fluorescence spectroscopy and the effect of the acetylation suggest that the reactive group for I is the lysine-199 residue located near the tryptophan-214 residue.

Reaction of human serum albumin and human erythrocytes with tritiated 2,4,6-trinitrobenzenesulfonic acid and tritiated picryl chloride

1. 1. Syntheses for tritiated 2,4,6-trinitrobenzenesulfonic acid (TNBS) and [ 3H]-picryl chloride (PC) are described. 2. 2. The time course of the reaction of [ 3H]TNBS and [ 3H]PC with human serum albumin was studied over a 4-h interval. At 30 min both reagents had reacted to the extent of 11.3 moles per mole of albumin. At 4 h approximately 17 moles of TNBS and approximately 33 moles of PC had reacted per mole of serum albumin. 3. 3. The time course of the reactions of [ 3H]TNBS and [ 3H]PC with normal human erythrocyte suspensions in phosphate buffer were studied. Distribution of label in the medium, in the cell membrane, and in the hemolysate hemoglobin was examined as a function of time for each reagent. The dynamic labeling system which emerges from this analysis discloses a source of reagent outside the membrane, reaction of reagent with the membrane while in transit, and exhaustion of free reagent by the hemoglobin sink inside the erythrocytes. The [ 3H]PC was found to enter the erythrocytes and label their membranes much more rapidly but the specific activity in the membranes was only about half of that achieved with the [ 3H]TNBS.

Reaction of Human Serum Albumin with Hæmatin and Hæm

IT was shown by Heilmeyer1 that the pigment in the blood serum of certain cases of pernicious anaemia accompanied by a severe "haematin jaundice" or "haematinaemia" differs from free alkaline haematin in that the absorption band is at about 620 mμ instead of at 610 mμ. As this pigment Was also obtained by Heilmeyer2 in vitro by adding haematin to blood serum, he concluded that "in serum the haematin combines with proteins which are only split off on adding an excess of concentrated alkali. Thus in serum we are concerned with the spectrum of a protein-haematin and not that of alkaline haematin".