Presence Of Human Serum Albumin Research Articles

Impact of human serum albumin on oritavancin in vitro activity against Staphylococcus aureus

Human serum albumin (HSA) did not affect oritavancin MICs against non–vancomycin-intermediate Staphylococcus aureus (non-VISA) strains. In time–kill assays, oritavancin bactericidal activity in the presence of HSA was significantly more rapid than comparators against non-VISA strains. HSA increased oritavancin MICs by 4-fold for VISA strains, reflective of reduced oritavancin activity in time–kill assays with HSA.

Voltammetric and spectroscopic investigations on the mechanism of interaction of buzepide methiodide with protein

Adsorption or immobilization of proteins on solid surfaces promotes the biological responses to materials. Using immobilization technique, we have prepared human serum albumin (HSA) modified glassy carbon electrode (GCE) and employed it to probe the mode of interaction between antidepressant drug, buzepide methiodide (BZP) and HSA. At HSA modified GCE, the peak potential of BZP appeared at more positive potential compared to that at bare electrode thereby indicating the hydrophobic mode of interaction between BZP and HSA. Peak currents of BZP decreased upon the addition of HSA at bare GCE with positive shift in peak potential. Further, no new peaks were observed in presence of HSA. From electrochemical data, the binding constant and binding ratio between HSA and BZP were calculated to be 9.33 × 10 6 M −1 and 1:2, respectively. FT-IR and circular dichroism (CD) studies revealed that the secondary structure of protein was perturbed upon interaction with BZP.

A New Gadolinium-Based MRI Zinc Sensor

The properties of a novel Gd(3+)-based MRI zinc sensor are reported. Unlike previously reported Gd(3+)-based MRI contrast agents, this agent (GdL) differs in that the agent alone binds only weakly with human serum albumin (HSA), while the 1:2 GdL:Zn(2+) ternary complex binds strongly to HSA resulting in a substantial, 3-fold increase in water proton relaxivity. The GdL complex is shown to have a relatively strong binding affinity for Zn(2+) (K(D) = 33.6 nM), similar to the affinity of the Zn(2+) ion with HSA alone. The agent detects as little as 30 microM Zn(2+) in the presence of HSA by MRI in vitro, a value slightly more than the total Zn(2+) concentration in blood (approximately 20 microM). This combination of binding affinity constants and the high relaxivity of the agent when bound to HSA suggests that this new agent may be useful for detection of free Zn(2+) ions in vivo without disrupting other important biological processes involving Zn(2+).

Photonucleophilic Addition of the ε‐Amino Group of Lysine to a Triflusal Metabolite as a Mechanistic Key to Photoallergy Mediated by the Parent Drug

A mechanism for triflusal-induced photoallergy involving complexation of 2-hydroxy-4-trifluoromethylbenzoic acid with site I of human serum albumin and subsequent formation of a covalent adduct by photoreaction between a metabolite and a neighboring lysine residue is proposed. This is supported by the observed photobinding to poly-L-lysine. Thereby, a photoantigen is generated, which is a likely trigger of the immune response.The goal of the work presented herein is to gain deeper insight into the molecular basis of photoallergy mediated by triflusal through its active metabolite, 2-hydroxy-4-trifluoromethylbenzoic acid (HTB). For this purpose, the interaction between HTB and human serum albumin (HSA) was investigated by fluorescence and laser flash photolysis to monitor inclusion into the protein binding sites through variation in the excited-state properties. A remarkable lengthening of HTB triplet lifetime in the presence of HSA was observed. The use of oleic acid as a displacement probe clearly suggests the preference for dark binding in site I. The mechanism of photobinding was studied by irradiation of HTB in the presence of amino acids, and, in the case of lysine, a photoadduct was detected that arises from nucleophilic attack by the epsilon-amino group to the trifluoromethyl substituent of HTB. Accordingly, photobinding of the metabolite to poly-L-lysine was also observed. Overall, these results are consistent with a mechanism for triflusal photoallergy involving complexation of HTB to site I of HSA and subsequent formation of a covalent photoadduct with one neighboring lysine residue.

In Situ Transient Spectroscopy for the Study of Glucuronidase Activity within Serum Albumin

Laser flash photolysis (LFP) has shown to be an efficient technique for in situ determination of the glucuronidase activity of human serum albumin (HSA). After incubation of the steroisomeric flurbiprofen glucuronides (FBPGluc) during regular time intervals at the selected temperatures, in the presence of protein, regression analysis was applied to the triplet decay at lambda=360 nm. This led to a satisfactory fitting when considering a set of four lifetimes; the corresponding preexponential coefficients AIFBP, AIIFBP, AFFBPGluc, and ABFBPGluc can be correlated with the presence of flurbiprofen (FBP) within the two known binding sites (I and II), together with FBPGluc free in solution (F) and bound (B) to the protein. The new methodology based on LFP of glucuronides in the presence of HSA is fast, experimentally straightforward, and does not involve any workup. This suggests the possibility of making use of the transient triplet-triplet absorption for investigating the enzymatic-like activity of different host biomolecules and at the same time determining the distribution of the generated drug between several compartments in the protein.

Impact of Human Serum Albumin on Oritavancin In Vitro Activity against Enterococci

Oritavancin is a lipoglycopeptide with activity against gram-positive pathogens including vancomycin-resistant enterococci. The impact of human serum albumin (HSA) on oritavancin activity against enterococci was compared to those of vancomycin, daptomycin, teicoplanin, and linezolid in vitro using MIC and time-kill methods. Oritavancin MICs increased between 0- and 8-fold in the presence of HSA. In time-kill assays with HSA, oritavancin retained activity, killing or inhibiting enterococci more rapidly than did comparators when peak concentrations were simulated.

Interaction of human serum albumin with charge transfer probeethyl ester of N,N-dimethylamino naphthyl acrylic acid: An extrinsic fluorescence probe for studying protein micro-environment

The charge transfer (CT) probe ethyl ester of N,N-dimethylamino naphthyl acrylic acid (EDMANA) bound to Human Serum Albumin (HSA) serves as an efficient reporter of the polarity and conformational changes of protein in aqueous buffer (Tris-HCl buffer, pH=7.03) and in presence of denaturant, quencher and reverse micelles. The change in fluorescence intensity and the position of emission maxima of EDMANA in presence of HSA well reflect the nature of binding and location of the probe inside the proteinous environment. The increase in steady state anisotropy values with increase of protein concentration indicate restriction imposed on the mobility of the probe molecules in the proteinous medium. The results of fluorescence quenching of EDMANA by acrylamide, Fluorescence Resonance Energy Transfer (FRET) and Red Edge Excitation Shift (REES) studies throw light on the accessibility to the probe bound to HSA and hence indicate the probable location of the probe within the hydrophobic cavity of HSA. The complicated nature of protein unfolding in presence of urea is well studied by change in the fluorescence properties of EDMANA bound to HSA protein.

Growth of β2-microglobulin-related amyloid fibrils by non-esterified fatty acids at a neutral pH

Abeta2M (beta(2)-microglobulin-related) amyloidosis is a frequent and serious complication in patients on long-term dialysis. Partial unfolding of beta2-m (beta(2)-microglobulin) may be essential to its assembly into Abeta2M amyloid fibrils in vivo. Although SDS around the critical micelle concentration induces partial unfolding of beta2-m to an alpha-helix-containing aggregation-prone amyloidogenic conformer and subsequent amyloid fibril formation in vitro, the biological molecules with similar activity under near-physiological conditions are still unknown. The effect of various NEFAs (non-esterified fatty acids), which are representative anionic amphipathic compounds in the circulation, on the growth of Abeta2M amyloid fibrils at a neutral pH was examined using fluorescence spectroscopy with thioflavin T, CD spectroscopy, and electron microscopy. Physiologically relevant concentrations of laurate, myristate, oleate, linoleate, and mixtures of palmitate, stearate, oleate and linoleate, induced the growth of fibrils at a neutral pH by partially unfolding the compact structure of beta2-m to an aggregation-prone amyloidogenic conformer. In the presence of human serum albumin, these NEFAs also induced the growth of fibrils when their concentrations exceeded the binding capacity of albumin, indicating that the unbound NEFAs rather than albumin-bound NEFAs induce the fibril growth reaction in vitro. These results suggest the involvement of NEFAs in the development of Abeta2M amyloidosis, and in the pathogenesis of Abeta2M amyloidosis.

Controlling Plasma Protein Binding: Structural Correlates of Interactions of Hydrophobic Polyamine Endotoxin Sequestrants with Human Serum Albumin

Hydrophobically substituted polyamine compounds, particularly N-acyl or N-alkyl derivatives of homospermine, are potent endotoxin (lipopolysaccharide) sequestrants. Despite their polycationic nature, the aqueous solubilites are limited owing to the considerable overall hydrophobicity contributed by the long-chain aliphatic substituent, but solubilization is readily achieved in the presence of human serum albumin (HSA). We desired first to delineate the structural basis of lipopolyamine-albumin interactions and, second, to explore possible structure-activity correlates in a well-defined, congeneric series of N-alkyl and -acyl homospermine lead compounds. Fluorescence spectroscopic and isothermal titration calorimetry (ITC) results indicate that these compounds appear to bind to HSA via occupancy of the fatty-acid binding sites on the protein. The acyl and carbamate compounds bind HSA the strongest; the ureido and N-alkyl analogues are significantly weaker, and the branched alkyl compound is weaker still. ITC-derived dissociation constants are weighted almost in their entirety by enthalpic deltaH terms, which is suggestive that the polarizability of the carbonyl groups facilitate, at least in large part, their interactions with HSA. The relative affinities of these lipopolyamines toward HSA is reflected in discernible differences in apparent potencies of LPS-sequestering activity under experimental conditions requiring physiological concentrations of HSA, and also of in vivo pharmacodynamic behavior. These results are likely to be useful in designing analogues with varying pharmacokinetic profiles.

Human serum albumin as a catalyst of RNA cleavage: N-Homocysteinylation and N-phosphorylation by oligonucleotide affinity reagent alter the reactivity of the protein

Kinetic parameters for the cleavage of UpA site in an oligonucleotide in the presence of human serum albumin (HSA) or one of its clinically relevant modification were measured. The RNA-hydrolyzing activity of HSA was decreased by its nonenzymatic N-homocysteinylation. According to 31P NMR data, Lys and Tyr residues were the labeling targets when a phosphorylating analog of oligoribonucleotide substrate was employed. The site of tyrosine modification was slowly dephosphorylated. Lys-directed affinity labeling suppressed oligonucleotide cleavage indicating that lysines took part in the reaction.

Chemiluminescence from singlet oxygen that was detected at two wavelengths and effects of biomolecules on it

We developed the detection apparatus that equipped with the two-photomultiplier tubes for chemiluminescence from singlet oxygen. Singlet oxygen was generated with reaction between sodium hypochlorite and hydrogen peroxide. The chemiluminescence from singlet oxygen, the dimol light emission (ca. 634 nm) and the monomol light emission (ca. 1270 nm), was observed simultaneously for the same reaction cell. The effects of sodium azide as an antioxidant, human serum albumin, and α-amino acids on the chemiluminescence based on the both emissions were examined; the observed chemiluminescence could provide direct information with regard to the reaction between singlet oxygen and antioxidant/biomolecules. The apparent rate constants for quenching singlet oxygen in the presence of human serum albumin were calculated to be ca. 3.3 × 10 9 and ca. 8.8 × 10 8 M −1 s −1 for the dimol and monomol light emissions, respectively, under the present conditions. The chemiluminescence intensities of the dimol emission decreased in the presence of His, Asp, Phe, Ser, and Tyr, and that of the monomol decreased in the presence of Cys and Trp. The chemiluminescence observed in the presence of biomolcules was discussed together with the reactivities of sodium hypochlorite and hydrogen peroxide to biomolecules.

Structural Effects of Biologically Relevant Rhodamines on Spectroscopy of Fluorescence Fluctuations

Exciton coupling in pi-pi complexes between the indole ring and other pi systems is known to enhance the efficiency of energy and electron transfer. Rhodamines' xanthylium rings allow the formation of weakly or nonfluorescent complexes with the amino acid tryptophan. Thus, because of the short distance of the participating electronic clouds, intrinsic electron transfer-induced fluorescence quenching occurs. In solution, the rate constant of electron transfer is known to be limited by collision interactions at the contact distance. By contrast, in protein local environments tryptophan residues can be either exposed or buried in hydrophobic regions. Herein, I report on the properties of aromatic derivatized rhodamines, among which is one with a bound phenylalanine amino acid group. Encompassed is the spectroscopic and kinetic information in bulk and at the single-molecule levels both in free solution and in the presence of human serum albumin. Spectroscopic characteristics are focused with special emphasis on enhanced fluorescence that is addressed considering optimized geometries and electronic spectra. The importance of the probes associated with peptides and metal ions both in condensed phase or interfaces and as substrates with proteins is put into perspective.

Role of Biophysical Parameters on ex Vivo and in Vivo Gene Transfer to the Airway Epithelium by Polyethylenimine/Albumin Complexes

Efficient gene transfer to the airways by nonviral vectors is a function of different parameters, among which the size and the charge of the transfecting particles. The aim of this study was to determine the transfection efficiency of polyethylenimine (PEI)/albumin polyplexes in ex vivo and in vivo models of respiratory epithelium and to correlate it with biophysical characteristics of the particles. Complexes were obtained by adding different amounts of human serum albumin (HSA) to PEI polyplexes preformed in saline. The presence of HSA caused the formation of bigger and more negative polyplexes and increased PEI transfection efficiency in primary respiratory epithelial cells by 4-6-fold. For in vivo administration to the lung, PEI polyplexes were formed in water and optimized with respect to the N/ P ratio. PEI/pC-Luc complexes gave the highest luciferase expression at N/ P 15 when administered through the trachea. At this N/ P ratio, the size and the surface charge of albumin-containing polyplexes were not different as compared with plain PEI polyplexes. Formulation of PEI polyplexes in the presence of HSA or murine serum albumin (MSA) resulted in a 2-fold increase in luciferase expression. In mice treated with PEI or PEI/MSA polyplexes containing the nuclear beta-gal gene, X-gal staining revealed that transfected cells localized at the bronchiolar epithelium and that PEI/MSA transfected four times as many cells as PEI ( p < 0.05). Finally, double administration of PEI/MSA polyplexes resulted in a further enhancement of transfection of the lung. Our data show that serum albumin enhances PEI-mediated gene transfer to airway epithelial cells in vivo, likely facilitating the uptake of polyplexes, and indicate that this formulation would fulfill the requirement of repeated administration, as necessary in chronic lung diseases like cystic fibrosis.

Spectroscopic Investigation on the Interaction of a Cyanine Dye with Serum Albumins

AbstractThe interactions of a cyanine dye with human serum albumin (HSA) and bovine serum albumin (BSA) have been investigated by using absorption and fluorescence spectra. Absorption spectral studies show that binding to the serum albumins leads to a bathochromic shift of the monomer band together with a notable intensity change. Furthermore, the number of binding sites (n) was identified by the absorption spectra. There is a constant enhancement of fluorescence quantum yield when the cyanine dye complexes with HSA or BSA. The apparent binding constant (Ka) and the free energy changes (ΔG) were obtained by analysis of fluorescence data of the cyanine dye in the absence and presence of HSA and BSA. Compared to BSA, HSA associates with the dye in a stronger way.

A Novel Chemiluminescence Technique for Quantitative Measurement of Low Concentration Human Serum Albumin

An efficient and highly sensitive chemiluminescence (CL) technique is proposed in the current study for detection of low levels of human serum albumin (HSA). Chemiluminescence (CL) produced during interaction between fluoresceinyl cypridina luciferin analog (FCLA)-1O2 can be modified with the presence of HSA. The conventional CL technique uses a quenching effect of HSA for its quantitative measurement. We are reporting here that the CL intensity can be enhanced, rather than quenched, by the addition of HSA. The CL signal can be linearly correlated with the HSA concentration over a clinically interesting range of 5 x 10(-9) - 8 x 10(-8) mol L(-1), with a detection limit of 2.5 x 10(-9) mol L(-1). The determination result was consistent with that obtained from conventional methods. One possible mechanism of HSA detection technique using CL enhancement approach is discussed. Intermolecular energy transfer in chemiluminescence systems and changes of microenvironment are likely to be contributors of the CL enhancement with HSA.

Characterization of Insulin Adsorption in the Presence of Albumin by Time-of-Flight Secondary Ion Mass Spectrometry and X-ray Photoelectron Spectroscopy

With a view to develop an encapsulation membrane for a bioartificial pancreas, we have studied the adsorption of insulin and human serum albumin (HSA) on it. The aim of this study was to determine the possibility of insulin detection on a polycarbonate membrane surface in the presence of HSA, an abundant blood protein. The first step of the work consisted in the identification of time-of-flight secondary ion mass spectrometry (ToF-SIMS) and X-ray photoelectron spectroscopy (XPS) specific signals for insulin and albumin. For this purpose, adsorption isotherms in physiological conditions (pH = 7.2, T = 37 degrees ) were established for the two proteins by looking at the SIMS intensity variations of the characteristic protein and substrate fragments when increasing the protein concentration in the solution. The CHS+ ToF-SIMS fragment and the S2p XPS peak were identified as representative insulin signals. The second step of the work consisted in performing simultaneous adsorption of the two proteins with increasing insulin concentration. We observed an increase of the insulin signal in ToF-SIMS and XPS for insulin concentration beyond 5 microg/mL. Principal component analysis (PCA) of the ToF-SIMS results permits us to obtain information about the protein layer composition. The results show that at low relative insulin concentration in solution, the mixed adsorbed layers are enriched in insulin compared to the solution.

Bioreversible derivatives of phenol. 1. The role of human serum albumin as related to the stability and binding properties of carbonate esters with fatty acid-like structures in aqueous solution and biological media.

With the overall objective of assessing the potential of utilizing plasma protein binding interactions in combination with the prodrug approach for improving the pharmacokinetics of drug substances, a series of model carbonate ester prodrugs of phenol, encompassing derivatives with fatty acid-like structures, were characterized in vitro. Stability of the derivatives was studied in aqueous solution, human serum albumin solution, human plasma, and rat liver homogenate at 37 degrees C. Stability of the derivatives in aqueous solution varied widely, with half-lives ranging from 31 to 1.7 x 10(4) min at pH 7.4 and 37 degrees C. The carbonate esters were subject to catalysis by plasma esterases except for the t-butyl and acetic acid derivatives, which were stabilized in both human plasma and human serum albumin solutions relative to buffer. In most cases, however, hydrolysis was accelerated in the presence of human serum albumin indicating that the derivatives interacted with the protein, a finding which was confirmed using the p-nitrophenyl acetate kinetic assay. Different human serum albumin binding properties of the phenol model prodrugs with fatty acid-like structure and neutral carbonate esters were observed. In the context of utilizing plasma protein binding in combination with the prodrug approach for optimizing drug pharmacokinetics, the esterase-like properties of human serum albumin towards the carbonate esters potentially allowing the protein to act as a catalyst of parent compound regenerations is interesting.

Irreversible Alkylation of Human Serum Albumin by Zileuton Metabolite 2-Acetylbenzothiophene-S-oxide: A Potential Model for Hepatotoxicity

2-acetylbenzothiophene-S-oxide (2-ABT-S-oxide or M1) is a reactive metabolite of zileuton, a drug used in the treatment of asthma and is capable of conjugating with glutathione in vitro. Human serum albumin (HSA) is the most abundant protein in plasma and plays a critical role in detoxifying reactive oxygen species. The current research is focused on understanding the interaction between M1 and HSA. The stability studies revealed the half-life of M1 to be about 0.85 h in HSA, 1.82 h in human plasma, and 4.48 h in phosphate-buffered saline (PBS) as determined by first-order approximation. The alkylation rate constant k for HSA was 20 M(-1) min(-1). After quenching with acetonitrile, the half-life of M1 did not change significantly, indicating that M1 is covalently bound to HSA. LC-MS and LC-MS/MS analysis of human plasma revealed the M1 alkylated peptide P (m/z 870) formed by HSA conjugation and concomitant water elimination. The specific amino acid on HSA bound to M1 was identified as Cys-34. This alkylation is observed to be concentration- and incubation-time-dependent in human plasma. HSA oxidized by N, N'-diacetyl-L-cystine exhibits a compromised ability of HSA to react with M1. The alkylated HSA diminished the binding affinity for warfarin. Furthermore, the alkylation was found to be irreversible in the dialysis experiment. In addition, M1 decomposes to 2-ABT in the presence of HSA, presumably acting as an oxidant. The formation of 2-ABT in the incubation and the self-condensation of M1 in PBS indicate that the alkylation of Cys-34 is only one of a number of reactions that occur in the presence of HSA. Irreversible protein modification may potentially lead to a loss of its function. HSA irreversible alkylation represents a model for other proteins to be potentially toxic and thus may help explain zileuton hepatotoxicity.

Synthesis and Characterization of a New Bioactivated Paramagnetic Gadolinium(III) Complex [Gd(DOTA-FPG)(H2O)] for Tracing Gene Expression

A smart contrast agent for magnetic resonance imaging (MRI) can be used to exploit an enzymatic activity specific to the tissue or disease state signified by converting an MRI-inactivated agent to an activated MRI agent. In this study, a beta-galactopyranose-containing gadolinium(III) complex [Gd(DOTA-FPG)(H 2O)] was designed, synthesized, and characterized as being potentially suitable for a bioactivated MRI contrast agent. The (17)O NMR experiments were conducted to estimate the water exchange rate k e x 298 and rotational correlation time tau R 298 . The k ex 298 value of [Gd(DOTA-FPG)(H 2O)] is similar to that of [Gd(DO3A-bz-NO 2)(H 2O)]. The rotational correlation time value of [Gd(DOTA-FPG)(H 2O)] is dramatically longer than that of [Gd(DOTA)(H 2O)] (-) Relaxometric studies show that the percentage change in the T 1 value of [Gd(DOTA-FPG)(H 2O)] decreases dramatically in the presence of beta-galactosidase and human serum albumin. The T(1) change percentage of [Gd(DOTA-FPG)(H 2O)] (60%) is significantly higher than those of Egad and gadolinium(III)-1-(4-(2-(1-(4,7,10-triscarboxymethyl-(1,4,7,10-tetraazacyclododecyl)))-ethylcarbamoyloxymethyl)-2-nitrophenyl)-beta- d-glucopyronuronate. The signal intensity of the MR image for [Gd(DOTA-FPG)(H 2O)] in the presence of human serum albumin and beta-galactosidase (2670 +/- 210) is significantly higher than that of [Gd(DOTA-FPG)(H 2O)] in the sodium phosphate buffer solution (1490 +/- 160). In addition, the MR images show a higher-intensity enhancement in CT26/beta-gal tumor with beta-galactosidase gene expression but not for the CT26 tumor without beta-galactosidase gene expression. We conclude that [Gd(DOTA-FPG)(H 2O)] is a suitable candidate for a bioactivated MRI contrast agent in tracing gene expression.

Characterization of the Myricetin−Human Serum Albumin Complex by Spectroscopic and Molecular Modeling Approaches

The interaction mechanism of flavonol myricetin (3,5,7,3',4',5'-hexahydroxyflavone) and human serum albumin (HSA) has been characterized by fluorescence, electronic absorption, and Fourier transform infrared (FT-IR) spectroscopic approaches and the molecular modeling method. The structural characteristics of myricetin and HSA were probed, and their binding affinities were determined under different pH conditions. The results showed that the binding abilities of the drug to protein decreased under lower pH conditions (pH 3.5 and 2.0) due to the alterations of the protein secondary and tertiary structures. The second derivative absorption spectra of myricetin after interacting with the protein showed that the drug existed as an anion form in the binding pocket. The fluorescence emission intensities of the normal and excited-state proton transfer (ESTP) tautomer of myricetin significantly enhanced in the presence of HSA with conspicuous shifts of the emission bands when excited with a wavelength of 370 nm, while the intensity ratios of the normal to ESTP tautomers rose rapidly with the increase of the HSA concentrations under different pH environments. This illustrated that the fluorescence emission of the normal tautomer (S1-S0, non-proton-transferred) predominated due to the interaction of drug and surrounding polar and ionic side chains of amino acid residues in the binding cavity. The similar spectroscopic properties of myricetin-HSA complex at pH 7.4 and 3.5 showed that the drug was located in subdomain IIA of the protein in the vicinity of the single Trp 214 because of the unfolding of the protein domain III in its F state. From the molecular modeling results, the drug-protein complex was stabilized by electrostatic force and hydrogen bonding with the amino acid residue in the binding pocket, which was consistent with the experimental results.