Showing papers on "Human serum albumin published in 1999"

Crystal structure of human serum albumin at 2.5 A resolution.

Abstract: A new triclinic crystal form of human serum albumin (HSA), derived either from pool plasma (pHSA) or from a Pichia pastoris expression system (rHSA), was obtained from polyethylene glycol 4000 solution. Three-dimensional structures of pHSA and rHSA were determined at 2.5 A resolution from the new triclinic crystal form by molecular replacement, using atomic coordinates derived from a multiple isomorphous replacement work with a known tetragonal crystal form. The structures of pHSA and rHSA are virtually identical, with an r.m. s. deviation of 0.24 A for all Calpha atoms. The two HSA molecules involved in the asymmetric unit are related by a strict local twofold symmetry such that the Calpha atoms of the two molecules can be superimposed with an r.m.s. deviation of 0.28 A in pHSA. Cys34 is the only cysteine with a free sulfhydryl group which does not participate in a disulfide linkage with any external ligand. Domains II and III both have a pocket formed mostly of hydrophobic and positively charged residues and in which a very wide range of compounds may be accommodated. Three tentative binding sites for long-chain fatty acids, each with different surroundings, are located at the surface of each domain.

Development of porous spherical hydroxyapatite granules: application towards protein delivery.

Abstract: A new method for the preparation of porous spherical hydroxyapatite granules is reported. It may be clinically applied towards orthopaedic or maxillofacial surgery as fillers or packing materials, and as biological chromatography supports. Its application towards delivery of macromolecules or protein drugs is discussed utilizing human serum albumin (HSA) as a model protein.

Fractionated Plasma Separation and Adsorption System: A Novel System for Blood Purification to Remove Albumin Bound Substances

Abstract: The removal of albumin bound substances has gained increasing interest in different diseases, especially in acute and chronic liver disease. Therefore, a new system, the fractionated plasma separation and adsorption (FPSA) system, was developed based on combined membrane and adsorbent blood purification techniques. The most important contribution to the FPSA system was the development of a new polysulfone hollow-fiber filter, which is characterized by a sieving coefficient of 0.89 for human serum albumin (HSA) but only of 0.17 for fibrinogen, and 0 (zero) for IgM immunoglobulins. Using a closed filtrate circuit connected to the new polysulfone filter which integrates 1 or 2 adsorption columns and also a high flux dialyzer adapted to a dialysis machine, the FPSA system opens excellent possibilities for the relatively specific removal of albumin bound substances from the blood such as albumin bound bilirubin or even tryptophan. In comparison to other systems (for example, the Molecular Adsorbent Recirculating System [MARS] and albumin dialysis systems), the FPSA system enables much higher elimination of strongly bound albumin substances. The first clinical investigations have recently started based on a modified dialysis machine designed with all necessary safety measures.

Protein conjugates of synthetic saccharides elicit higher levels of serum IgG lipopolysaccharide antibodies in mice than do those of the O-specific polysaccharide from Shigella dysenteriae type 1.

Abstract: Our development of vaccines to prevent shigellosis is based on the hypothesis that a critical (protective) level of serum IgG to the O-specific polysaccharide (O-SP) domain of Shigella lipopolysaccharide (LPS) confers immunity The O-SP is a hapten and must be conjugated to a protein to induce serum antibodies The O-SP of Shigella dysenteriae type 1 (≈27 tetrasaccharide repeat units), prepared by acid hydrolysis of the LPS, was bound to human serum albumin (HSA) by multiple point attachment (O-SP-HSA): The molar ratio of HSA to O-SP was 10 Synthetic saccharides, composed of one or multiples of the O-SP tetrasaccharide, equipped with a spacer at their reducing end, were bound to HSA by a single point attachment: The average molar ratios of the saccharides to HSA ranged from 4 to 24 Serum IgG anti-LPS, elicited in mice by O-SP-HSA or synthetic tetra-, octa-, dodeca-, and hexadecasaccharide fragments, was measured by ELISA Outbred 6-week-old female mice were injected sc three times at biweekly intervals with 25 μg of saccharide as a conjugate and were bled 7 days after the second and third injections Excepting the tetramer, conjugates of the octamer, dodecamer and hexadecamer elicited IgG LPS antibodies after the second injection, a statistically significant rise (booster) after the third injection, and higher levels than those vaccinated with O-SP-HSA (P = 00001) The highest geometric mean levels of IgG anti-LPS were elicited by the hexadecamer with 9 chains or 9 moles of saccharide/HSA (155 ELISA units) followed by the octamer with 20 chains (111 ELISA units) and the dodecamer with 10 chains (952 ELISA units) Clinical evaluation of these synthetic saccharides bound to a medically useful carrier is planned

Binding of propofol to blood components: implications for pharmacokinetics and for pharmacodynamics

Abstract: Aims Propofol is a widely used iv anaesthetic agent However, its binding properties to blood components have not been fully studied Methods We studied the binding of propofol to erythrocytes, to human serum and to isolated serum proteins Because propofol bound to ultrafiltration and equilibrium dialysis membranes, we used a co-binding technique with dextran coated charcoal and with erythrocytes Results Propofol free fraction in blood was 12–17% at total concentrations ranging from 280 to 179 μm (05 to 32 μg ml−1 ) Fifty percent was bound to erythrocytes and 48% to serum proteins, almost exclusively to human serum albumin In the clinical range of concentrations (05–16 μg ml−1 ) 40% of the molecules bound to erythrocytes are on the red blood cells membranes No binding to lipoproteins occurred and binding to α1-acid glycoprotein was less than 15% Conclusions We conclude that hypoalbuminaemia may increase propofol free fraction particularly during prolonged administration Since propofol is non-restrictively cleared, no change in clearance is expected to occur, and the increase in free fraction will not be compensated by a parallel increase in clearance It is also noted that many in vitro studies used concentrations 50 to 500 times the concentration expected to be encountered in the immediate cellular environment

Amadori albumin in type 1 diabetic patients: correlation with markers of endothelial function, association with diabetic nephropathy, and localization in retinal capillaries.

Abstract: Nonenzymatic glycation is increased in diabetes. Most studies so far have focused on the role of advanced glycation end products (AGEs) in vascular complications, whereas the role of early glycation Amadori-modified proteins, which is the predominant form of glycated proteins, has not been systemically investigated in humans. We developed an antiserum against glycated human serum albumin (HSA) and used this to study the role of early glycation products in vascular complications in type 1 diabetic patients. Amadori albumin was determined to be the recognition epitope of the antiserum. The antibody recognized a specific glucose adduct and a conformational component specific for human albumin in Amadori albumin, with no recognition of AGEs. Plasma Amadori albumin levels were significantly higher in type 1 diabetic patients (n = 55) than in healthy control subjects (n = 60) (39.2+/-9.9 vs. 20.9+/-4.0 U/ml, P < 0.0005). Amadori albumin correlated with levels of plasma markers of endothelial function von Willebrand factor (r = 0.29, P < 0.05) and vascular cell adhesion molecule-1 (r = 0.41, P < 0.005), but not soluble E-selectin. In addition, Amadori albumin immunoreactivity was detected in the capillaries of retinas of diabetic patients. Plasma levels of Amadori albumin were determined in a second group of type 1 diabetic patients with long-standing diabetes with (n = 199) or without (n = 192) diabetic nephropathy. Patients with nephropathy had higher Amadori albumin levels than did those without it (50.9+/-9.5 vs. 45.1+/-6.3 U/ml, P < 0.0005). Age-, sex-, and diabetes duration-adjusted analyses showed that nephropathy was significantly associated with Amadori albumin with an odds ratio (OR [95% CI]) of 1.11 [1.08-1.15] per U/ml increase. After additional adjustment for levels of creatinine, glycated hemoglobin, cholesterol, triglycerides, blood pressure, preexistent retinopathy, and cardiovascular disease, Amadori albumin continued to be significantly associated with nephropathy (OR 1.06 [1.01-1.11]) per U/ml increase. Our results are consistent with a proposed pathophysiological role of Amadori albumin in microvascular complications of type 1 diabetic patients.

Alkylation of human serum albumin by sulfur mustard in vitro and in vivo: mass spectrometric analysis of a cysteine adduct as a sensitive biomarker of exposure.

Abstract: To develop a mass spectrometric assay for the detection of sulfur mustard adducts with human serum albumin, the following steps were performed: quantitation of the binding of the agent to the protein by using [14C] sulfur mustard and analysis of acidic and tryptic digests of albumin from blood after exposure to sulfur mustard for identification of alkylation sites in the protein. The T5 fragment containing an alkylated cysteine could be detected in the tryptic digest with micro-LC/tandem MS analysis. Attempts to decrease the detection limit for in vitro exposure of human blood by analysis of the alkylated T5 fragment were not successful. After Pronase treatment of albumin, S-[2-[(hydroxyethyl)thio]ethyl]Cys-Pro-Phe was analyzed by means of micro-LC/tandem MS, allowing a detection limit for in vitro exposure of human blood of 10 nM, which is 1 order of magnitude lower than that obtained by means of modified Edman degradation. The analytical procedure could be successfully applied to the analysis of albumin samples from Iranian victims of the Iran-Iraq war.

Erythropoietin analog human serum albumin fusion protein

Abstract: Erythropoietin analog-human serum albumin (EPOa-hSA) fusion protein and methods of making and using the fusion protein.

Molten globule-like state of human serum albumin at low pH.

Abstract: Human serum albumin (HSA), under conditions of low pH, is known to exist in two isomeric forms, the F form at around pH 4.0 and the E form below 3.0. We studied its conformation in the acid-denatured E form using far-UV and near-UV CD, binding of a hydrophobic probe, 1-anilinonaphthalene-8-sulfonic acid (ANS), thermal transition by far-UV and near-UV CD, tryptophan fluorescence, quenching of tryptophan fluorescence using a neutral quencher, acrylamide and viscosity measurements. The results show that HSA at pH 2.0 is characterized by a significant amount of secondary structure, as evident from far-UV CD spectra. The near-UV CD spectra showed a profound loss of tertiary structure. A marked increase in ANS fluorescence signified extensive solvent exposure of non-polar clusters. The temperature-dependence of both near-UV and far-UV CD signals did not exhibit a co-operative thermal transition. The intrinsic fluorescence and acrylamide quenching of the lone tryptophan residue, Trp214, showed that, in the acid-denatured state, it is buried in the interior in a non-polar environment. Intrinsic viscosity measurements showed that the acid-denatured state is relatively compact compared with that of the denatured state in 7 m guanidine hydrochloride. These results suggest that HSA at pH 2.0 represents the molten globule state, which has been shown previously for a number of proteins under mild denaturing conditions.

Adsorption of serum albumins at the air/water interface

Abstract: The adsorption of bovine serum albumin and human serum albumin (HSA) at the air/water interface has been studied by neutron specular reflection. All the neutron measurements were performed in null reflecting water (D2O:H2O ≃ 1:11), and the specular reflectivity at this water contrast is entirely from the adsorbed protein layer at the air/water interface. Accurate measurement of surface excesses and layer thicknesses has allowed us to infer the possible structural conformation of the two protein molecules on the surface of water. The effect of bulk protein concentration on the adsorbed amount and the total thickness of the adsorbed layer was examined at pH 5, close to the isoelectric point of 4.8 for the two albumins. The surface excess (Γ) of both proteins increased sharply over the concentration range of 5 × 10-4 to 5 × 10-2 g dm-3, beyond which Γ tended to the respective saturation limits. The saturation value of surface excess of BSA at 1 g dm-3 was found to be 2.8 ± 0.3 mg m-2 as compared with 2.1 ± 0...

Contrast agents for magnetic resonance angiographic applications: 1H and 17O NMR relaxometric investigations on two gadolinium(III) DTPA-like chelates endowed with high binding affinity to human serum albumin.

Abstract: -pentaacetic acid) bearing different substituents for binding to human serum albumin (HSA) are compared. In spite of the structural differences of the recognition synthon and of the residual electric charge, the two chelates display an analogous binding affinity for the serum protein. Upon formation of the adducts with HSA, the exchange rates of the coordinated water appear slowed down by an amount corresponding to ca. 50% of the rates found for the free complexes. The relaxivity of [Gd(BOM)3DTPA (H2O)]2 − is significantly higher than that of MS-325 either in the free complex or in the macromolecular adduct. Finally, the effect of pH on the stability of the HSA adducts and on the values of their relaxivities has been investigated.

Interaction of curcumin with human serum albumin--a spectroscopic study.

Abstract: Curcumin (diferuloyl methane) has a wide range of physiological and pharmacological actions. Curcumin interaction with human serum albumin (HSA) has been followed by fluorescence quenching and circular dichroism (CD) measurements. Based on fluorescence measurements, the equilibrium constant for the interaction is 2.0+/-0.2x10(5) M(-1). Binding of curcumin to HSA induces an extrinsic CD band in the visible region. From the induced CD band measurements, the equilibrium constant has a value of 2.1+/-0.3x10(4) M(-1). Thus, HSA has two kinds of affinity sites for curcumin, one with high affinity and the other with lower affinity. Job's plot indicated a binding stoichiometry of 1:1 for the high-affinity site. The equilibrium constant was invariant with temperature in the range of 15 to 45 degrees C, suggesting the role of hydrophobic interactions in the binding of curcumin to HSA. Curcumin does not change the conformation of the HSA molecule. These measurements have implications in the understanding of the curcumin transport under physiological conditions.

Photophysics of hypericin and hypocrellin A in complex with subcellular components: interactions with human serum albumin.

Abstract: — Time-resolved fluorescence and absorption measurements are performed on hypericin complexed with human serum albumin, HSA (1:4, 1:1 and ∼5:1 hypericin: HSA complexes). Detailed comparisons with hypocrellin A/HSA complexes (1:4 and 1:1) are made. Our results are consistent with the conclusions of previous studies indicating that hypericin binds to HSA by means of a specific hydrogen-bonded interaction between its carbon-yl oxygen and the N1-H of the tryptophan residue in the HA subdomain of HSA. (They also indicate that some hypericin binds nonspecifically to the surface of the protein.) A single-exponential rotational diffusion time of 31 ns is measured for hypericin bound to HSA, indicating that it is very rigidly held. Energy transfer from the tryptophan residue of HSA to hypericin is very efficient and is characterized by a critical distance of 94 A, from which we estimate a time constant for energy transfer of ∼3 × 10–15 s. Although it is tightly bound to HSA, hypericin is still capable of executing excited-state intramolecular proton (or hydrogen atom) transfer in the ∼5:1 complex, albeit to a lesser extent than when it is free in solution. It appears that the proton transfer process is completely impeded in the 1:1 complex. The implications of these results for hypericin (and hypocrellin A) are discussed in terms of the mechanism of intramolecular excited-state proton transfer, the mode of binding to HSA and the light-induced antiviral and antitumor activity.

The Fluorescent Probe Prodan Characterizes the Warfarin Binding Site on Human Serum Albumin

Abstract: — The fluorescent probe Prodan (6-propionyl-2-dimethyl-aminonaphthalene) binds with high affinity to human serum albumin (HSA). The spectral characteristics of the Prodan bound to the protein are very different from the free Prodan in solution. These differences allowed the spectra to be deconvoluted into log-normal bands in order to quantify the bound and unbound ligand and to calculate the binding constant at different temperatures. From such temperature dependence, we found the binding to be exothermic with a van't Hoff enthalpy of -22.8 kJ mol-1. Thermodynamic analysis suggests that the in teraction may be mainly caused by hydrophobic forces and electrostatic interactions. The above analysis of the spectra and the measures of the fluorescence polarization during the successive presence of six specific drugs suggest that the Prodan binding site corresponds with the warfarin binding site on HSA, whereas under the present experimental conditions the other characteristic binding sites of HSA were not affected. Thus, this fluorescent probe provides a rapid and simple means for the characterization of a specific binding site on HSA and also for detecting potential or nonspecific drug-protein interactions.

Re-formation of the Helical Structure of Human Serum Albumin by the Addition of Small Amounts of Sodium Dodecyl Sulfate after the Disruption of the Structure by Urea. A Comparison with Bovine Serum Albumin

Abstract: Upon the addition of small amounts of sodium dodecyl sulfate (SDS), the helicity of human serum albumin (HSA), lost in the urea denaturation, was mostly recovered. The profile of the recovery differed depending on the urea concentration. Then the urea concentrations were divided into three ranges: [1] a range below 3 M where the helicity only decreased as in the absence of urea (the helicity decreased down to 49% in the SDS solution); [2] a range between 4 and 8 M where the helicity initially increased up to 66% (this was the same as in the native state) and then sharply decreased; [3] a range above 9 M where the helicity only increased with an increase in added SDS concentration. When SDS was added prior to the urea denaturation, the same helicity was obtained at each surfactant concentration. Thus the SDS denaturation finally predominates over the urea denaturation. In the middle range, profiles of the structural change were rather complicated. The increase and decrease of helicity were accomplished be...

Formation of an Intrapolymer Complex from Human Serum Albumin and Poly(ethylene glycol)

Abstract: Complexation between human serum albumin (HSA) and poly(ethylene glycol) (PEG) was studied using different experimental techniques: quasi-elastic light scattering (QELS), static light scattering (SLS), electrophoretic light scattering (ELS), dialysis, and fluorescence spectroscopy. The QELS study for aqueous HSA-PEG mixtures at different levels of pH and ionic strength (NaCl) showed the formation of a watersoluble complex, the size of which varied depending on both the ionic strength and the molecular weight of PEG but remained unaltered when the mixing ratio of PEG to HSA was varied. The study of the complexation in the presence and absence of 1 M urea as a function of pH by QELS and fluorescence spectroscopy strongly suggested that hydrogen bonding plays an important role in the complex formation. A combination of SLS and dialysis at pH 2 and at the ionic strength 0.1 demonstrated that the complexation yielded an “intrapolymer” complex in which several HSA molecules bound to a PEG chain. In addition, ELS indicated that the resulting intrapolymer complex behaves like a free draining coil during electrophoresis.

Improved two-dimensional gel electrophoresis representation of serum proteins by using ProtoClear.

Abstract: ProtoClear is a proprietary technique for clearing albumin and immunoglobulin G (IgG) from human serum samples. Albumin constitutes 57-71% of total serum protein and IgG ranges from 8-26%. Removal of these two proteins alone clears approximately 75% of the total protein present in serum and allows the detection of the remaining proteins that are present in far lower concentrations. ProtoClear effectively removed >95% of human serum albumin (HSA) and >97% of human IgG as measured by an anti-HSA competitive immunoassay and a radial immunodiffusion assay, respectively. ProtoClear was far more specific at removing albumin and IgG than Cibracon Blue Dye chromatography (Cibracon Blue), the typically utilized alternative. Comparing two-dimensional (2-D) gels of serum cleared by either Cibracon Blue or by ProtoClear, it was apparent that Cibracon Blue removed a number of proteins in addition to albumin. Following removal of albumin and IgG from serum, we found a significant improvement in the resolution of polypeptide spots detected on two-dimensional gels.

Interaction of Acrylodan with Human Serum Albumin. A Fluorescence Spectroscopic Study

Abstract: The binding of the fluorescent probe acrylodan (AC) to human serum albumin (HSA) was studied by fluorescence spectroscopy. The binding isotherms could be fitted to two types of sites. Competition experiments using io-doacetamide suggested that AC binds tightly on HSA by the cysteine-34. Attempts were made to find the location of the second site using high concentrations of warfarin, phenylbutazone, diazepam, indomethacin, palmitic acid or bilirubin in order to displace the bound AC to the HSA. Bilirubin was the only ligand able to displace the bound AC. This result suggests that AC, which is a very hydrophobic molecule also capable of labeling lysine residues, should also bind the human albumin in the primary site of bilirubin, but with less affinity than to the cysteine-34.

Altered Pharmacokinetics of a Novel Anticancer Drug, UCN-01, Caused by Specific High Affinity Binding to α1-Acid Glycoprotein in Humans

Abstract: The large species difference in the pharmacokinetics/pharmacodynamics of 7-hydroxystaurosporine (UCN-01) can be partially explained by the high affinity binding of UCN-01 to human α 1 -acid glycoprotein (AGP) (Fuse et al. , Cancer Res., 58: 3248–3253, 1998). To confirm whether its binding to human AGP actually changes the in vivo pharmacokinetics, we have studied the alteration in its pharmacokinetics after simultaneous administration of human AGP to rats: ( a ) the protein binding of UCN-01 was evaluated by chasing its dissociation from proteins using dextran-coated charcoal. The UCN-01 remaining 0.1 h after adding dextran-coated charcoal to human plasma or AGP was ∼80%, although the values for other specimens, except monkey plasma (∼20%), were b ) the pharmacokinetics of UCN-01 simultaneously administered with human AGP has been determined. The plasma concentrations after i.v. administration of UCN-01 with equimolar human AGP were much higher than those after administration of UCN-01 alone. The steady-state distribution volume and the systemic clearance were reduced to about 1/100 and 1/200, respectively. Human AGP thus reduced the distribution and elimination of UCN-01 substantially. On the other hand, dog AGP, which has a low binding affinity for UCN-01, did not change the pharmacokinetics of UCN-01 so much. Furthermore, human AGP markedly reduced the hepatic extraction ratio of UCN-01 from 0.510 to 0.0326. Also, human AGP (10 μm) completely inhibited the initial uptake of UCN-01 (1 μm) into isolated rat hepatocytes, whereas the uptake of UCN-01 was unchanged in the presence of human serum albumin (10 μm). In conclusion, the high degree of binding of UCN-01 to human AGP causes a reduction in the distribution and clearance, resulting in high plasma concentrations in humans.

Characterization of Minor Site Probes for Human Serum Albumin by High-Performance Affinity Chromatography

Abstract: This study used high-performance affinity chromatography (HPAC) and immobilized human serum albumin (HSA) columns to examine the specificity and cross-reactivity of various compounds that have been proposed as markers for the minor binding sites of HSA. These agents included acetyldigitoxin and digitoxin as probes for the digitoxin site, phenol red as a probe for the bilirubin site, and cis- or trans-clomiphene as markers for the tamoxifen site. None of these probes showed any significant binding at HSA's indole−benzodiazepine site. However, phenol red did bind at the warfarin−azapropazone site of HSA, and cis/trans-clomiphene gave positive allosteric effects caused by the binding of warfarin to HSA. Digitoxin and acetyldigitoxin were found to bind to a common, unique region on HSA; cis- and trans-clomiphene also appeared to interact at a unique site, although trans-clomiphene displayed additional direct competition with phenol red. From these results it was possible to develop a model that described the ...

Adsorption of human serum albumin: Dependence on molecular architecture of the oppositely charged surface

Abstract: We contrast the adsorption of human serum albumin (HSA) onto two solid substrates previously primed with the same polyelectrolyte of net opposite charge to form one of two alternative structures: randomly adsorbed polymer and the “brush” configuration. These structures were formed either by the adsorption of quaternized poly-4-vinylpyridine (QPVP) or by end-grafting QPVP chains of the same chemical makeup and the same molecular weight to surfaces onto which QPVP segments did not adsorb. The adsorption of HSA was quantified by using Fourier transform infrared spectroscopy in attenuated total reflection (FTIR-ATR). The two substrates showed striking differences with regard to HSA adsorption. First, the brush substrate induced lesser perturbations in the secondary structure of the adsorbed HSA, reflecting easier conformational adjustment for longer free segments of polyelectrolyte upon binding with the protein. Second, the penetration of HSA into the brush substrate was kinetically retarded relative to the r...