Showing papers on "Human serum albumin published in 2002"

Practical Aspects of the Ligand-Binding and Enzymatic Properties of Human Serum Albumin

Abstract: Recent work with approaches like recombinant mutants and X-ray crystallography has given much new information about the ligand-binding properties of human serum albumin (HSA). The information increases the understanding of this unique transport and depot protein and could give a structural basis for the possible construction of therapeutic agents with altered HSA-binding properties. A tabulation of high-affinity binding sites for both endogenous and exogenous compounds has been made; it could be useful for the above-mentioned purpose, but it could also be of value when trying to predict potential drug interactions at the protein-binding level. Drug displacement is not always a complication to therapy; it can be used to increase the biological effect of a drug. However, due to rebinding at other sites, the increase in the free concentration of a displaced ligand can be less than expected. Drugs and drug metabolites can also interact covalently with HSA; thiol-containing drugs often bind to the single free cysteine residue of HSA, and glucuronidated drugs react irreversibly with other residues of the protein. Reversible binding of ligands is often stereospecific, and therefore immobilized HSA can be used to separate drug isomers. Albumin-containing dialysates are useful for extracorporeal removal of endogenous toxins and in the treatment of drug overdoses. HSA has different types of hydrolytic activities, which also can be stereospecific. The esterase-like property seems especially useful in converting prodrugs to active drugs in plasma.

The interaction of MS-325 with human serum albumin and its effect on proton relaxation rates.

Abstract: MS-325 is a novel blood pool contrast agent for magnetic resonance imaging currently undergoing clinical trials to assess blockage in arteries. MS-325 functions by binding to human serum albumin (HSA) in plasma. Binding to HSA serves to prolong plasma half-life, retain the agent in the blood pool, and increase the relaxation rate of water protons in plasma. Ultrafiltration studies with a 5 kDa molecular weight cutoff filter show that MS-325 binds to HSA with stepwise stoichiometric affinity constants (mM(-1)) of K(a1) = 11.0 +/- 2.7, K(a2) = 0.84 +/- 0.16, K(a3) = 0.26 +/- 0.14, and K(a4) = 0.43 +/- 0.24. Under the conditions 0.1 mM MS-325, 4.5% HSA, pH 7.4 (phosphate-buffered saline), and 37 degrees C, 88 +/- 2% of MS-325 is bound to albumin. Fluorescent probe displacement studies show that MS-325 can displace dansyl sarcosine and dansyl-L-asparagine from HSA with inhibition constants (K(i)) of 85 +/- 3 microM and 1500 +/- 850 microM, respectively; however, MS-325 is unable to displace warfarin. These results suggest that MS-325 binds primarily to site II on HSA. The relaxivity of MS-325 when bound to HSA is shown to be site dependent. The Eu(III) analogue of MS-325 is shown to contain one inner-sphere water molecule in the presence and in the absence of HSA. The synthesis of an MS-325 analogue, 5, containing no inner-sphere water molecules is described. Compound 5 is used to estimate the contribution to relaxivity from the outer-sphere water molecules surrounding MS-325. The high relaxivity of MS-325 bound to HSA is primarily because of a 60-100-fold increase in the rotational correlation time of the molecule upon binding (tau(R) = 10.1 +/- 2.6 ns bound vs 115 ps free). Analysis of the nuclear magnetic relaxation dispersion (T(1) and T(2)) profiles also suggests a decrease in the electronic relaxation rate (1/T(1e) at 20 MHz = 2.0 x 10(8) s(-1) bound vs 1.1 x 10(9) s(-1) free) and an increase in the inner-sphere water residency time (tau(m) = 170 +/- 40 ns bound vs 69 +/- 20 ns free).

Reversible and Covalent Binding of Drugs to Human Serum Albumin: Methodological Approaches and Physiological Relevance

Abstract: Human serum albumin (HSA) plays a fundamental role in the transport of drugs, metabolites, and endogenous ligands. Binding to HSA controls the free, active concentration of a drug, provides a reservoir for a long duration of action, and ultimately affects drug absorption, metabolism, distribution and excretion. The free concentration of a drug can also be affected by interaction with co-administered drugs or by pathological conditions that can modify to a significant extent the binding properties of the carrier, resulting in important clinical impacts for drugs that have a relatively narrow therapeutic index. This manuscript will review the physiological role of albumin in the human body and the pharmacological consequences of drug-albumin binding, and then focus on the structure and the properties of the protein binding sites, as studied by different methodologies. Among these, biochromatography on immobilized albumin has been shown to be a rapid and effective tool for the characterization of albumin binding sites and their enantioselectivity, and for the study of the changes in the binding properties of the protein arising by interaction between different ligands. We will discuss the potential offered by the combined use of circular dichroism on the same protein/drug system in solution, not only for the determination of binding parameters and the detection of displacement phenomena, but also for the identification of conformational features underlying binding stereoselectivity. In particular, the essential role of these methodologies in the study of the enantioselective phenomena occurring in the HSA binding of chiral drugs will be addressed. The effect of reversible or covalent binding of drugs will also be discussed and examples of physiological relevance reported.

Pharmaceutical Strategies Utilizing Recombinant Human Serum Albumin

Abstract: Gene manipulation techniques open up the possibility of making recombinant human serum albumin (rHSA) or mutants with desirable therapeutic properties and for protein fusion products. rHSA can serve as a carrier in synthetic heme protein, thus reversibly carrying oxygen. Myristoylation of insulin results in a prolonged half-life because of self aggregation and increased albumin binding. Preferential albumin uptake by tumor cells serves as the basis for albumin-anticancer drug conjugate formulation. Furthermore, drug targeting can be achieved by incorporating drugs into albumin microspheres whereas liver targeting can be achieved by conjugating drug with galactosylated or mannosylated albumin. Microspheres and nanoparticles of different sizes can, with or without drugs and/or radioisotopes, be used for drug delivery or diagnostic purposes. In vivo implantation of albumin fusion protein expressing cells encapsulated in HSA-alginate coated beads showed promising results compared to organoids in rats. Chimeric peptide strategy with cationized albumin as the transport can deliver drugs via receptor mediated transcytosis through the blood brain barrier. Gene bearing, albumin microbubbles containing ultrasound contrast agents can non-invasively deliver gene after destruction by ultrasound. Various site-directed mutants of HSA can be tailor made depending on the application required.

Protein adsorption on supported phospholipid bilayers.

Abstract: Quartz crystal microbalance with dissipation (QCM-D) measurements were used to investigate the adsorption of human fibrinogen, human serum albumin, bovine hemoglobin, horse heart cytochrome c, human immunoglobulin (hIgG), and 10% fetal bovine serum on supported bilayers of egg-phosphatidylcholine (eggPC) lipids. For comparison the adsorption of fibrinogen and hIgG to eggPC bilayers was also studied with surface plasmon resonance (SPR). The supported bilayers were formed in situ by vesicle adhesion and spontaneous fusion onto a SiO(2) surface. The supported lipid bilayer is highly protein resistant: The irreversible adsorption measured with the QCM-D technique was below the detection level, while reversible protein adsorption was detected for all the proteins in the range 0.3-4% of the saturation coverage on a hydrophobic thiol monolayer on gold. The adsorbed amounts were slightly higher for the SPR measurements. Possible mechanisms for the protein resistance of eggPC bilayers are briefly discussed.

Interaction of Ochratoxin A with Human Serum Albumin. Preferential Binding of the Dianion and pH Effects

Abstract: Ochratoxin A (OTA), a fungal metabolite produced by several strains of Aspergillus and Penicillium, binds to serum albumin with high affinity only in the completely deprotonated form (dianion). The pKa of the phenolic group of OTA decreased by more than three units when it was bound to human serum albumin (HSA). Optical spectroscopy provided evidence that HSA has at least two binding sites for OTA, each being able to accommodate one dianion. These two sites were characterized by the binding constants of 5.2 × 106 and 1.0 × 105 M-1. The binding constant for the monoanion of OTA was estimated to be ∼103 M-1. Fluorescence polarization spectroscopy confirmed weak interaction of the monoanion with the protein in the F and E forms (pH 8) compared to the N form (pH ∼ 7). Fluorescence anisotropy decay of the dianion of OTA bound to HSA (36.6 ns) was much longer than its emission lifetime (5.2 ns) and was close to reported values for the rota...

Chromatographic assay of glycation adducts in human serum albumin glycated in vitro by derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl-carbamate and intrinsic fluorescence

Abstract: Glycation of proteins leads to the formation of advanced glycation endproducts (AGEs) of diverse molecular structure and biological function. Serum albumin derivatives modified to minimal and high extents by methylglyoxal and glucose in vitro have been used in many studies as model AGE proteins. The early and advanced glycation adduct contents of these proteins were investigated using the 6-aminoquinolyl-N-hydroxysuccinimidyl-carbamate (AQC) chromatographic assay of enzymic hydrolysates. AGEs derived from methylglyoxal, glyoxal and 3-deoxyglucosone, the hydroimidazolones N(delta)-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (MG-H1), N(delta)-(5-hydro-4-imidazolon-2-yl)ornithine (G-H1) and N(delta)-[5-(2,3,4-trihydroxybutyl)-5-hydro-4-imidazolon-2-yl]ornithine (3DG-H1), bis(lysyl)imidazolium cross-links methylglyoxal-derived lysine dimer (MOLD), glyoxal-derived lysine dimer (GOLD), 3-deoxyglucosone-derived lysine dimer (DOLD), monolysyl adducts N(epsilon)-(1-carboxyethyl)lysine (CEL), N(epsilon)-carboxymethyl-lysine (CML) and pyrraline, other AGEs, N(delta)-(4-carboxy-4,6-dimethyl-5,6-dihydroxy-1,4,5,6-tetrahydropyrimidin-2-yl)ornithine (THP), argpyrimidine and pentosidine, and fructosyl-lysine were determined. AGEs with intrinsic fluorescence (argpyrimidine and pentosidine) were assayed without derivatization. Human serum albumin (HSA) glycated minimally by methylglyoxal in vitro contained mainly MG-H1 with minor amounts of THP and argpyrimidine. Similar AGEs were found in prothrombin glycated minimally by methylglyoxal and in N(alpha)-t-butyloxycarbonyl-arginine incubated with methylglyoxal. HSA glycated highly by methylglyoxal contained mainly argpyrimidine, MG-H1 and THP, with minor amounts of CEL and MOLD. HSA glycated minimally by glucose in vitro contained mainly fructosyl-lysine and CML, with minor amounts of THP, MG-H1, G-H1, 3DG-H1, argpyrimidine and DOLD. HSA glycated highly by glucose contained these AGEs and pyrraline, and very high amounts ( approximately 8 mol/mol of protein) of fructosyl-lysine. Most AGEs in albumin glycated minimally by methylglyoxal and glucose were identified. Significant proportions of arginine and lysine-derived AGEs in albumin modified highly by methylglyoxal, and lysine-derived AGEs in albumin modified highly by glucose, remain to be identified.

Serum albumin binding moieties

Abstract: Compositions comprising non-naturally occurring serum albumin binding moieties are described, together with methods of use thereof, e.g., for detecting or isolating serum albumin molecules in a solution, for blood circulation imaging, and for linking therapeutics or other molecules to albumin. Preferred serum albumin binding peptides having a high affinity for human serum albumin are particularly disclosed.

Albumin selectively inhibits TNFα-induced expression of vascular cell adhesion molecule-1 in human aortic endothelial cells

Abstract: Objective: Leukocyte adhesion to, and transmigration across, the vascular endothelium are critical initiating steps in inflammation and atherosclerosis. We hypothesized that albumin, the major plasma protein, acts as an anti-inflammatory agent towards endothelial cells. Methods and Results: To test the hypothesis, we studied the effects of bovine serum albumin (BSA) on TNFα-induced expression of adhesion molecules in cultured human aortic endothelial cells (HAEC). We found that incubation of HAEC for 16 h with BSA (0.5–5%, w/v) dose-dependently inhibited TNFα-induced mRNA and protein expression of vascular cell adhesion molecule-1 (VCAM-1), but not intercellular adhesion molecule-1 nor E-selectin. Yeast recombinant human serum albumin exerted similar inhibitory effects on VCAM-1 expression, whereas γ-globulin was ineffective. BSA also significantly inhibited TNFα-induced adhesion of monocytic THP-1 cells to HAEC in a dose-dependent manner. Furthermore, BSA strongly inhibited activation and nuclear translocation of the transcription factor, nuclear factor-κB (NF-κB). For example, the physiologically relevant concentration of 5% BSA inhibited NF-κB activation by 90±7%, VCAM-1 mRNA and protein expression by 81±4 and 80±13%, respectively, and THP-1 adhesion by 73±9% ( n = 3). The inhibitory effect of BSA on TNFα-induced VCAM-1 expression was not attenuated by inhibition of intracellular GSH synthesis. Conclusions: Our data show that physiological concentrations of albumin selectively inhibit TNFα-induced upregulation of VCAM-1 expression and monocyte adhesion, most likely by inhibiting NF-κB activation in a GSH-independent manner.

The effect of intestinal ischemia and reperfusion injury on ICAM-1 expression, endothelial barrier function, neutrophil tissue influx, and protease inhibitor levels in rats

Abstract: Multiple organ dysfunction syndrome (MODS) is mediated by complex mechanisms in which interactions between activated leukocytes and endothelial cells play a central role. ICAM-1 (intercellular adhesion molecule-1) mediates firm adhesion and transendothelial migration of activated leukocytes from postcapillary venules into the tissue. The present study evaluated the ICAM-1 expression in various organs after 40 min of intestinal ischemia and 1, 3, 6, 12 h of reperfusion (I/R) in the rat, using a dual monoclonal antibody technique (n = 36). Endothelial barrier permeability, using the vascular leakage of radiolabeled human serum albumin was also assessed (n = 12). Neutrophil sequestration in the lungs was quantitated by myeloperoxidase activity and plasma protease inhibitor levels were measured with electroimmunoassay. Significant regional differences were found in ICAM-1 expression between organs, both constitutively and after I/R-injury. The highest constitutive levels were observed in the liver and lungs, followed by the kidneys. The constitutive ICAM-1 expression in the intestines and in the heart was about 1/20 compared with that found in the liver and lungs. The brain and muscle had levels of about 1/150 of that in the liver and lungs. After intestinal I/R, significant increases (17-45%) were found in the lungs, intestines, brain, heart, and muscle. Albumin leakage index (ALI) in all examined organs and myeloperoxidase activity in the lungs increased after I/R-injury. Serum levels of albumin and most protease inhibitors decreased significantly after I/R challenge. Intestinal I/R results in an increase of systemic ICAM-1 expression with marked organ variability. The upregulation of ICAM-1 could represent a crucial step in the adherence- and migration process of activated leukocytes and potentially in the development of tissue injury.

Determination of drug-plasma protein binding kinetics and equilibria by chromatographic profiling: exemplification of the method using L-tryptophan and albumin.

Abstract: Drug-plasma protein binding may greatly influence the bioavailability and metabolism of a plasma-borne drug, the bound form being partially protected from the metabolic fate of the unbound drug. Traditionally, equilibrium values (e.g., percentage binding) for drug-protein binding have been measured to rationalize in vivo phenomena. However, such studies overlook the influence of kinetics. A rapid method of simultaneously determining kinetic rate constants and equilibrium constants from chromatographic profiles has been developed, based on the use of immobilized protein columns and HPLC. By measuring the chromatographic profiles (the position and width) of a retained and an unretained compound one can directly determine both the rate and equilibrium constants. Results are presented for the binding of L-tryptophan to human serum albumin to exemplify the method. The association equilibrium constant (Ka) and the association and dissociation rate constants (k(a) and k(d), respectively) were thereby measured in an aqueous pH 7.4 environment at 37 degrees C as 0.84 10(4) M(-1), 5.8 10(4) M(-1) s(-1), and 6.9 s(-1), respectively. These compare favorably with previously published results. The described method may be used in quantitative structure-property relationship-based rational drug discovery or for the rationalization of drug pharmacokinetics.

Targeted expression of human serum albumin to potato tubers.

Abstract: Complementary DNA expression of mature human serum albumin was engineered into potato plants under the transcriptional control of patatin B33 promoter and potato proteinase inhibitor II terminator. Protein secretion was achieved by using the signal sequence from potato proteinase inhibitor II. Recombinant albumin accumulated up to 0.2% of total soluble tuber protein in single transformant lines, regardless of the potato cultivar used. Electrophoretic mobility and N-terminal amino acid sequence analysis of partially purified recombinant albumin confirmed proper processing of an immune responsive recombinant albumin, and revealed that the proteinase inhibitor II signal sequence was correctly removed. No further optimisation of these yields was obtained by HSA expression in patatin antisense plants (line Pas58). Subcellular localisation showed that recombinant protein was successfully targeted to the apoplast. Potato tubers may be used, by applying this technology, to produce other heterologous proteins of interest in the biopharmaceutical industry.

S-Nitroso Human Serum Albumin Treatment Reduces Ischemia/Reperfusion Injury in Skeletal Muscle via Nitric Oxide Release

Abstract: Background— Peroxynitrite generated from nitric oxide (NO) and superoxide (O2−) contributes to ischemia/reperfusion (I/R) injury. Feedback inhibition of endothelial NO synthase by NO may inhibit O2− production generated also by endothelial NO synthase at diminished local l-arginine concentrations accompanying I/R. Methods and Results— During hindlimb I/R (2.5 hours/2 hours), in vivo NO was monitored continuously (porphyrinic sensor), and high-energy phosphates, reduced and oxidized glutathione (chromatography), and I/R injury were measured intermittently. Rabbits receiving human serum albumin (HSA) (controls) were compared with those receiving S-nitroso human serum albumin (S-NO-HSA) beginning 30 minutes before reperfusion for 1 hour or 30 minutes before ischemia for 3.5 hours (0.1 μmol · kg−1 · h− 1). The onset of ischemia led to a rapid increase of NO from its basal level (50±12 nmol/L) to 120±20 and 220±15 nmol/L in the control and S-NO-HSA–treated groups, respectively. In control animals, NO dropped b...

Development of mammalian serum albumin affinity purification media by peptide phage display.

Abstract: Several phage isolates that bind specifically to human serum albumin (HSA) were isolated from disulfide-constrained cyclic peptide phage-display libraries. The majority of corresponding synthetic peptides bind with micromolar affinity to HSA in low salt at pH 6.2, as determined by fluorescence anisotropy. One of the highest affinity peptides, DX-236, also bound well to several mammalian serum albumins (SA). Immobilized DX-236 quantitatively captures HSA from human serum; mild conditions (100 mM Tris, pH 9.1) allow release of HSA. The DX-236 affinity column bound HSA from human serum with a greater specificity than does Cibacron Blue agarose beads. In addition to its likely utility in HSA and other mammalian SA purifications, this peptide media may be useful in the proteomics and medical research markets for selective removal of mammalian albumin from serum prior to mass spectrometric and other analyses.

Beyond Expansion: Structural Studies on the Transport Roles of Human Serum Albumin

Abstract: Introduction Clinically, human serum albumin (HSA) is best known as a volume expander but its prominent role in this capacity perhaps overshadows the protein’s other major talent: transport. As a plasma transporter molecule HSA displays truly bewildering binding capabilities. Its primary physiological ligands are non-esterified long-chain fatty acids but HSA also binds toxic metabolites such as bilirubin and CMPF, steroid hormones, thyroxine, tryptophan and several vitamins and metal ions, facilitating their movement around the body or delivery to sites of disposal’. The natural capacity of HSA for a range of endogenous compounds means that the protein is also able to bind a wide variety of drug molecules including chlorpropamide, diazepam, ibuprofen, indomethacin, naproxen and warfarin. The protein can thus have a dramatic effect on the pharmacokinetics and pharmacodynamics of such agents. The broad binding specificity of albumin has intrigued researchers for decades and stimulated wide-ranging investigations in both basic and clinical sciences which aimed to probe the interactions of HSA with its endogenous ligands and to better understand the impact of the protein on various drug therapies. More recently, efforts have been made to exploit the binding properties of HSA to develop it as a novel delivery vehicle for drugs2 or oxygen’, to modulate drug pharmac~dynamics~ or to use the protein to effect chiral separations’. In order to understand and exploit the binding capacity of HSA more fully, detailed structural information on ligand binding to the protein is required. In recent years my group has been applying protein crystallographic methods in order to determine the structure of HSA and a range of HSA-ligand complexes at essentially atomic resolutionbii. Our work to date has focused on structural analysis of HSA complexes with saturated and unsaturated fatty acids, the hormone thyroxine and a range of drug molecules. These investigations have yielded a range of new insights into the way that HSA accommodates endogenous and exogenous ligands; one of the most interesting issues that we can address with this approach is the extent to which different ligands may variously compete or cooperate in their binding to sites on the protein.

Development of an in vitro screening model for the biosynthesis of acyl glucuronide metabolites and the assessment of their reactivity toward human serum albumin.

Abstract: An in vitro screening model was developed to determine the reactivity of acyl glucuronide metabolites from carboxylic drugs. This assay is composed of two phases. The first is a phase of biosynthesis of acyl glucuronides by human liver microsomes (HLM). The second, during which acyl glucuronides are incubated with human serum albumin (HSA), consists of assessing the reactivity of acyl glucuronides toward HSA. Both phases are performed successively in the same experiment. This model was validated using eight carboxylic drugs that were well known for their reactivity, their extent of covalent binding, and their immunological potential. These products were representative of the scale of reactivity. Each compound was incubated with HLM at 400 microM and metabolized into acyl glucuronide to different extents, ranging from 5.6% (tolmetin) to 89.4% (diclofenac). The first-order aglycone appearance rate constant and the extent of covalent binding to proteins were assayed during the incubation of acyl glucuronides formed with HSA for 24 h. Extensive isomerization phenomenon was observed for each acyl glucuronide between the two phases. An excellent correlation was observed (r(2), 0.94) between the extent of drug covalent binding to albumin and the aglycone appearance constant weighted by the percentage of isomerization. This correlation represents an in vitro reactivity scale, which will be helpful in drug discovery support programs to predict the covalent binding potential of new chemical entities. This screening model will also allow the comparison of acyl glucuronide reactivity for related structure compounds.

Investigation into the interaction of recombinant human serum albumin with Re-lipopolysaccharide and lipid A:

Abstract: The interaction of bacterial endotoxins, deep rough mutant lipopolysaccharide LPS Re and the 'endotoxic principle' lipid A, with recombinant human serum albumin (rHSA) was investigated with a variety of physical techniques and biological assays. With Fourier-transform infrared spectroscopy and differential scanning calorimetry, the influence of albumin on the acyl chain melting behavior of the endotoxins was measured. Also, the effect on the functional groups of the endotoxins, in particular with respect to their orientation, was studied, including competition experiments with polymyxin B. Furthermore, the influence of endotoxin binding to rHSA on the protein's secondary structure was investigated. The results indicate a non-electrostatic binding with no change of the backbone orientation of LPS and only a slight change of the secondary structure of rHSA. Correspondingly, the amount of charge neutralization of the endotoxins due to rHSA measured by the electrophoretic mobility exhibited only a slight reduction of the surface potential. From these measurements and isothermal titration calorimetry, the lipid:protein binding stoichiometry was estimated to [LPS]:[rHSA], 10:1 molar. The determination of the aggregate structure of the endotoxins by X-ray small-angle scattering exhibited a complex change of a cubic into a non-lamellar structure. No influence of rHSA on endotoxin intercalation into phospholipid liposomes induced by lipopolysaccharide-binding protein could be detected by fluorescence resonance energy transfer. Finally, the LPS-induced cytokine production of human mononuclear cells was only slightly increased at high molar rHSA excess, while the coagulation of amebocyte lysate in the Limulus test yielded a complex change due to rHSA binding of LPS.

Evalution of capillary electrophoresis-frontal analysis for the study of low molecular weight drug-human serum albumin interactions.

Abstract: Capillary electrophoresis frontal analysis was applied to 12 low molecular weight compounds including 8 drug substances displaying a range of different properties with respect to binding affinity, binding location, structure, lipophilicity, charge at physiological pH, and electrophoretic mobility. It was found that capillary electrophoresis frontal analysis can be used as a general method to study and quantify drug-human serum albumin interactions. The binding parameters obtained were consistent with literature values. Dextran was in some cases added to the run buffer to improve separation of the drug and human serum albumin plateau peaks. Results indicate that mobility differences between free and complexed human serum albumin give rise to only minor errors. Capillary electrophoresis frontal analysis was also found applicable to the study of human serum albumin drug displacement reactions. Low sensitivity of the UV-detection system was found to be the major limitation of capillary electrophoresis frontal analysis. The method is simple, and minimal effort has to be put into method development, which makes it well suited for screening in early drug development.

How do fatty acids cause allosteric binding of drugs to human serum albumin

Abstract: Purpose. This study was undertaken to investigate how fatty acids cause the allosteric binding of drugs to human serum albumin (HSA). The influence of fatty acids on the binding of ketoprofen (KP), an NSAID, to HSA was examined by using a photoaffinity labeling technique.

Characteristics of bovine hemoglobin as a potential source of hemoglobin-vesicles for an artificial oxygen carrier

Abstract: Hemoglobin-vesicles (HbV) have been developed for use as artificial O(2) carriers in which a purified Hb solution is encapsulated within a phospholipid bilayer membrane. In this study, bovine Hb (BHb) was tested as a source of HbV instead of human Hb (HHb). We compared the preparation process and characteristics of BHbV with those of HHbV. The purification of BHb was effectively performed simply with an ultrafiltration system including a process for removing virus and scrapie reagent. The removal ratio of the phospholipid components of bovine red blood cells was over 99.99%, and the protein purity was over 99.9%. The deoxygenated and carbonylated BHb showed denaturation transition temperatures at 83 and 87 degrees C, respectively, which are higher than those of HHb (80 and 78 degrees C, respectively), and resistant to pasteurization (60 degrees C, 10 h). The purified BHb was concentrated to over 40 g/dl, and encapsulated in a phospholipid bilayer membrane to form BHbV with a diameter of about 280 nm. The O(2) affinity (P(50)) of the BHbV was regulated by coencapsulation of an appropriate amount of Cl(-) (as NaCl), which binds to BHb as an allosteric effector, in the range 16-28 Torr, comparable to human blood (P(50) = 28 Torr). This is quite simple in comparison with HHb which requires phosphate derivatives such as pyridoxal 5'-phosphate as a replacement for 2,3-diphoshoglyceric acid. The viscosity and colloid osmotic pressure of the BHbV when suspended in 5% human serum albumin are 3.5 cP and 20 Torr, respectively, comparable to those of human blood. In conclusion, BHb can be used as a source for the production of HbV, not only because of its abundance in the cattle industry, but also because of the physicochemical advantages of the purification process, thermal stability, and regulation of O(2) affinity in comparison with HHb.

The Effect of a Phosphodiester Linking Group on Albumin Binding, Blood Half-Life, and Relaxivity of Intravascular Diethylenetriaminepentaacetato Aquo Gadolinium(III) MRI Contrast Agents

Abstract: Amphiphilic gadolinium complexes were investigated as potential magnetic resonance imaging (MRI) contrast agents. A series of complexes was synthesized in order to study the effect of hydrophilic phosphodiester groups on albumin binding, relaxivity, and blood half-life in rats. Thus, compound 11a, a diethylenetriaminepentaacetato aquo gadolinium(III) (Gd-DTPA) derivative with an octyl substituent, was synthesized and compared to 5b, the analogous octyl derivative containing a phosphodiester linkage between the gadolinium chelate and the alkyl chain. Likewise, 11b, a naphthyl Gd-DTPA derivative, was compared to the naphthyl phosphodiester derivative 5c. A direct comparison is not available for 5a, a 4,4-diphenylcyclohexyl phosphodiester Gd-DTPA derivative; however, its pharmacokinetic properties mirror those of the other phosphodiester derivatives. Although the introduction of the phosphodiester moiety decreased log P by approximately 1.7 units, albumin binding data obtained in 4.5% human serum albumin (HS...

Negligible depletion solid-phase microextraction with radiolabeled analytes to study free concentrations and protein binding : an example with [3H]Estradiol

Abstract: A new method is presented that enables sensitive measurement of free concentrations of radiolabeled ligands. Additionally, protein binding of radiochemicals in complex matrixes can be determined with this new technique that combines negligible depletion solid-phase microextraction (nd-SPME) with liquid scintillation counting (LSC) as detection. [3H]Estradiol was taken as an example compound. Possible matrix effects of protein on fiber uptake kinetics were studied. No matrix effect was found, either by fouling of the fiber, or by changed uptake kinetics. The validity of the method was shown in the determination of the affinity constant (Ka) of estradiol for human serum albumin (HSA). The Ka was estimated at 8.9 x 104 M-1, which corresponds well with literature values. This study shows that nd-SPME is suitable to study the free concentration and protein binding of [3H]-estradiol. The method described in this paper combines the advantages of nd-SPME with the advantages of radiolabeled analytes, creating a timesaving, simple, and sensitive analytical tool that will be particularly useful in complex matrixes containing many potential interferences for chromatographic methods. Chemicals/CAS: estradiol, 50-28-2; human serum albumin, 9048-49-1

Fluorescence for the determination of protein with functionalized nano-ZnS

Abstract: ZnS nanoparticles have been prepared and modified with sodium thioglycolate. The functionalized nanoparticles are water-soluble. They were used as fluorescence probes in the determination of proteins, which was proved to be a simple, rapid and specific method. In comparison with single organic fluorophores, these nanoparticle probes are brighter, more stable against photobleaching, and do not suffer from blinking. Under optimum conditions, linear relationships were found between the enhanced intensity of fluorescence at 441 nm and the concentration of protein in the range 0.1–4.0 μg mL−1 for human serum albumin (HSA), 0.2–3.0 μg mL−1 for bovine serum albumin (BSA) and 0.1–4.5 μg mL−1 for γ-globulin (γ-G). The limits of detection were 0.015 μg mL−1 for HSA, 0.024 μg mL−1 and 0.017 μg mL−1 for BSA and γ-G, respectively. The method has been applied to the analysis of human serum samples collected from the hospital and the results were in good agreement with those reported by a hospital, indicating that the method presented here is not only sensitive and simple, but also reliable and suitable for practical application.

Fatty acids carried on albumin modulate proximal tubular cell fibronectin production: a role for protein kinase C.

Abstract: Background. Proteinuric renal disease is associated with accumulation of tubulointerstitial matrix proteins. Human proximal tubular cells (PTCs) produce fibronectin in response to serum proteins but not albumin alone. It has been suggested that renal toxicity of filtered albumin depends on its lipid moiety. We therefore investigated the functional consequences of different fatty acids (FAs) carried on human albumin after exposure to human PTCs in culture. Methods. Confluent human PTCs were exposed to recombinant human serum albumin (rHSA) or palmitate (P)-, stearate (S)-, oleate (O)-, and linoleate (L)-complexed rHSA. In all experimental conditions, test media contained 1 mg'ml rHSA alone or carrying 100 mmol FAs. Mitogenic response was assessed by [ 3 H]thymidine incorporation. Cell culture supernatants were assayed for fibronectin. Protein kinase C (PKC) activity was assessed in cell lysates. Results. Apical exposure to rHSA alone or the O-rHSA complex stimulated a significant increase in [ 3 H]thymidine incorporation, whereas the L-rHSA complex was markedly inhibitory to human PTC growth. The L-rHSA complex was associated with severe cytotoxicity as assessed by lactate dehydrogenase release. Among all conditions, O-rHSA was the only test media that significantly increased fibronectin levels over control conditions (150.1 ± 10.6% over control, P<0.05, n=3). Pre-treatment of PTCs with PKC inhibitors before O-rHSA exposure resulted in a dose-dependent decrease in fibronectin secretion. O-rHSA activated PKC significantly compared with controls. Conclusions. We conclude that rHSA has a mitogenic effect on human PTCs, but fibronectin secretion was only induced by O-complexed rHSA and the O-rHSA effect was mediated via PKC activation. Involvement of PKC signal transduction pathway may be a novel therapeutic target for ameliorating proteinuria-induced tubular injury.