Monomeric Albumin Research Articles

Molecular heterogeneity of urinary albumin in glomerulonephritis: Comparison of cardiovascular disease with albuminuria

Background Despite the unstable structure of urinary albumin in kidney diseases, urinary albumin fragments have been identified by denaturing methods such as two-dimensional electrophoresis. This study examined the relationship between the structural heterogeneity of urinary albumin and protease effects. Methods Urine samples from patients with glomerulonephritis (GN), cardiovascular diseases (CVD), and healthy subjects were analyzed by non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE), Western blot, diagonal 2-dimensional non-reducing/reducing (d2D) SDS PAGE, and albumin zymography. Results The major band was monomer albumin in CVD and healthy subjects; however, 13 urinary albumin bands ranging from 55 to 172 kDa were identified by non-reducing SDS PAGE in GN. The results from d2D SDS PAGE showed urinary albumin polymerization between disulfide bridges, interactions with other proteins, and reduction induced degradation in GN patients. The results from albumin zymography showed that low-molecular mass forms of albumin did not necessarily correspond to high protease activity. Furthermore, concentrated healthy urine showed similar protease digestion as in GN without low-molecular mass of albumin. Conclusions The molecular alterations observed cannot be explained only by urinary proteases. The specific alteration of urinary albumin molecules in GN can be attributed to different mechanisms to CVD.

Albuminuria in Hematopoietic Cell Transplantation Patients: Prevalence, Clinical Associations, and Impact on Survival

Chronic kidney disease (CKD) is common after hematopoietic cell transplantation (HCT). We prospectively measured the urinary albumin:creatinine ratio (ACR) in 142 patients. Total (intact) monomeric albumin was determined by liquid chromatography of untreated urine samples collected weekly to day 100 after HCT. Albuminuria was defined as ACR (mg/g creatinine) > 30; proteinuria, as ACR >300. Cox and logistic regression analyses evaluated ACR as a risk factor for clinical events. The prevalence of albuminuria was 37% at baseline, 64% at day 100, and 50% at 1 year. Proteinuria occurred in 4% of patients at baseline, in 15% at day 100, and in 4% at 1 year. Characteristics associated with albuminuria include age, sex, donor type, hypertension, and sinusoidal obstruction syndrome (SOS). Albuminuria was associated with an increased risk of acute graft-versus-host disease (aGVHD) and bacteremia, but not acute kidney injury (AKI). Albuminuria at day 100 was associated with CKD at 1 year (odds ratio = 4.0; 95% confidence interval [CI] = 1.1 to 14.6). Nonrelapse mortality (NRM) risk was elevated (hazard ratio = 6.8; 95% CI = 1.1 to 41.5) in patients with overt proteinuria at day 100. Albuminuria occurs frequently after HCT and is correlated with aGVHD, bacteremia, hypertension, and progression of renal disease. Proteinuria at day 100 is associated with an 6-fold increased risk of NRM by 1 year after HCT.

Albumin molecularly imprinted polymer with high template affinity — Prepared by systematic optimization in mixed organic/aqueous media

An approach using systematic optimization for the formation of an albumin molecularly imprinted polymer (MIP), able to separate albumin from proteins in solution, has been prepared by imprinting albumin using a copolymer comprising 3-dimethylaminopropyl methacrylate and tetraethylene glycol dimethacrylate in a mole ratio of 1 to 8. Cytochrome c, lysozyme and myoglobin were used in competitive re-binding experiments to compete with the polymer's native template with all protein species present at 0.0004 g mL − 1 . The effects of: monomer to crosslinker mole ratio, polymerization temperature and time were investigated. It was found that the addition of water 6.04%, into the pre-polymerization albumin–monomer complex enhanced the adsorption capacity and selectivity of the resulting MIP from 2.18 × 10 − 3 to 6.02 × 10 − 3 g g-MIP − 1 and 83.5% to 98.7%, respectively. These results also showed that the MIP possessed high selectivity and adsorption capacity with respect to albumin in comparison with interfering species also present in solution. Polymerization temperature, time and the water content of the pre-polymerization mixture were all shown to have significant effects on the resulting albumin-MIP's performance. However, their influence on the polymer's affinity for the potentially interfering species was negligible. Additionally, higher polymerization temperatures (> 38 °C) and extended polymerization times (> 60 h) increased monomer conversion as determined by HPLC, but decreased the selectivity and adsorption capacity of the MIP. An optimized MIP, with very high selectivity and 6.37 × 10 − 3 g g-MIP − 1 template re-adsorption capacity was obtained using the following polymerization conditions: 0.125 mole ratio of monomer to crosslinker, 6.04 wt.% water content with respect to the mass of the monomer complex, 60 h polymerization time at 38 °C, and with 0.47% albumin in the pre-polymerization monomer complex. Finally, the functions of polymerization temperature, time and the significance of the water content in the albumin–monomer complex are also discussed.

Phosphorylation Relieves Autoinhibition of the Kinetochore Motor Cenp-E

During mitosis, chromosome alignment depends on the regulated dynamics of microtubules and on motor protein activities. At the kinetochore, the interplay between microtubule-binding proteins, motors, and kinases is poorly understood. Cenp-E is a kinetochore-associated kinesin involved in chromosome congression, but the mechanism by which this is achieved is unclear. Here, we present a study of the regulation of Cenp-E motility by using purified full-length (FL) Xenopus Cenp-E protein, which demonstrates that FL Cenp-E is a genuine plus-end-directed motor. Furthermore, we find that the Cenp-E tail completely blocks the motility of Cenp-E in vitro. This is achieved through direct interaction between its motor and tail domains. Finally, we show that Cenp-E autoinhibition is reversed by MPS1- or CDK1-cyclin B-mediated phosphorylation of the Cenp-E tail. This suggests a model of dynamic control of Cenp-E motility, and hence chromosome congression, dependent upon phosphorylation at the kinetochore.

Reversible and irreversible membrane fouling during in-line microfiltration of concentrated protein solutions

In-line microfiltration which is widely used for sterilization of therapeutic proteins prior to formulation is frequently operated at constant flux by pumping purified protein solution through a membrane. Increase in transmembrane pressure (TMP) due to membrane fouling is a major problem in such processes. This study examines how resistance to filtration increases during in-line microfiltration of concentrated protein solutions. The TMP was found to increase in three distinct phases, each characterized by its own rate and extent of reversibility. The major portion of the TMP increase was found to be reversible upon front washing with buffer without any kind of physical or chemical cleaning. There was no evidence of concentration polarization in the processes studied. Size exclusion chromatographic analysis of front wash samples suggested bovine serum albumin (BSA) monomer as the major foulant accountable for the reversible fouling. Aggregate free protein solutions prepared by pre-filtration showed negligible membrane fouling. We hypothesize that trace amounts of protein aggregates present in untreated feed solutions were responsible for initiating membrane fouling. However, the actual fouling was caused by the protein monomer which was sequestered on and within the membrane by association with the retained aggregates. The location of foulant on and within the membrane was examined by confocal laser scanning microscopy. The deposition of the foulant was found to be restricted to a zone very close to the membrane surface. Intermittent protein filtration experiments were carried out in an effort to gain better insight on the membrane fouling process and to identify new strategies to maximize product throughput.

Reduced immunoreactivity of urinary albumin in patients with cardiovascular diseases: Analysis of immunochemically nonreactive albumin

We analyzed 55 spot urine samples from patients with cardiovascular diseases. Urinary albumin concentrations were measured with size exclusion high performance liquid chromatography (HPLC), turbidimetric immunoassay (TIA) using anti human serum albumin polyclonal antibody, and enzyme-linked immunosorbent assay (ELISA) using anti human serum albumin monoclonal antibody. Fractionated urine samples from the HPLC were also analyzed with the immunoprecipitation reactions using as same monoclonal antibody as ELISA. As a result, the urinary albumin concentration analyzed by the HPLC was systematically higher than that of immunoassays, however, ‘albumin peak’ from the HPLC contained other urinary proteins. Result of immunoprecipitation reaction showed the presence of monomer albumin that could not react with the monoclonal antibody. These results suggest that not only contamination of other proteins, the albumin fraction from the HPLC included albumin with reduced its reactivity to the specific monoclonal antibody.

Separation of biomacromolecules by electrofiltration through gel layers

A straightforward method for concomitant separation and isolation of biomacromolecules from a mixture in solution was developed. Three gel layers that comprise a middle separation layer of 10% polyacrylamide gel were constructed. This gel system was formed in an electroconcentration apparatus above a collection chamber surrounded at the bottom by a dialysis membrane. The mixture is applied over the gel layers where biomacromolecules are caused to migrate by electrophoresis through the gel system, where they are separated into discrete bands and electroeluted into the collection chamber without dismantling the apparatus. The isolated biomacromolecules are removed from the chamber in a highly pure and concentrated form ready for further investigations. Cooling can be applied throughout the whole process, and the setup and conditions of run can be modified according to the characteristics of the biomacromolecules to be purified. The components of a mixture containing the glycoprotein ovalbumin and bovine serum albumin monomer, dimer, and tetramer were successfully isolated as concentrated and highly pure fractions with good recoveries ranging from 70 to 89%. Other proteins were successfully isolated under denaturing conditions in the presence of sodium dodecyl sulfate (SDS) or 6 M urea.

Thermal stabilization of human albumin by medium‐ and short‐chain n‐alkyl fatty acid anions

A comprehensive study of the thermal stabilization of defatted human albumin monomer by n-alkyl fatty acid anions (FAAs), formate through n-decanoate, was carried out by differential scanning calorimetry (DSC). The concentration of each ligand affording maximum thermal stabilization was determined; n-nonanoate provides the greatest stabilization but is only marginally better than n-octanoate and n-decanoate. The use of reversible thermodynamics and a two-state denaturation model for albumin has been validated. Standard free energies of binding, calculated from increases in free energy of denaturation, for n-butanoate and longer FAAs, are linear with n-alkyl chain length whereas those for formate, acetate, and n-propionate deviate from linearity; those for acetate and n-propionate are even greater than that of n-butanoate, thereby suggesting, in addition to the common class of sites available to all such ligands, the presence of an additional class of lower affinity binding sites available only to these shortest ligands. Competition experiments involving acetate and n-octanoate and involving n-pentanoate and n-octanoate confirmed the binding of acetate to lower affinity sites unavailable to n-octanoate and n-pentanoate. Furthermore, an equation is provided, allowing computation of the transition temperature as a function of the free energy for any reversible process causing a change in thermal stability of a protein undergoing reversible, two-state denaturation. With this equation, modeling the competition experiments by using the binding parameters determined by DSC provides additional support for the class of lower affinity sites, which play a significant role in thermal stabilization of albumin at higher concentrations of these shortest FAAs.

A covalently linked recombinant albumin dimer is more rapidly cleared in vivo than are wild-type and mutant C34A albumin

Mammalian albumins are abundant plasma proteins that exhibit a relatively slow terminal clearance. For this reason they have been fused to potentially therapeutic proteins with rapid terminal clearance to produce fusion proteins with more desirable clearance profiles. A disulfide-linked albumin dimer has been described, but its abundance and stability in plasma are uncertain. To determine whether an obligatory albumin dimer incapable of dissociation would clear less rapidly than monomeric albumin, we expressed 3 recombinant rabbit serum albumin (RSA) polypeptides: H 6RSA, RSA modified by the addition of an N-terminal hexahistidinyl tag; H 6RSA(C34A), H 6RSA with a single cysteine (Cys) 34–to–alanine (Ala) substitution (C34A); and DiRSA, H 6RSA(C34A) joined by way of its C-terminus to RSA(C34A) through an intervening hexaglycine spacer. The C34A mutation was introduced to eliminate the possibility of disulfide bond–mediated dimerization. We expressed the proteins with the use of the yeast Pichia pastoris and purified them using nickel-chelate, ion exchange, and gel-filtration chromatography. After radioiodination and injection into rabbits, H 6RSA and H 6RSA(C34A) exhibited indistinguishable terminal catabolic half-lives (4.9 ± 0.7 and 4.8 ± 0.5 days, mean ± SD), whereas that of DiRSA was reduced to 3.0 ± 0.3 days (p < .05). The three proteins circulated in intact form, and their distributions in liver, lung, kidney, heart, and spleen did not differ 24 hours after injection. Although more DiRSA than H 6RSA(C34A) was present in urine, in both cases it was in acid-soluble form. Ethyl palmitate treatment reduced the relative acceleration of DiRSA clearance compared with that of H 6RSA(C34A), suggesting a role for the reticuloendothelial system in the differential clearance of the larger protein. Our results suggest that an albumin fusion protein should include only a single copy of albumin; that if the fusion protein exceeds a certain size, it may not acquire the slow clearance profile of native albumin; and that albumin dimerization through Cys34 probably does not contribute substantially to albumin metabolism in vivo.

The cytochrome bc1 and cytochrome c oxidase complexes associate to form a single supracomplex in yeast mitochondria.

The mitochondrial electron transport chain complexes are large multisubunit complexes embedded in the inner membrane. We report here that in the yeast Saccharomyces cerevisiae, the cytochrome bc(1) and cytochrome c oxidase complexes co-exist as a larger complex of approximately 1000 kDa in the mitochondrial membrane. Following solubilization with a mild detergent, the cytochrome bc(1)-cytochrome c oxidase complex remains stable. It was analyzed using the techniques of gel filtration and blue native-polyacrylamide gel electrophoresis. Direct physical association of subunits of the cytochrome bc(1) complex with those of the cytochrome c oxidase complex was verified by co-immunoprecipitation analysis. Our data indicate that the cytochrome bc(1) complex is exclusively in association with the cytochrome c oxidase complex in yeast mitochondria. We term this complex the cytochrome bc(1)-cytochrome c oxidase supracomplex.

Checking the integrity of albumin after pasteurisation: A fluorescent assay for quantitating monomeric albumin using anthroyloxy-fatty acids analogs

This study presents a new assay based on the binding of anthroyloxy-stearates to albumin and the change in the fluorescence intensity when these fluorescent probes are bound to the protein. This assay permits the quantification of the albumin monomers present in a mixture of monomers and polymers, and allows to check the integrity of albumin after pasteurisation.

Alterations in renal degradation of albumin in early experimental diabetes in the rat: a new factor in the mechanism of albuminuria

1. Albumin is normally excreted as a mixture of intact protein and fragments that are produced during renal passage. The purpose of this study was to investigate the ratio of intact versus degraded forms of excreted albumin to ascertain whether changes in this ratio could account for the apparent increase in albumin excretion seen in diabetes, as measured by standard radioimmunoassay techniques. 2. Four-week male Sprague-Dawley rats with streptozotocin-induced diabetes and age-matched control rats were intravenously injected with [3H]albumin. Urine collected over 2 h was analysed by size exclusion chromatography and radioimmunoassay. A standard radioimmunoassay found a 7-fold increase in albumin excretion rate in diabetic rats, whereas there was only a 2-fold increase in albumin excretion (intact plus fragments). Urine analysed by size exclusion chromatography showed severe degradation for control rats (% monomer=4+/-2%); in diabetic rats there was a significant amount of monomer albumin excreted, along with moderately degraded and heavily degraded albumin (% monomer=17+/-5%). 3. This study has shown that the radioimmunoassay, which specifically detects intact albumin, considerably underestimates the amount of total urinary albumin which consists of intact and degraded material. The increase in albumin excretion rate observed in diabetes as measured by radioimmunoassay is mainly due to a change in the amount of intact albumin excreted and this is specifically due to the inhibition of albumin degradation at a post-glomerular site and not due to the onset of any type of glomerular 'shunt' pathway.

Alterations in renal degradation of albumin in early experimental diabetes in the rat: a new factor in the mechanism of albuminuria

1.Albumin is normally excreted as a mixture of intact protein and fragments that are produced during renal passage. The purpose of this study was to investigate the ratio of intact versus degraded forms of excreted albumin to ascertain whether changes in this ratio could account for the apparent increase in albumin excretion seen in diabetes, as measured by standard radioimmunoassay techniques. 2.Four-week male Sprague–Dawley rats with streptozotocin-induced diabetes and age-matched control rats were intravenously injected with [3H]albumin. Urine collected over 2 h was analysed by size exclusion chromatography and radioimmunoassay. A standard radioimmunoassay found a 7-fold increase in albumin excretion rate in diabetic rats, whereas there was only a 2-fold increase in albumin excretion (intact plus fragments). Urine analysed by size exclusion chromatography showed severe degradation for control rats (% monomer = 4±2%); in diabetic rats there was a significant amount of monomer albumin excreted, along with moderately degraded and heavily degraded albumin (% monomer = 17±5%). 3.This study has shown that the radioimmunoassay, which specifically detects intact albumin, considerably underestimates the amount of total urinary albumin which consists of intact and degraded material. The increase in albumin excretion rate observed in diabetes as measured by radioimmunoassay is mainly due to a change in the amount of intact albumin excreted and this is specifically due to the inhibition of albumin degradation at a post-glomerular site and not due to the onset of any type of glomerular ‘shunt' pathway.

The RNA molecule CsrB binds to the global regulatory protein CsrA and antagonizes its activity in Escherichia coli.

The RNA-binding protein CsrA (carbon storage regulator) is a new kind of global regulator, which facilitates specific mRNA decay. A recombinant CsrA protein containing a metal-binding affinity tag (CsrA-H6) was purified to homogeneity and authenticated by N-terminal sequencing, matrix-assisted laser desorption/ionization time of flight mass spectrometry, and other studies. This protein was entirely contained within a globular complex of approximately 18 CsrA-H6 subunits and a single approximately 350-nucleotide RNA, CsrB. cDNA cloning and nucleotide sequencing revealed that the csrB gene is located downstream from syd in the 64-min region of the Escherichia coli K-12 genome and contains no open reading frames. The purified CsrA-CsrB ribonucleoprotein complex was active in regulating glg (glycogen biosynthesis) gene expression in vitro, as was the RNA-free form of the CsrA protein. Overexpression of csrB enhanced glycogen accumulation in E. coli, a stationary phase process that is repressed by CsrA. Thus, CsrB RNA is a second component of the Csr system, which binds to CsrA and antagonizes its effects on gene expression. A model for regulatory interactions in Csr is presented, which also explains previous observations on the homologous system in Erwinia carotovora. A highly repeated nucleotide sequence located within predicted stem-loops and other single-stranded regions of CsrB, CAGGA(U/A/C)G, is a plausible CsrA-binding element.

Advanced glycation end product (AGE): characterization of the products from the reaction between D-glucose and serum albumin.

We incubated bovine serum albumin (BSA) with glucose in an attempt to study how the advanced glycation end products (AGEs) are formed and what methods can be used for their identification and isolation. The reaction was monitored by boronated affinity gel, size exclusion and ion exchange chromatography, and chromatofocusing. Reaction products were also characterized by fluorescence measurement, fructosamine assay, and polyacrylamide gel electrophoresis (PAGE). Based on the measurement of AGE-associated fluorescence (excitation, 370 nm; emission, 440 nm) we found that the AGEs could be detected as early as after 3 days incubation. The fluorescence was always associated with the larger molecules of cross-linking product resulting from the reaction between BSA and glucose. The overall fluorescence intensity increased with incubation time and fluorescence of the highest intensity was found with the AGE product largest in size. As with the Amadori product, AGEs also bind to the boronated gel column but with an even higher affinity. Compared to the original albumin monomer AGE molecules are not only larger in size but also have lower isoelectric points and carry more negative charges. Both the size and the negative charges of AGEs continue to increase over time during incubation. This results in a group of cross-linking molecules heterogeneous in size and charge. These results will aid in both the isolation and selection of appropriate AGE molecules for the preparation of anti-AGE antibodies, calibrator, and control in the development of an AGE immunoassay.

Kinetic analysis of TNF-α oligomer-monomer transition by surface plasmon resonance and immunochemical methods

In this work we have studied the kinetic parameters of oligomeric tumour necrosis factor alpha (TNF-alpha) dissociation using biospecific interaction analysis (BIA), based on surface plasmon resonance (SPR) of TNF-alpha immobilized on a sensor chip, and by an ELISA technique able to detect TNF-alpha oligomers in solution. Validation studies, carried out with sensor chips bearing TNF-alpha oligomers or bovine albumin monomers, verified that: (a) TNF-alpha can be immobilized in the oligomeric form onto sensor chips; (b) the covalent linkage between TNF-alpha and sensor chips is stable under the experimental conditions; (c) TNF-alpha monomers are present on the sensor chips after dissociation; (d) immobilization and dissociation rate constant (kdiss) measurements are reproducible. The kdiss of recombinant TNF-alpha, measured under non denaturing conditions at pH 7.4 by BIA and ELISA were in good agreement, being 0.92 x 10(-5)/s and 1.1 x 10(-5)/s respectively (corresponding to a half life of about 20.9 h and 17.5 h, respectively). The dissociation rate was found to be significantly affected by the presence of detergents and by the pH of the solution, suggesting that TNF-alpha, at low concentrations, exists in solution with different molecular forms depending on the time of storage and buffer composition. Real-time BIA is rapid and does not require particular antibodies or reagents. Thus, the stability of the quaternary structure of natural or recombinant TNF-alpha from human or animal species as well as that of other oligomeric cytokines can probably be studied using this method.

Recognition of anthracycline binding domains in bovine serum albumin and design of a free fatty acid sensor protein

The mode of binding of N-acylated doxorubicin derivatives to bovine serum albumin (BSA) has been determined by spectrophotometric analysis. A water-insoluble derivative containging a N-hydroxysuccinimide ester moiety on the sugar amino group side-chain is found to react very rapidly with a specific domain (BSA A600) located in the NH 2-terminal half of the BSA molecule. A stable covalent protein-anthracycline complex with 1:1 molar stoichiometry and containing the albumin monomer is formed. This specific association between albumin and doxorubicin derivative is accompanied by large changes in the spectral characteristics of the anthracycyline chromophore. A new strong absortpion band at 600 nm is associated to ionization of the chromophore phenolic groups. Titration experiments indicate that the p K a of the protein bound anthracycline is about 3 pH units lower than the p K a of free doxorubicin in aqueous buffer indicating chromophore localization in a basic microenvironment on the albumin molecule. For N-acylated doxorubicin derivatives which associate non-covalently to the BSA A600 domain, the strength of binding is found to be controlled by ionic as well as hydrophobic protein-anthracycline interactions. Water-soluble derivatives containing a side-chain carboxylic group bind with K d ⇔ lde 10 μM, which is at least 100-fold more strongly than doxorubicin. The anthracycline chromophore is displaced from the BSA A600 domain in a non-competitive manner by fatty acids ranging in chain length from C 6 to C 18 and at a fatty acid/BSA molar ratio < 2. We therefore propose a model for the anthracycline binding domain in which the chromophore resides near the opening of the hydrophobic channel into which the fatty acid hydrocarbon chain is inserted. The clusters of basic amino acids residues located at this site may form the basic anthracycline microenvironment. Our results demonstrate that doxorubicin derivatives with a sugar amino group side-chain are well suited as probes for investigations on protein-anthracycline interactions. The practical application of the covalent BSA-DOX complex as a free fatty acid sensor protein for detection of enzymatic release of fatty acids in liposomal and cell membranes is suggested.

Adsorption of BSA from seawater at differently charged mercury surfaces

The adsorption of bovine serum albumin (BSA) monomer from 0.7 M NaCl, a model solution for seawater, and from genuine seawater has been measured as a function of time and BSA bulk concentration. The method of out-of-phase alternating current (ac) voltammetry has been used for the measurement of BSA adsorption at three differently charged surfaces of a hanging mercury drop electrode (HMDE). The capacity current depression is read off at the time where no further decrease in the capacity current occurs. The equilibrium adsorption data are treated according to the Langmuir isotherm, the adsorption constant B being evaluated. The value of B for BSA adsorption from 0.7 M NaCl solution of pH 7.0 increases from (3.4 to 7.5 to 13.6) × 10 8 dm 3 mol −1, as the charge of the HMDE surface changes from positive to uncharged to negative, respectively. The value of B for BSA adsorption from seawater of pH 7.8 is (7.0, 5.5 and 9.1) × 10 8 dm 3mol −1, as the HMDE surface charge varies from positive to uncharged to negative, respectively.

Significance of natural polymerized albumin and its receptor in hepatitis B infection of hepatocytes

Lack of information regarding the presence of native albumin polymer in serum and its structural similarity to the one produced by glutaraldehyde treatment casts doubt on the postulate that hepatitis B virus attachment to hepatocytes is mediated through polymerized albumin. We used a sandwich enzyme-linked immunosorbent assay with murine monoclonal antibodies raised against glutaraldehyde-polymerized albumin to detect native albumin polymer in human serum and its cross-reactivity with other albumin polymers. Presence of polymerized albumin receptor on the HepG2 cell was studied by radioreceptor assay. Purified hepatitis B virus and synthetic peptide analogous to part of pre-S2 sequence (120-145) were used to study polymerized albumin-dependent attachment of the virus to HepG2 cells. Antibodies raised against pre-S2 peptide were used to inhibit the pre-S2 and hepatitis B virus attachment to HepG2 cells. Glutaraldehyde-treated polymerized albumin was found to be immunologically cross-reactive with native albumin polymer. Its levels were found to be significantly raised in sera of patients with liver diseases. Polymerized albumin has specific saturable receptor on HepG2 cells with two classes of binding sites of different equilibrium dissociation constant (Kd1 = (16 +/- 9.6)pmol/L and Kd2 = (1,019 +/- 172)pmol/L. Albumin monomer was unable to compete for the polymerized albumin receptor sites on HepG2 cells. Anti-pre-S2 antibodies inhibit hepatitis B virus and pre-S2 binding to hepatocyte by 40% and 70%, respectively. Added extraneous polymerized albumin and the antibody against it did not interfere with virus attachment to HepG2 cells.

Significance of natural polymerized albumin and its receptor in hepatitis B infection of hepatocytes

Lack of information regarding the presence of native albumin polymer in serum and its structural similarity to the one produced by glutaraldehyde treatment casts doubt on the postulate that hepatitis B virus attachment to hepatocytes is mediated through polymerized albumin. We used a sandwich enzyme-linked immunosorbent assay with murine monoclonal antibodies raised against glutaraldehyde-polymerized albumin to detect native albumin polymer in human serum and its cross-reactivity with other albumin polymers. Presence of polymerized albumin receptor on the HepG2 cell was studied by radioreceptor assay. Purified hepatitis B virus and synthetic peptide analogous to part of pre-S2 sequence (120–145) were used to study polymerized albumin-dependent attachment of the virus to HepG2 cells. Antibodies raised against pre-S2 peptide were used to inhibit the pre-S2 and hepatitis B virus attachment to HepG2 cells. Glutaraldehyde-treated polymerized albumin was found to be immunologically cross-reactive with native albumin polymer. Its levels were found to be significantly raised in sera of patients with liver diseases. Polymerized albumin has specific saturable receptor on HepG2 cells with two classes of binding sites of different equilibrium dissociation constant (Kd1) = (16 ± 9.6)pmol/L and Kd2 = (1,019 ± 172)pmol/L. Albumin monomer was unable to compete for the polymerized albumin receptor sites on HepG2 cells. Antipre-S2 antibodies inhibit hepatitis B virus and pre-S2 binding to hepatocyte by 40% and 70%, respectively. Added extraneous polymerized albumin and the antibody against it did not interfere with virus attachment to HepG2 cells. (HEPATOLOGY 1991;13:134–142).