Alteration of immunological properties of bovine serum albumin by covalent attachment of polyethylene glycol.

Methoxypolyethylene glycols of 1900 and 5000 daltons have been attached covalently to bovine serum albumin using cyanuric chloride as the coupling agent. When sufficient polymer is attached, the modified bovine serum albumin appears to lose its immunogenicity in the rabbit and, on intramuscular or intravenous injection, elicits antibodies neither to itself nor to native bovine serum albumin. It does not react with antibodies raised against native bovine serum albumin. Bovine serum albumin to which methoxypolyethylene glycol has been attached exhibits a blood circulating life in the rabbit rather similar to native bovine serum albumin, except that it is not removed from circulation by the eventual development of antibodies. Modified bovine serum albumins which had been iodinated with 125I, or prepared with [14C]cyanuric chloride, were injected intravenously in rabbits. Both labels appeared almost quantitatively in the urine after 30 days. The modified bovine serum albumins showed substantial changes in properties, such as solubility, electrophoretic mobility in acrylamide gel, ion exchange chromatography, and sedimentation, as compared with the unmodified protein.     

Activation of apoalkaline phosphatase by serum albumin with Zn2+ in rat hepatoma cells.

Reuber rat hepatoma cells (R-Y121B) cultured at 0.5% serum accumulated apoalkaline phosphatase in intact cells. When R-Y121B cells were cultured in the presence of bovine serum albumin, alkaline phosphatase activity increased in the cells, and the associated increase in enzyme activity differed amongst bovine serum albumin preparations. The treatment of bovine serum albumin with activated charcoal not only enhanced the effect of serum albumin on alkaline phosphatase activity, but also cancelled the differences due to different preparations of serum albumin. In contrast, no effect from serum albumin was observed in the increase of alkaline phosphatase activity in R-Y121B cell homogenates incubated at 37 degrees C. The activated-charcoal treatment of bovine serum albumin increased the amount of Zn2+ bound to the protein. When R-Y121B cells were cultured with bovine serum albumin, the concentration of Zn2+ in the cytosol fraction slightly increased. However, the effect of serum albumin on Zn2+ concentration in the cytosol fractions was independent of charcoal treatment. It was concluded that serum albumin with Zn2+ induces the activation of apoalkaline phosphatase due to Zn2+ binding.     

Microcalorimetric study of the domain organization of serum albumin

Scanning microcalorimetry was used for studying the melting of the structure of human and bovine serum albumins and their fragments. It was shown that the melting of the native structure of serum albumin observed by the excessive heat absorption is a complex process which is described by three simple transitions overlapping in temperature. This means that the serum albumin molecule consists of three more or less independent cooperative structures, domains.     

Investigation of Bovine Serum Albumin (BSA) Attachment onto Self-Assembled Monolayers (SAMs) Using Combinatorial Quartz Crystal Microbalance with Dissipation (QCM-D) and Spectroscopic Ellipsometry (SE)

Understanding protein adsorption kinetics to surfaces is of importance for various environmental and biomedical applications. Adsorption of bovine serum albumin to various self-assembled monolayer surfaces including neutral and charged hydrophilic and hydrophobic surfaces was investigated using in-situ combinatorial quartz crystal microbalance with dissipation and spectroscopic ellipsometry. Adsorption of bovine serum albumin varied as a function of surface properties, bovine serum albumin concentration and pH value. Charged surfaces exhibited a greater quantity of bovine serum albumin adsorption, a larger bovine serum albumin layer thickness, and increased density of bovine serum albumin protein compared to neutral surfaces at neutral pH value. The quantity of adsorbed bovine serum albumin protein increased with increasing bovine serum albumin concentration. After equilibrium sorption was reached at pH 7.0, desorption of bovine serum albumin occurred when pH was lowered to 2.0, which is below the isoelectric point of bovine serum albumin. Our data provide further evidence that combinatorial quartz crystal microbalance with dissipation and spectroscopic ellipsometry is a sensitive analytical tool to evaluate attachment and detachment of adsorbed proteins in systems with environmental implications.     

Concentrations and partial volumes of proteins

The amount of egg albumin and bovine serum albumin in water has been studied as a function of temperature and time. The temperature selected for heating was 102 °C. The proteins appear to decompose above this temperature. The suitable length of time of drying is 24 h at 102 °C. Four modifications of the method of dry weight have been explored. Glass paper in the weighing bottle increases the area available for evaporation. The densities of solutions of egg albumin and bovine serum albumin have been measured at 30.00 °C as a function of concentration with a Mettler/Paar density meter and the apparent specific volumes calculated. The apparent specific volume of egg albumin is independent of concentration and is 0.7463 ± 0.00016. The apparent specific volume of bovine serum albumin is constant from zero concentration up to about 0.2 g/g of solution and in this concentration range the apparent specific volume is 0.7348 ± 0.0001. Beyond this concentration, in agreement with the results of Bernhardt and Pauly, the apparent specific volume drops sharply with increasing protein concentration.     

Inactivation by detergents of the proline transport system in membrane vesicles from Escherichia coli and its reactivation by bovine serum albumin

The proline transport system of membrane vesicles from Escherichia coli was inactivated by a low concentration of detergents such as deoxycholate, dodecyl sulfate and Triton X-100. The addition of a large amount of bovine serum albumin to membrane vesicles which had been treated with one of these detergents resulted in the restoration of the proline transport activity. The restoration of the transport activity by bovine serum albumin was most remarkable with the deoxycholate-inactivated membrane vesicle. 80% inactivation of the transport system with 0.005% deoxycholate was completely overcome by the addition of albumin. The degree of restoration was dependent on the concentration of albumin. Although albumin stimulated the proline transport activity itself, the stimulatory effect could not account for the restoration transport activity. The binding of deoxy[¹⁴C]cholate to the membrane vesicle was roughly proportional to the amount of detergent added. Deoxycholate once bound to the membrane vesicle was removed almost completely by the incubation with albumin. It is concluded that the removal of detergent from the membrane vesicle by bovine serum albumin results in the restoration of the proline transportactivity.     

Different interactions of human and bovine serum albumin with Cibacron Blue and Blue Dextran

The interaction of human and bovine serum albumin with Cibacron Blue and Blue Dextran in aqueous solution was studied by means of difference spectroscopy. Both human and bovine albumin interact strongly with underivatized Cibacron Blue in three independent binding sites (K = 105). On the contrary, Blue Dextran interacts strongly only with human albumin, but does not bind appreciably to bovine albumin. These results suggest that the binding sites are exposed and easily accessible in human albumin, while in bovine albumin they are sterically hindered and therefore not accessible to the bulky Blue Dextran.     

A stoichiometric analysis of bovine serum albumin-gossypol interactions: a fluorescence quenching study.

The interaction of gossypol with bovine serum albumin, human serum albumin and n-bromosuccinimide-modified bovine serum albumin has been followed by fluorescence quenching measurements. The presence of a high affinity site (association constant K = 2.2 x 10(6) M-1) for gossypol on bovine serum albumin and human serum albumin is indicated. The stoichiometry of binding for the high affinity site was evaluated using Job's method of continuous variation, thereby suggesting the formation of 1:1 complex. Modification of the tryptophan residues on bovine serum albumin does not affect the binding of gossypol to either high or low affinity site of albumin.     

Diffusion-controlled association of a dye, 1-anilinonaphthalene-8-sulfonic acid, to a protein, bovine serum albumin, using a fast-flow microsecond mixer and stopped-flow

The kinetic constants of the two fastest reactions of 1-anilinonaphthalene-8-sulfonic acid binding to bovine serum albumin are derived from the results of experiments with a microsecond fast-flow mixing technique and a stopped-flow method. The experiments are interpreted in terms of rapid bimolecular diffusion-controlled associations to two independent regions on the protein surface; this reaction mechanism contrasts with previous kinetic studies of ligand binding to bovine serum albumin which have not demonstrated the fastest kinetic processes.     

The paradoxical effect of fatty acid on steroid-albumin interaction.

Careful investigation of the influence of palmitic and lauric acid on the interaction of progesterone and testosterone with several batches of untreated and defatted bovine and human serum albumins have revealed that, by contrast with published data for studies with progesterone as well as nonsteroid ligands, there is a surprising stimulation rather than inhibition of binding, albeit with a reduction of the apparent number of binding sites in almost all instances. Furthermore, fatty acid tends to minimize or eliminate the well-known differences in affinity between bovine and human albumin for interactions with these two steroids. The values for binding affinity in the interaction of testosterone with these batches of human serum albumin are significantly higher than those previously published by us and other authors and the value for progesterone-bovine albumin interaction is not in accordance with the "polarity rule". Studies of these same interactions by ultraviolet difference spectroscopy give further evidence of the augmentation in binding but, in the case of defatted bovine albumin only, the aromatic difference troughs are indicative of tyrosine perturbation whereas refatted bovine albumin, defatted and refatted human albumin manifest tryptophan perturbation. Quantitative correlation of perturbation with level of bound steroid suggests that fatty acid alters the ratio (possibly hydrogen-bonded to non hydrogen-bonded) of two forms of bound steroid. There is also further evidence that the binding sites for testosterone and progesterone are not identical.     

Biocompatible glucose sensor prepared by modifying protein and vinylferrocene monomer composite membrane

This paper proposes a very simple procedure for preparing a biocompatible sensor based on a protein (bovine serum albumin, BSA), enzyme and vinylferrocene (VF) composite membrane modified electrode. The membrane was prepared simply by first casting vinylferrocene and then coating it with BSA and glucose oxidase immobilised with glutaraldehyde. The sensor response was independent of dissolved oxygen concentration from 3 to 10 ppm and showed good stability for serum sample measurement, unlike the commonly used BSA/enzyme modified electrode. The sensor response was almost unchanged over the measurement time (>10 h) whereas the responses of a BSA and glucose oxidase modified platinum electrode and an osmium-polyvinylpyridine wired horseradish peroxidase modified electrode (Ohara et al., 1993) fell to 68% of their initial value in a serum sample containing 10mM glucose.     

The Role of Histidine Residues in the Non-Enzyme Covalent Attachment of Glucose and Ascorbic Acid to Protein

Copper ions have been suggested to play a role in the non-covalent glycosylation (glycation) of proteins via transition metal-catalysed oxidations. We have further investigated “autoxidative glycosylation” by comparison of the behaviour of dog and bovine serum albumin with respect to the oxidative reactions of glucose and ascorbate. The proteins possess similar numbers of total amino residues available for glucose attachment but dog serum albumin contains fewer histidine groups and also lacks a high affinity copper-binding site. We find that the higher copper-binding capacity of bovine serum albumin is reflected in a lower rate of ascorbate oxidation as well as less protein oxidative damage than is the case for dog serum albumin. We also observe that modification of bovine serum albumin histidine groups by diethylpyrocarbonate enhances ascorbate-mediated protein fluorophore formation.     

Kinetics of formation of hydrophobic regions during refolding of bovine serum albumin

The rate of formation of hydrophobic regions during refolding of bovine serum albumin was studied using 1-anilinonaphthalene-8-sulfonate as the hydrophobic fluorescent probe. The refolding of serum albumin exhibited a sigmoidal behavior. The exhibition of a lag phase followed by a faster kinetic phase suggested that the refolding is a cooperative, sequential process. Refolding under reducing conditions almost completely inhibited the regeneration of hydrophobic binding regions, suggesting that the formation of disulfide bonds plays an important role in the refolding of serum albumin. The rate and the extent of refolding was apparently maximum at about 20 degrees; at 37 degrees the extent of refolding was very low compared to that at the other temperatures studied. Based on the results, the mechanism of albumin refolding is interpreted in terms of domain structures and interdomain interactions.     

Preparation and properties of the water-soluble chlorophyll-bovine serum albumin complexes

By mixing chlorophyll (Chl) a or b with a dense bovine serum albumin solution, the water-soluble Chl-bovine serum albumin complexes were prepared. These complexes, eluted near the void volume on a gel filtration, were separated well from unreacted bovine serum albumin, indicating an aggregation of such molecules in the complexes. Preparation of chlorophyllide (Chlide) a- or Chlide b-bovine serum albumin complex was unsuccessful, while the phytol-, and beta-carotene-bovine serum albumin complexes could be obtained. Chls in the Chl-bovine serum albumin complexes had the following characteristics. Main absorption peak of Chl a or b in the red region occurred at 675 nm or 652 nm, respectively. The Chl a-bovine serum albumin complex having absorption peak at 740 nm was also prepared. As compared with the stabilities of Chl a and b in Triton X-100. Both Chls in the bovine serum albumin-complexes were stable against oxidative stresses, such as photobleaching, Fenton reagent, peroxidase-H2O2 system. But they were easily hydrolyzed by chlorophyllase. These properties of Chls in the bovine serum albumin-complexes were similar to those of Chls in the isolated light-harvesting Chl a/b protein complex. A possible localization of Chls within the bovine serum albumin complexes was suggested that the porphyrin moiety of Chl was buried in bovine serum albumin; however, the hydrophilic edge of porphyrin ring, adjacent to the phytol group, occurred in the hydrophilic region of a bovine serum albumin molecule.     

Elimination of Homologous And Heterologous (Bovine) Serum Albumin from the Blood Circulation in the Dog)

The elimination of intravenously administered I131-labelled bovine serum albumin has been compared to the elimination rate of relabelled homologous serum albumin in normal and bled dogs, which had lost considerable blood volumes. The investigation shows that during the first four to five days after the administration the elimination is similar of heterologous and homologous serum albumin. This proves that bovine serum albumin can be regarded to be an equivalent plasma expander to homologous serum albumin in the dog. Elimination of homologous as well as heterologous serum albumin follows a simple exponential curve during four to five days after administration. The intravascular half-lives for homologous serum albumin were 6.4 ±1.5 days and 6.4 ± 0.6 days respectively in control and bled dogs. Corresponding values for heterologous (bovine) serum albumin were 5.0 ± 0.3 and 4.8 ± 0.4 days respectively. The quote for cencentrations of homologous and heterologous serum albumin in different tissues was found to be relatively constant approximately 1.4. An exception was the stomach wall in bled dogs which had a quote of 1.1 only.     

Development of sheep embryos in vitro in a medium supplemented with different batches of serum albumin

Variability in different lots of commercial serum albumin affects mammalian embryo development in culture. The composition of commercial preparations of ovine, bovine and defatted bovine serum albumin and a fraction of ovine serum containing proteins with a mean molecular weight of 65 kDa (fraction 3) was examined by polyacrylamide gel electrophoresis. All preparations were heavily contaminated with serum proteins other than albumin. Day-6 sheep morulae were cultured for 48 h in a basal bicarbonate-buffered salt solution supplemented with the commercial preparations of ovine, bovine or defatted bovine serum albumin. These three albumin preparations differed in their abilities to support the development of morulae into expanded blastocysts, but these differences disappeared when the basal medium was also supplemented with a component of ovine serum containing substances with molecular weights of less than 10 kDa. In the latter case, the three commercial albumin preparations and fraction 3 of ovine serum all supported full development in about 40-60% of morulae.     

Detection of protein orientation on the silica microsphere surface using transverse electric/transverse magnetic whispering gallery modes

The state of adsorbed protein molecules can be examined by comparing the shifts in a narrow line resonance wavelength of transverse electric (TE) and transverse magnetic (TM) whispering gallery modes (WGM) when the molecules adsorb onto a transparent microsphere that houses WGM. In adsorption of bovine serum albumin (BSA) onto an aminopropyl-modified silica microsphere, the TM/TE shift ratio indicated highly anisotropic polarizability of BSA in the direction normal to the surface, most likely ascribed to anchoring the heart-shaped protein molecule by one of its tips. The polarization-dependent resonance shift was confirmed when the surrounding refractive index was uniformly changed by adding salt, which would simulate adsorption of large objects.     

Preparation and properties of the water-soluble chlorophyll-bovine serum albumin complexes

By mixing chlorophyll (Chl) a or b with a dense bovine serum albumin solution, the water-soluble Chl-bovine serum albumin complexes were prepared. These complexes, eluted near the void volume on a gel filtration, were separated well from unreacted bovine serum albumin, indicating an aggregation of such molecules in the complexes. Preparation of chlorophyllide (Chlide) a- or Chlide b-bovine serum albumin complex was unsuccessful, while the phytol-, and β-carotene-bovine serum albumin complexes could be obtained. Chls in the Chl-bovine serum albumin complexes had the following characteristics. (i) Main absorption peak of Chl a or b in the red region occurred at 675 nm or 652 nm, respectively. The Chl a-bovine serum albumin complex having absorption peak at 740 nm was also prepared. As compared with the stabilities of Chl a and b in Triton X-100. (ii) Both Chls in the bovine serum albumin-complexes were stable against oxidative stresses, such as photobleaching, Fenton reagent, peroxidase-H₂O₂ system. But (iii) they were easily hydrolyzed by chlorophyllase. These properties of Chls in the bovine serum albumin-complexes were similar to those of Chls in the isolated light-harvesting Chl a/b protein complex. A possible localization of Chls within the bovine serum albumin complexes was suggested that the porphyrin moiety of Chl was buried in bovine serum albumin; however, the hydrophilic edge of porphyrin ring, adjacent to the phytol group, occurred in the hydrophilic region of a bovine serum albumin molecule.     

Urea denaturation of bovine serum albumin at pH 9

The action of 5 m urea on bovine serum albumin has been studied at pH 9.0 and 25°C. Analysis by the acrylamide gel electrophoresis revealed the presence of a few components 1, 1′, 2, 3, 4 and 5. The components 1 and 1′ are monomers, component 2 is a dimer, and components 3, 4 and 5 are aggregates. In presence of SH blocking reagent, bovine serum albumin gave only the zone 1, indicating that the components 1′-5 were formed by the SH to S-S exchange reactions. Component 1′ was formed by the intramolecular SH to S-S exchange reaction, and components 2–5 were formed by the intermolecular exchange reaction. Addition of cysteine either to bovine serum albumin or to the SH-blocked bovine serum albumin increased the percent of zone 1′, indicating that a complex bovine serum albumin-cysteine was formed or that the SH-catalyzed structural alteration occurred in bovine serum albumin. Components 1, 1′, 2 and 3 were isolated separately by the preparative disc gel electrophoresis. The sedimentation coefficients 1 and 1′ differed slightly indicating that they were different monomers, and values were slightly smaller than the normal value of bovine serum albumin, indicating that these components were in slightly expanded state. Isolated component 1 was exposed to 5 m urea again, but no further change occurred. This supports the concept of microheterogeneity of bovine serum albumin.     

o-Quinones formed in plant extracts. Their reaction with bovine serum albumin

1. The reactions between chlorogenoquinone, the o-quinone formed during the oxidation of chlorogenic acid, and bovine serum albumin depend on the ratio of reactants. 2. When the serum albumin is in excess, oxygen is not absorbed and the products are colourless. This reaction probably involves the thiol group of bovine serum albumin; it does not occur with bovine serum albumin which has been treated with p-chloromercuribenzoate, iodoacetamide or Ellman's reagent. 3. When bovine serum albumin reacts with excess of chlorogenoquinone, oxygen is absorbed and the products are red. The red colour is probably formed by reaction of the lysine in-amino groups of bovine serum albumin, as it is prevented by treating the protein with formaldehyde, succinic anhydride or O-methylisourea. 4. Bovine serum albumin modified by a 1.5-fold (BSA-Q) and a fivefold (BSA-Q2) excess of chlorogenoquinone were separated by chromatography on DEAE-Sephadex A-50, and some of their properties observed. 5. Reaction of BSA-Q2 with fluorodinitrobenzene suggests that the terminal alpha-amino group, as well as lysine in-amino groups, are combined with chlorogenoquinone.