Showing papers on "Serum albumin published in 2011"
TL;DR: The adsorption and conformation of bovine serum albumin (BSA) on gold nanoparticles (AuNPs) were interrogated both qualitatively and quantitatively via complementary physicochemical characterization methods.
Abstract: The adsorption and conformation of bovine serum albumin (BSA) on gold nanoparticles (AuNPs) were interrogated both qualitatively and quantitatively via complementary physicochemical characterization methods. Dynamic light scattering (DLS), asymmetric-flow field flow fractionation (AFFF), fluorescence spectrometry, and attenuated total reflectance−Fourier transform infrared (ATR-FTIR) spectroscopy were combined to characterize BSA−AuNP conjugates under fluid conditions, while conjugates in the aerosol state were characterized by electrospray-differential mobility analysis (ES-DMA). The presence of unbound BSA molecules interferes with DLS analysis of the conjugates, particularly as the AuNP size decreases (i.e., below 30 nm in diameter). Under conditions where the γ value is high, where γ is defined as the ratio of scattering intensity by AuNPs to the scattering intensity by unbound BSA, DLS size results are consistent with results obtained after fractionation by AFFF. Additionally, the AuNP hydrodynamic s...
370 citations
TL;DR: The impact of glycation on the structure of albumin and its various activities, especially its antioxidant and binding capacities is reported, specifically the role of the protein as a biological marker of diabetes.
364 citations
TL;DR: The accurate and full basic data in the work is beneficial to clarifying the binding mechanism of TC with HSA in vivo and understanding its effect on protein function during the blood transportation process.
274 citations
TL;DR: In vivo antitumor tests revealed that CCM-HSA-NPs had a greater therapeutic effect than CCM (50% or 66% tumor growth inhibition vs. PBS-treated controls) in tumor xenograft HCT116 models without inducing toxicity.
259 citations
TL;DR: The fluorescence data showed that tartrazine could bind to the two SAs to form a complex, and the binding process was a spontaneous molecular interaction procedure, in which van der Waals and hydrogen bond interactions played a major role.
Abstract: Tartrazine is an artificial azo dye commonly used in food products. The present study evaluated the interaction of tartrazine with two serum albumins (SAs), human serum albumin (HSA) and bovine serum albumin (BSA), under physiological conditions by means of fluorescence, three-dimensional fluorescence, UV-vis absorption, and circular dichroism (CD) techniques. The fluorescence data showed that tartrazine could bind to the two SAs to form a complex. The binding process was a spontaneous molecular interaction procedure, in which van der Waals and hydrogen bond interactions played a major role. Additionally, as shown by the UV-vis absorption, three-dimensional fluorescence, and CD results, tartrazine could lead to conformational and some microenvironmental changes of both SAs, which may affect the physiological functions of SAs. The work provides important insight into the mechanism of toxicity of tartrazine in vivo.
219 citations
TL;DR: Results indicate that the binding of baicalein to BSA causes apparent change in the secondary structure of BSA, but does not affect the polarity around the chromophore molecule.
198 citations
TL;DR: Serum albumin predicts all-cause, cardiovascular, and infection-related mortality in both peritoneal dialysis patients and HD patients, but the threshold at which risk of death increases varies by dialysis modality, and this difference should be considered by agencies or organizations that set quality standards.
197 citations
TL;DR: The production of Oryza sativa recombinant HSA (OsrHSA) from transgenic rice seeds is reported, which displays similar in vitro and in vivo immunogenicity as pHSA and can help to satisfy an increasing worldwide demand for human serum albumin.
Abstract: Human serum albumin (HSA) is widely used in clinical and cell culture applications. Conventional production of HSA from human blood is limited by the availability of blood donation and the high risk of viral transmission from donors. Here, we report the production of Oryza sativa recombinant HSA (OsrHSA) from transgenic rice seeds. The level of OsrHSA reached 10.58% of the total soluble protein of the rice grain. Large-scale production of OsrHSA generated protein with a purity >99% and a productivity rate of 2.75 g/kg brown rice. Physical and biochemical characterization of OsrHSA revealed it to be equivalent to plasma-derived HSA (pHSA). The efficiency of OsrHSA in promoting cell growth and treating liver cirrhosis in rats was similar to that of pHSA. Furthermore, OsrHSA displays similar in vitro and in vivo immunogenicity as pHSA. Our results suggest that a rice seed bioreactor produces cost-effective recombinant HSA that is safe and can help to satisfy an increasing worldwide demand for human serum albumin.
192 citations
TL;DR: The objective of this study was to investigate the plasma‐protein binding of the most abundant bioflavonoid, quercetin, using 14C‐labelled quercets, and to report on the effects of these dietary components in man.
Abstract: Although the bioflavonoids, a large group of polyphenolic natural products, exert chemopreventive effects in cardiovascular disease and cancer, there is little information about the disposition of these dietary components in man. The objective of this study was to investigate the plasma-protein binding of the most abundant bioflavonoid, quercetin, using 14C-labelled quercetin. An ultracentrifugation assay (170,000 g for 16 h at 20 degrees C) was shown to sediment plasma proteins. Binding of quercetin to normal plasma was extensive (99.1+/-0.5%, mean +/- s.d., n = 5). The unbound fraction varied as much as 6-fold, 0.3-1.8%, between subjects. This high binding was independent of quercetin concentration over the range 1.5-15 microM (0.5-5 microg mL(-1)). Human serum albumin was the primary protein responsible for the binding of quercetin in plasma (99.4+/-0.1%). Binding by alpha1-acid glycoprotein (39.2+/-0.5%) and very-low-density lipoproteins (<0.5% of total quercetin) did not make substantial contributions to overall plasma binding. The equilibrium association constant for the binding of quercetin to serum albumin was 267+/-33 x 10(3) M(-1) (n = 15). Thermodynamic data for the binding of quercetin to serum albumin indicated spontaneous, endothermic association. Displacement studies suggested that in man the 'IIA' subdomain binding site of human serum albumin was the primary binding site for quercetin. Association of quercetin with erythrocytes was significantly (P < 0.001) reduced by plasma protein binding. These data indicate poor cellular availability of quercetin because of its extensive binding to plasma proteins.
185 citations
TL;DR: Molecular evidence is presented that a minimal albumin binding domain (ABD) derived from streptococcal protein G can be used for efficient half-life extension by indirect targeting of FcRn and it is shown that ABD, and ABD recombinantly fused to an Affibody molecule, in complex with albumin does not interfere with the strictly pH-dependent F cRn-albumin binding kinetics.
175 citations
TL;DR: Differences between black and white persons in glycated albumin, fructosamine, and 1,5-anhydroglucitol levels parallel differences between these groups in HbA(1c) values, suggesting racial differences in hemoglobin glycation and erythrocyte turnover cannot explain racial disparities in these serum markers.
Abstract: In a major change to clinical guidelines, glycated hemoglobin A1c (HbA1c) has recently been recommended for use as a diagnostic test for diabetes in the United States (1). However, there is on-going debate about the interpretation of HbA1c values among black persons and the possible need for race-based HbA1c cut points (2–14). Black persons are well known to have higher HbA1c values than their white counterparts in both the presence and absence of diabetes (2, 4, 15–21) and even in the setting of a low fasting glucose measurement (13, 17). It is unclear whether this disparity stems from racial differences in pre- or postprandial glycemia (6, 13, 22), the tendency of hemoglobin to undergo glycation (5), erythrocyte turnover, or erythrocyte permeability to glucose. Serum glycemic markers, such as fructosamine, glycated albumin, and 1,5-anhydroglucitol offer ways to evaluate racial disparities in glycemia that are biologically independent of erythrocyte turnover and hemoglobin glycation.
Hemoglobin A1c results from the glycation of hemoglobin in erythrocytes and represents long-term (2- to 3-month) glycemia. In contrast, fructosamine and glycated albumin reflect the modification of serum proteins (mainly albumin) by glucose and are markers of 2- to 4-week endogenous glucose exposure. 1,5-Anhydroglucitol is a marker of glycemia-induced glycosuria because reabsorption of filtered 1,5-anhydroglucitol in the proximal tubule is competitively inhibited by glucose (25, 26). Lower serum 1,5-anhydroglucitol reflects high circulating glucose and the occurrence of glycosuria over the previous 1 to 2 weeks (26–31).
We compared nontraditional serum glycemic markers (glycated albumin, fructosamine, and 1,5-anhydroglucitol) with standard markers (HbA1c and fasting glucose) in participants in the Atherosclerosis Risk in Communities (ARIC) Study to determine if the documented higher values of HbA1c in black persons were also observed for serum measures of glycemia. We hypothesized that HbA1c, fructosamine, and glycated albumin values would be higher in black persons, and 1,5-anhydroglucitol would be lower in black persons, as compared with white persons before and after adjustment for fasting glucose concentration.
TL;DR: The findings suggest that the conformational conversion occurs in the oligomers that serve as precursors to amyloid fibrils and precedes the overall fibrillar growth.
Abstract: We have investigated the fibrillation propensity of different conformational isomers of an archetypal, all α-helical protein, namely, bovine serum albumin (BSA), under different pH conditions and ionic strengths using fluorescence and circular dichroism (CD) spectroscopy. At low pH and higher protein concentration, the partially folded conformers associate to form oligomers that are converted into ordered amyloid-like fibrils when incubated at elevated temperature. We have elucidated the mechanism of fibril formation, especially the early steps, by monitoring the kinetics of structural changes during the aggregation process. Various structural probes in tandem were utilized to decipher the temporal evolution of both conformational and size changes by measuring the time dependence of fluorescence intensity and anisotropy of intrinsic tryptophans and several extrinsic fluorophores during the aggregation. Additionally, CD spectroscopy was utilized to monitor the changes in protein secondary structural content during fibrillation. Our findings suggest that the conformational conversion occurs in the oligomers that serve as precursors to amyloid fibrils and precedes the overall fibrillar growth.
TL;DR: The influence of different tumor phenotypes on the EPR effect is investigated, hypothesizing that a baseline level of uptake must be exceeded to visualize high and specific uptake of a targeted macromolecular radiotracer.
Abstract: The enhanced permeability and retention (EPR) effect is the mechanism by which high-molecular-weight nontargeted drugs and prodrugs accumulate in tissues with increased vascular permeability, such as in sites of inflammation or cancer. Attributes of the EPR effect were first described in 1986 by Matsumura and Maeda (1). In their landmark study, the increased tumor uptake and retention of the proteinaceous anticancer drug styrene-maleic acid polymer-functionalized neocarzinostatin (molecular weight [MW], ~16 kDa) was explained by the binding of styrene-maleic acid polymer-functionalized neocarzinostatin to plasma albumin (MW, ~67 kDa). Compared with the native small-molecule neocarzinostatin, this albumin-bound macromolecular drug (MW, ~83 kDa) displayed increased delivery to, and concentration in, the tumor, with prolonged duration of action and increased therapeutic efficacy. The generality of the EPR effect and its dependence on molecular size was demonstrated further by the encapsulation of styrene-maleic acid polymer-functionalized neocarzinostatin in Lipiodol (ethiodized poppyseed oil) liposomes (2)—a result that fueled research interest in liposomal formulations as drug delivery vehicles for anticancer chemotherapeutics (3–8).
From a physiologic perspective, the EPR effect is specific in that it occurs only in tissues that display abnormal vasculature such as the microenvironment of tumors. Yet in terms of biochemistry, the EPR effect depends only on global properties including molecular size, shape, charge, and polarity and is independent of any targeted or specific binding properties such as ligand–receptor interactions.
It is important to note that the EPR effect is different from the nontargeted, passive uptake of small molecules by diffusion in diseased tissues (9). The principal difference between EPR and passive localization lies in the characteristics of retention and tissue clearance rather than uptake. Small molecules rapidly penetrate the interstitial space of tumors, but in the absence of specific binding to cellular proteins, cellular uptake, or metabolism, the drug is not retained and may be free to diffuse out of the tissue back into the blood pool or lymphatic system. In contrast, macromolecules have smaller diffusion constants that, on the one hand, reduce the initial rate of tumor uptake but, on the other, also tend to increase the half-life of blood-pool circulation, enhance tissue retention, and greatly decrease the rate of clearance. By way of example, the vascular permeability dye Evans blue (which forms a noncovalent, macromolecular complex with albumin) was shown to have a decreased rate of clearance from tumor tissue (>3–4 wk), compared with healthy tissue (<1 wk) (1).
Despite advances over the last 25 years in our understanding of the nature of the EPR effect, important questions remain. What is the influence of tumor size and phenotype on the EPR effect? To what extent does tissue perfusion, vascularity, and necrosis influence drug uptake by the EPR effect? For a given molecule, how does the EPR effect contribute to accumulation of the drug in the target tissue over time? Furthermore, with the growth of nanotechnology for potential use in biomedical applications, there is an immediate requirement to ensure rigorous evaluation of the efficacy of these new constructs. Any given class of macromolecule with characteristic size, shape, charge, polarity, and lipophilicity will show tumor uptake to a certain extent via the EPR effect. In molecular targeting, this lower limit has to be exceeded to visualize specific binding of the radiotracer. Numerous multimodality nanoparticle-based agents such as functionalized carbon nanotubes, liposomes, macromolecular proteins, nucleic acid polymers, dendrimers, aptamers, iron-oxide particles, and quantum dots have been reported to accumulate in tumors. However, the question of whether this uptake is specific for the intended target is not always addressed.
Answering all of the aforementioned questions is clearly beyond the scope of a single research article. Rather, in this work we examined whether the magnitude of the EPR effect with reference to radiotracer uptake in tumors correlates with different tumor sizes and phenotypes. We investigated the in vivo distribution of 89Zr-labeled mouse serum albumin (89Zr-mAlb) because albumin is, in general, known to accumulate in inflamed tissues and tumors via the EPR effect (10,11). Therefore, mAlb can serve as a macromolecular radiopharmaceutical displaying quantifiable uptake in 3 well-established xenograft models of human prostate cancer. Tumor models focus on 3 commonly used human prostate cancer lines of different origins, CWR22rv1, DU-145, and PC-3. Experiments include the use of PET, ex vivo biodistribution, contrast-enhanced ultrasound (CEUS) imaging, histology, and autoradiography to probe the influence of tumor size and phenotype on the magnitude of the EPR effect.
TL;DR: In this paper, a biomarker for diagnosing or prognosing childhood MR Nephropathy (MN) was proposed, which is cationic Bovine Serum Albumin (BSA) and an antibody that binds to a polypeptide of sequence SEQ ID NO: 3.
Abstract: The invention concerns a biomarker for diagnosing or prognosing childhood Membranous Nephropathy (MN), said biomarker is (i) cationic Bovine Serum Albumin (BSA), and/or (ii) an antibody that binds to a polypeptide of sequence SEQ ID NO: 3. The invention further concerns an antibody or antibody fragment or a composition comprising such an antibody or antibody fragment, wherein said antibody or antibody fragment is specific to an amino acid sequence SEQ ID NO: 3. The invention also concerns a foodstuff likely to contain BSA or cow milk or cow milk extracts, wherein said foodstuff is depleted in BSA.
TL;DR: Evidence is provided that after inhalation of nanoparticles, a different protein coating and thus different biological behavior may result compared to direct administration to the bloodstream, which is significant for nano-toxicology.
01 Jul 2011
TL;DR: The SVM model was integrated to a free, web-based prediction platform and the potential offered by the combined use of in silico calculation methods and experimental binding data is illustrated.
Abstract: Motivation: Human serum albumin (HSA), the most abundant plasma protein is well known for its extraordinary binding capacity for both endogenous and exogenous substances, including a wide range of drugs. Interaction with the two principal binding sites of HSA in subdomain IIA (site 1) and in subdomain IIIA (site 2) controls the free, active concentration of a drug, provides a reservoir for a long duration of action and ultimately affects the ADME (absorption, distribution, metabolism, and excretion) profile. Due to the continuous demand to investigate HSA binding properties of novel drugs, drug candidates and drug-like compounds, a support vector machine (SVM) model was developed that efficiently predicts albumin binding. Our SVM model was integrated to a free, web-based prediction platform ( http://albumin.althotas.com). Automated molecular docking calculations for prediction of complex geometry are also integrated into the web service. The platform enables the users (i) to predict if albumin binds the query ligand, (ii) to determine the probable ligand binding site (site 1 or site 2), (iii) to select the albumin X-ray structure which is complexed with the most similar ligand and (iv) to calculate complex geometry using molecular docking calculations. Our SVM model and the potential offered by the combined use of in silico calculation methods and experimental binding data is illustrated.
Contact: eszter.hazai@virtuadrug.com
Supplementary information:Supplementary data are available at Bioinformatics online.
TL;DR: The binding of model drugs to human blood and individual blood components has been determined by equilibrium dialysis and expressed in terms of classes of binding sites, association constants and binding capacities.
Abstract: The binding of model drugs to human blood and individual blood components has been determined by equilibrium dialysis and expressed in terms of classes of binding sites, association constants and binding capacities. Chlorpromazine and imipramine are bound to three major components: membranes of red cells, albumin, and lipoproteins. The affinity and capacity of lipoprotein binding is at least as high as that of albumin and is equally distributed on HDL, LDL, VLDL, and on the chylomicrons. White blood cells and platelets are of minor importance in terms of binding capacity. No binding was detected with gamma-globulins or alpha- or beta-globulins other than lipoproteins. In contrast, salicylic acid was not bound to red cells or lipoproteins.
TL;DR: The co-crystal structures of HSA in complex with six dansylated amino acids that are specific for either drug site 1 or drug site 2 are presented and the structural basis of the site-specificity of different dansyated amino acid compounds is explained.
TL;DR: Fluorescence titrations support evidence that an observed dependence of PFAA‐BSA binding on pH is attributable to conformational changes in the protein, suggesting that physiological implications of strong binding to albumin may be important for short‐chain PFAAs.
Abstract: Interactions of perfluoroalkyl acids (PFAAs) with tissue and serum proteins likely contribute to their tissue distribution and bioaccumulation patterns. Protein-water distribution coefficients (K(PW) ) based on ligand associations with bovine serum albumin (BSA) as a model protein were recently proposed as biologically relevant parameters to describe the environmental behavior of PFAAs, yet empirical data on such protein binding behavior are limited. In the present study, associations of perfluoroalkyl carboxylates (PFCAs) with two to 12 carbons (C₂-C₁₂) and perfluoroalkyl sulfonates with four to eight carbons (C₄, C₆, and C₈) with BSA are evaluated at low PFAA:albumin mole ratios and various solution conditions using equilibrium dialysis, nanoelectrospray ionization mass spectrometry, and fluorescence spectroscopy. Log K(PW) values for C₄ to C₁₂ PFAAs range from 3.3 to 4.3. Affinity for BSA increases with PFAA hydrophobicity but decreases from the C₈ to C₁₂ PFCAs, likely due to steric hindrances associated with longer and more rigid perfluoroalkyl chains. The C₄-sulfonate exhibits increased affinity relative to the equivalent chain-length PFCA. Fluorescence titrations support evidence that an observed dependence of PFAA-BSA binding on pH is attributable to conformational changes in the protein. Association constants determined for perfluorobutanesulfonate and perfluoropentanoate with BSA are on the order of those for long-chain PFAAs (K(a) ∼10⁶/M), suggesting that physiological implications of strong binding to albumin may be important for short-chain PFAAs.
TL;DR: The experimental results revealed the static quenching mechanism in the interaction of TRP with protein and the hydrogen bond and van der Waals forces played the major role in the binding of TRp to proteins.
TL;DR: Results showed that albumin layering is important toward improving insulin transport across the intestinal membrane, possibly by stabilizing insulin in the intestinal conditions and suggests a potential carrier system in delivering protein-based drugs by the oral route.
TL;DR: GDC-0449 levels strongly correlated with AAG levels, showing parallel fluctuations of AAG and total drug over time and consistently low, unbound drug levels, different from previously reported AAG-binding drugs.
Abstract: Purpose: In a phase I trial for patients with refractory solid tumors, hedgehog pathway inhibitor vismodegib (GDC-0449) showed little decline in plasma concentrations over 7 days after a single oral dose and nonlinearity with respect to dose and time after single and multiple dosing. We studied the role of GDC-0449 binding to plasma protein alpha-1-acid glycoprotein (AAG) to better understand these unusual pharmacokinetics. Experimental Design: Sixty-eight patients received GDC-0449 at 150 ( n = 41), 270 ( n = 23), or 540 ( n = 4) mg/d, with pharmacokinetic (PK) sampling at multiple time points. Total and unbound (dialyzed) GDC-0449 plasma concentrations were assessed by liquid chromatography/tandem mass spectrometry, binding kinetics by surface plasmon resonance–based microsensor, and AAG levels by ELISA. Results: A linear relationship between total GDC-0449 and AAG plasma concentrations was observed across dose groups ( R 2 = 0.73). In several patients, GDC-0449 levels varied with fluctuations in AAG levels over time. Steady-state, unbound GDC-0449 levels were less than 1% of total, independent of dose or total plasma concentration. In vitro , GDC-0449 binds AAG strongly and reversibly ( K D = 13 μmol/L) and human serum albumin less strongly ( K D = 120 μmol/L). Simulations from a derived mechanistic PK model suggest that GDC-0449 pharmacokinetics are mediated by AAG binding, solubility-limited absorption, and slow metabolic elimination. Conclusions: GDC-0449 levels strongly correlated with AAG levels, showing parallel fluctuations of AAG and total drug over time and consistently low, unbound drug levels, different from previously reported AAG-binding drugs. This PK profile is due to high-affinity, reversible binding to AAG and binding to albumin, in addition to solubility-limited absorption and slow metabolic elimination properties. Clin Cancer Res; 17(8); 2512–20. ©2011 AACR .
TL;DR: Results indicate that all the nanoparticles presented a mean size and a polydispersity lower than 300nm and 0.33nm, respectively, were spherical shaped and negatively charged even after drug loading, and the presence of the different beta cyclodextrins in the polymeric network affected drug loading and could differently modulate nanoparticle mucoadhesiveness and drug permeation behaviour.
TL;DR: The method was specific for measuring glycated amino acids in albumin and had good basic performance characteristics, and may be a useful indicator for diabetes control.
Abstract: Background:Glycated albumin (GA) is a medium-term glycemic control marker of diabetes and may be more sensitive to changes in plasma glucose than hemoglobin A1c. We studied where and how many fructosyl groups bind to albumin, and which glycation sites are measured by the enzymatic method for GA. We also studied the basic performance of the enzymatic method for GA.Methods:Glycated albumin was measured using an enzymatic method (Lucica®GA-L, Asahi Kasei Pharma) on a biochemical autoanalyzer. Molecular weights of purified GA and nonglycated albumin were measured by a mass spectrometry system. Two hundred one healthy volunteers with normal results of oral glucose tolerance testing were recruited to determine the reference range in Americans.Results:The present method measured only glycated amino acids from albumin in serum protein. We estimate that the number of glycated amino acids measured by this method was approximately two per molecule of albumin. The general performance (sensitivity, specificity, reprod...
TL;DR: Data suggest that covalent linkage of folic acid can specifically increase cancer cell HSA NP uptake into cancer cells but not into HFFs.
TL;DR: In this article, X-ray photoelectron spectroscopy was used to study the interaction of two different sized proteins, bovine serum albumin (BSA) and fibrinogen, with an aluminosilicate system containing yttrium and iron that is a potential biomaterial.
TL;DR: The results showed that chlorination, especially at position 3', had significant effects on the binding constant value of chlorinated genistein derivatives to BSA; however, the binding site and the binding number were slightly affected.
Abstract: Chlorination can significantly enhance the antioxidant and antitumor activity of genistein. In this paper, genistein, 8-chlorogenistein, and 3',8-dichlorogenistein were selected to investigate the binding to bovine serum albumin (BSA) using fluorescence spectroscopy and circular dichroism (CD). The results showed that chlorination, especially at position 3', had significant effects on the binding constant value of chlorinated genistein derivatives to BSA; however, the binding site and the binding number were slightly affected. The thermodynamic parameters indicated that hydrophobic and electrostatic forces played important roles in the binding process and the enhanced binding affinity mainly associated with the increase of the hydrophobicity caused by the chlorine atom substitution. Furthermore, the CD data demonstrated that the conformation of BSA was slightly altered in the presence of genistein, 8-chlorogenistein, and 3',8-dichlorogenistein, with different reduced α-helix contents. The results obtained herein will be of biological significance in toxicology investigation and genistein derivative drug design.
TL;DR: PA was found to be more potent than BP with respect to stimulation of T cells from patients with penicillin hypersensitivity, illustrating the functional relevance of diastereoisomeric hapten formation.
Abstract: Covalent binding to proteins to form neoantigens is thought to be central to the pathogenesis of penicillin hypersensitivity reactions. We have undertaken detailed mass spectrometric studies to define the mechanism and protein chemistry of hapten formation from benzylpenicillin (BP) and its rearrangement product, benzylpenicillenic acid (PA). Mass spectrometric analysis of human serum albumin exposed to BP and PA in vitro revealed that at low concentrations (drug protein molar ratio 0.001:1) and during short time incubations BP and PA selectively target different residues, Lys199 and Lys525, respectively. Molecular modeling showed that the selectivity was a function of noncovalent interaction before covalent modification. With increased exposure to higher concentrations of BP and PA, multiple epitopes were detected on albumin, demonstrating that the multiplicity of hapten formation is a function of time and concentration. More importantly, we have demonstrated direct evidence that PA is a hapten accounting for the diastereoisomeric BP antigen formation in albumin isolated from the blood of patients receiving penicillin. Furthermore, PA was found to be more potent than BP with respect to stimulation of T cells from patients with penicillin hypersensitivity, illustrating the functional relevance of diastereoisomeric hapten formation.
TL;DR: The results suggest the reversible covalent modification of EGCg via Schiff-base formation, and that the immobilization of E GCg to HSA, through the formation of a stable complex, prevented the polymerization and decomposition ofEGCg in human serum.
Abstract: Human serum albumin (HSA) contributes to the stabilization of (-)-epigallocatechin gallate (EGCg) in serum. We characterize in the present study the mechanisms for preventing EGCg oxidation by HSA. EGCg was stable in human serum or buffers with HSA, but (-)-epigallocatechin (EGC) was unstable. We show by comparing EGCg and EGC in a neutral buffer that EGCg had a higher binding affinity than EGC. This indicates that the galloyl moiety participated in the interaction of EGCg with HSA and that this interaction was of critical importance in preventing EGCg oxidation. The binding affinity of EGCg for HSA and protein carbonyl formation in HSA were enhanced in an alkaline buffer. These results suggest the reversible covalent modification of EGCg via Schiff-base formation, and that the immobilization of EGCg to HSA, through the formation of a stable complex, prevented the polymerization and decomposition of EGCg in human serum.
TL;DR: Warfarin and phenprocoumon binding to human serum albumin was studied by equilibrium dialysis to establish an understanding of the mechanism of action of these drugs.
Abstract: Warfarin and phenprocoumon binding to human serum albumin was studied by equilibrium dialysis. The first stoichiometric binding constant was 1.89 x 10(5) M-1 for warfarin and 2.40 x 10(5) M-1 for phenprocoumon. The affinity of warfarin was markedly increased on addition of up to 3 mol mol-1 albumin of palmitic, stearic, oleic or linoleic acids with energetic couplings for co-binding of one molecule of each of the fatty acids and one molecule of warfarin of 0.9, 1.1, 0.7 and 0.6 kJ mol-1, respectively. The affinity of phenprocoumon was only increased slightly on addition of palmitate with an energetic coupling of 0.3 kJ mol-1. Six consecutive serum samples were obtained from each of 14 patients undergoing surgery. The serum affinity of the drugs varied considerably corresponding to free drug concentrations between 0.7 and 2.7% for warfarin and between 0.8 and 4.9% for phenprocoumon. The affinity of warfarin but not of phenprocoumon was correlated to the increasing plasma fatty acid concentration. Anticoagulant therapy with phenprocoumon may thus be less sensitive than warfarin to changes in the fatty acid concentration of plasma.