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.     

Localization and verification by synthesis of five antigenic sites of bovine serum albumin.

Recently we have shown that the major antigenic sites of bovine serum albumin exhibit functional equivalence progessively increasing with the time at which antibodies are obtained after the first immunization. Analysis of our recent immunochemical findings and the known covalent structure of bovine serum albumin have enabled us to predict the locations of five antigenic sites of bovine serum albumin. The predicted locations were synthesized, and immunochemical studies with late-course antisera showed them to constitute antigenic sites of native bovine serum albumin.     

Protein-cationic detergent interaction. Equilibrium dialysis study of the interaction of bovine serum albumin and other proteins with alkylpyridinium bromide.

The binding isotherms of native bovine serum albumin with cationic detergents, such as octyl, decyl, dodecyl and tetradecylpyridinium bromides were determined at pH 6.8 and 3.4 at 25 degrees C. The isotherms for dodecyl and tetradecylpyridinium bromides were also determined at 3 degrees C. The average number of detergent cations bound increased with increasing hydrocarbon chain length. At low detergent concentration the binding of all alkylpyridinium bromides was smaller at pH 3.4 than at pH 6.8. Dodecylpyridinium bromide was bound to native beta-lactoglobulin, aldolase, ovalbumin, haemoglobin, myoglobin, lysozyme, trypsin and ribonuclease at pH 6.8. No binding occurred to alpha-chymotrypsin and chymotrypsinogen. The free enthalpy change, --delta G degrees, calculated from intrinsic association constants K was determined.     

Thermal denaturation of globular proteins. Fourier transform-infrared studies of the amide III spectral region.

Fourier transform-infrared (FT-IR) spectra are reported for the amide III spectral region of the native and thermally denatured forms of chymotrypsinogen, ribonuclease, bovine serum albumin, and lysozyme. Chymotrypsinogen denatures into structures containing substantial contributions from beta-sheets, while lysozyme and bovine serum albumin show increased amounts of random-coil forms. The changes observed for ribonuclease are quite small. Bovine serum albumin shows at least six bands in the 1,260-1,320 cm-1 region which undergo large intensity changes upon thermal denaturation, and hence are assignable to alpha-helical amide III modes. The large number of observed bands suggests that slight variations in helical geometry, symmetry, or interactions result in changed amide III frequencies, so that simple correlations between narrow frequency ranges and secondary structures may not be applicable for this mode. A widened frequency range is suggested as diagnostic for helical structures.     

Identification of macrophage cell-surface binding sites for cationized bovine serum albumin.

Autoimmune diseases are characterized by the presence of autoantibodies often restricted to host proteins exhibiting charge rich domains. Charged polypeptides elicit strong immune responses, and cationized bovine serum albumin and other cationic proteins are significantly more immunogenic than their less charged counterparts. These phenomena may involve enhanced protein uptake by macrophages, resulting in greater processing and presentation of antigenic peptide-MHC complexes to T-cells. We compared macrophage cell-surface binding and uptake of native and cationized bovine serum albumin. Specific binding of [125I]cationized bovine serum albumin to THP-1 macrophages in vitro was 11-16 fold greater than for native albumin. Half-maximal inhibition of [125I]cationized albumin binding was observed at 10-7M ligand. The specificity of [125I]cationized bovine serum albumin binding and uptake was further studied in terms of competitive inhibition of proteolysis by proteins of varying charge content. Cationized bovine serum albumin, but not native albumin, inhibited proteolysis of [125I]cBSA. Calf thymus histones also inhibited cBSA degradation. High concentration of myelin basic protein was moderately effective at blocking cBSA degradation, while myoglobin and beta lactalbumin showed no inhibition. These results indicate that specific cell-surface binding sites which occur on macrophages may mediate selective uptake of certain proteins with highly charged domains including some autoantigens.     

The adsorption of proteins and protein--dodecyl sulphate complexes on N-(3-carboxypropionyl)aminodecyl-Sepharose.

Equilibrium and kinetic aspects of the binding of several proteins to N-(3-carboxypropionyl)aminodecyl-Sepharose, an amphiphilic ampholytic adsorbent, were studied at 22 degrees C, pH 7.0, I 0.10--0.12. In the absence of detergents Scatchard plots are linear for human haemoglobin and soya-bean trypsin inhibitor, but non-linear for bovine serum albumin, which is also adsorbed more tightly than the other two proteins. The introducion of 3.5mM-sodium dodecyl sulphate causes dramatic increases in the amounts and affinities of serum albumin and haemoglobin adsorbed, but has relatively little effect on the trypsin inhibitor. At concentrations of sodium dodecyl sulphate greater than about 10mM there is a fall in the binding of all proteins, owing to competition from the detergent for binding sites on the adsorbent, and a tendency towards more uniform behaviour by different proteins. Kinetic experiments suggest that in the absence of the detergent haemoglobin and serum albumin are adsorbed initially by mainly ionic forces, but that subsequently hydrophobic forces become dominant. Addition of 3.5 mM-sodium dodecyl sulphate causes pronounced changes in the time course of adsorption of haemoglobin and serum albumin, the nature of the changes being different for each protein. The significance of these results is discussed.     

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.     

Effect of albumin on the in vitro conjugation of bilirubin by rat liver microsomes

These studies were carried out to determine whether bovine serum albumin (BSA), which is usually included in the incubation mixture for the in vitro determination of bilirubin-UDP-glucuronyl transferase (GT) activity, affects GT activity. Using bilirubin as substrate, addition of BSA to the enzyme reaction mixture at concentrations varying from 2 to 30 mg/ml resulted in a dose-related inhibition of "native" GT activity of rat liver microsomes. When detergent-activated enzyme was employed, increasing concentrations of BSA also required higher concentrations of deoxycholate, digitonin, or Triton X-100 to produce maximal bilirubin conjugation. Low BSA concentrations (2 mg/ml) prevented enzyme activation by both detergents and UDP-N-acetyl glucosamine. When BSA was omitted and bilirubin dissolved in dimethyl sulfoxide, UDP-N-acetyl glucosamine failed to enhance GT activity, and activation by detergents was only 15-25% of that observed in the presence of optimal concentrations of BSA. When rat albumin was substituted for BSA, a similar dose-related inhibition of in vitro bilirubin conjugation by untreated microsomes was observed, although at any given albumin concentration, GT activity was lower with rat than with bovine albumin. Additionally, both detergents and UDP-N-acetyl glucosamine produced similar GT activation regardless of the rat albumin concentration. Finally, these effects of BSA and rat albumin could not be reproduced when beta-lactoglobulin was employed and/or when p-nitrophenol was the acceptor substrate of GT. These findings indicate that albumin, in particular BSA, profoundly and selectively influences the in vitro activity of microsomal GT toward bilirubin as the acceptor substrate.     

Phosphatidyl choline interaction with bovine serum albumin: effect of the physical state of the lipid on protein-lipid complex formation.

Radioactively labeled [¹⁴C]phosphatidyl choline dispersed on Celite was equilibrated with bovine serum albumin solutions buffered at pH 8.0. Phosphatidyl choline was rapidly solubilized in the presence of serum albumin, and formed stable protein-lipid complexes which were isolated by gel-filtration through a Sepharose 4B column. Under similar conditions, equilibration of the protein with phosphatidyl choline liposome dispersions in buffer did not result in complex formation. The altered physical state of phosphatidyl choline on the weakly adsorbing Celite surface appears to be essential for binding by native bovine serum albumin. This work reports the first observation of phosphatidyl choline binding to native serum albumin in bulk phase and suggests the possibility of exposing monodisperse lipids, under controlled conditions, to proteins having lipid binding properties.     

血清白蛋白在多聚阳离子－壳聚糖共混材料表面的构象变化及其与细胞生长的关系

用L-多聚赖氨酸、聚乙烯亚胺及L-多聚鸟氨酸三种多聚阳离子对壳聚糖进行共混修饰,制备了三种共混材料.在这些材料表面吸附了血清白蛋白,并利用圆二色(CD)光谱研究了白蛋白吸附到材料表面后的构象变化.结果显示,与天然状态相比,白蛋白吸附到共混材料表面后,其α-螺旋、β-折叠及无规则卷曲的含量均发生了明显改变.通过研究MC3T3-E1细胞在这些材料表面的生长情况,发现细胞的增殖与血清白蛋白的构象变化有一定关系,在吸附的白蛋白构象与天然构象最接近的共混材料表面,MC3T3-E1细胞增殖水平最高.     

Effects of protein conformational changes on separation performance in electrostatic interaction chromatography: unfolded proteins and PEGylated proteins

As it is important to understand how protein conformational changes affect the separation performance in ion exchange chromatography (IEC), we investigated two model systems, unfolded proteins (lysozyme and bovine serum albumin) with urea and dithiothreitol, and PEGylated proteins (lysozyme attached with polyethyleneglycol molecular weight 5000). Linear gradient elution IEC experiments were carried out and the data were analysed by our model previously presented in order to obtain the binding site value B and the peak salt concentration I(R). Unfolded proteins (bovine serum albumin and lysozyme) with urea and dithiothreitol showed weaker retention and larger binding site values compared with the values for native proteins. Multiple PEGylated lysozyme peaks were separated, and eluted earlier than the native peak appeared. There is a good correlation between B and I(R) for PEGylated lysozymes.     

The recBC enzyme of Escherichia coli K12: premature cessation of catalytic activities in vitro and reactivation by potassium ions.

It is shown that in vitro the degradation of native and single-stranded DNA as well as the hydrolysis of ATP by purified recBC enzyme ceases 2-3 min after the start of the reaction. The presence of potassium ions (60-100 mM), bovine serum albumin (1 mg/ml) or protein from cell-free Escherichia coli extract (10 microgram/ml) prevents the cessation of the activity. Once the cessation has occurred, the activity of the enzyme can be completely restored by the addition of potassium ions, but not by bovine serum albumin. Sedimentation studies revealed that, in contrast to the active recBC enzyme, the 'silent' enzyme is no longer associated with substrate DNA of high molecular weight. On the basis of these results and other observations it is hypothesized that during the degradation of DNA in the absence of potassium ions or bovine serum albumin the recBC enzyme is subject to an alteration of its molecular conformation which results in an inactive form.     

Protein-detergent interactions studied by capillary isotachophoresis

The interactions of sodium dodecyl sulphate with bovine serum albumin and ovalbumin have been studied by capillary isotachophoresis. This method makes it possible to determine very accurately the number of ligands bound to the high-affinity binding sites of the native protein. Bovine serum albumin was found to have seven high-affinity binding sites whereas ovalbumin in its native state was found to lack high-affinity binding sites for dodecyl sulphate.     

Deuteroporphyrin-albumin binding equilibrium. The effects of porphyrin self-aggregation studied for the human and the bovine proteins.

The binding equilibrium of deuteroporphyrin IX to human serum albumin and to bovine serum albumin was studied, by monitoring protein-induced changes in the porphyrin fluorescence and taking into consideration the self-aggregation of the porphyrin. To have control over the latter, the range of porphyrin concentrations was chosen to maker dimers (non-covalent) the dominant aggregate. Each protein was found to have one high-affinity site for deuteroporphyrin IX monomers, the magnitudes of the equilibrium binding constants (25 degrees C, neutral pH, phosphate-buffered saline) being 4.5 (+/- 1.5) X 10(7) M-1 and 1.7 (+/- 0.2) X 10(6) M-1 for human serum albumin and for bovine serum albumin respectively. Deuteroporphyrin IX dimers were found to bind directly to the protein, each protein binding one dimer, with high affinity. Two models are proposed for the protein-binding of porphyrin monomers and dimers in a porphyrin system having both species: a competitive model, where each protein molecule has only one binding site, which can be occupied by either a monomer or a dimer; a non-competitive model, where each protein molecule has two binding sites, one for monomers and one for dimers. On testing the fit of the data to the models, an argument can be made to favour the non-competitive model, the equilibrium binding constants of the dimers, for the non-competitive model (25 degrees C, neutral pH, phosphate-buffered saline), being: 8.0 (+/- 1.8) X 10(8) M-1 and 1.2 (+/- 0.6) X 10(7) M-1 for human serum albumin and bovine serum albumin respectively.     

Cationization of protein antigens. III. Abrogation of oral tolerance

The immunoregulatory effects of oral pretreatment of BDF1 mice with cationized bovine serum albumin (cBSA) and native bovine serum albumin (nBSA) have been compared. Oral administration of nBSA suppressed the antibody response to both forms of the antigen. In contrast, oral pretreatment with cBSA greatly enhanced the anti-BSA response in animals subsequently challenged i.p. with either the cationized or native form of the molecule. The enhancement observed with cBSA pretreatment was more pronounced than the suppression observed with the same amount and number of feedings of nBSA. As little as a single oral dose of 10 mg of cBSA produced a significant increase in antibody concentration. Cell transfer of spleen cells into irradiated syngeneic recipients demonstrated that both T cells and B cells were involved in the generation of the response, with a greater degree of enhancement provided by cBSA-pretreated T cells. These data extend our previous findings and demonstrate that administration of cBSA by a normally "tolerogenic" route results in enhancement rather than suppression of the immune response.     

1H- and 2H-NMR study of bovine serum albumin solutions

Frozen, native and denatured bovine serum albumin solutions have been studied with a wide-band NMR pulse spectrometer. Both macromolecular and water protons spin-spin and spin-lattice relaxation times--t2m, t1m, t2w, t1w--have been measured between 170 and 360 K. In the native sample, the t2m process is the tumbling rate of the bovine serum albumin molecules. It gives to the spin-lattice relaxation an omega 0(-2) frequency dependence at room temperature in the studied frequency range, 6-90 MHz. An additional process contributes to t1m-1; it arises from internal backbone or segmental motions and provides a lower frequency behaviour. On denaturation, bovine serum albumin molecules lose their tumbling motion and form a rigid network, while internal backbone motions seem unaffected. Calorimetric Cp measurement confirms the occurrence of a phase transition upon denaturation. 1H and 2H spin-lattice relaxation times of water protons depend mainly on bound water mobility. 1H and 2H t2w depend also on the tertiary structure of bovine serum albumin and on its mobility, because of a fast exchange process between water and some protein protons (or deutons), while a cross-relaxation process between protein and water protons contributes to 1H t1w. Denaturation has no influence on bound water motional properties and bound water population.     

Static and dynamic quenching of protein fluorescence. II. lysozyme.

The fluorescence parameters—lifetime, relative quantum yield, wavelength of maximum fluorescence intensity, half-width, and polarization—of 0.01% lysozyme were measured at 15°C in aqueous solution, in glycerol–water mixtures (0–90% v/v glycerol), in aqueous urea (0–8M) solutions, and in aqueous guanidine hydrochloride (0–6.4M) solutions. The changes in the static and dynamic quenching of lysozyme fluorescence, monitored by the quantum yield and lifetime measurements, were correlated with the other fluorescence parameters and compared with our earlier results with bovine serum albumin. The results were interpreted in terms of induced conformational changes. The various perturbants altered the fluorescence parameters of lysozyme and bovine serum albumin very differently. The differences were shown to be entirely consistent with our earlier conclusion that bovine serum albumin fluorophores are nonsurface residues and with the conclusion of others that lysozyme fluorophores are surface residues. Unlike their effects on bovine serum albumin, urea and guanidine hydrochloride affect lysozyme structure quite differently, both in nature and degree. We have suggested that the affect of urea on lysozyme fluorescence is an indirect result of reduction in the size of the cleft brought about by the structure-breaking action of urea on water in the cleft. 4M Urea is sufficient for this reaction. Large decreases in the polarization of the fluorescence of lysozyme in the 0.8–1.6M and 3.2–4.8M guanidine hydrochloride ranges demonstrated two guanidine hydrochloride-induced conformation changes. A red shift of the fluorescence maximum to 354 nm indicated that the second transition completely exposes all fluorescing tryptophan residues of lysozyme to mobile solvent water. However, even 6.4M guanidine hydrochloride did not completely unravel the lysozyme molecule at 15°C, as evidenced by its failure to cause any of the tyrosine residues to become fluorescent.     

Metabolic interactions between animal cells through permeable intercellular junctions.

The isolated perfused rat liver was used to study the degradation of ¹²⁵I-labelled protein supplied in the perfusion medium. Formaldehyde-denatured proteins (human serum albumin, bovine serum albumin and especially rat liver phosphoenolpyruvate carboxykinase (GTP)) were taken up by the liver and degraded at high rates. Native human serum albumin was not degraded at significant rates by the perfused liver, while native phosphoenolpyruvate carboxykinase (GTP) was catabolised at about one-fourth the rate of the denatured enzyme. The degradation rate of denatured human serum albumin increased markedly as protein was added up to 0.7 mg, and more gradually with further increases in added protein. The biphasic nature of concentration dependence probably reflects the contribution of different cell types in the liver. Autoradiographic examination of serial biopsies taken during perfusion of the liver with formaldehyde-denatured, ¹²⁵I-labelled bovine serum albumin showed that at the cellular level the radioactivity was located predominantly in Kupffer and other non-parenchymal cells; and at the subcellular level the radioactivity was largely in endocytic vesicles, lysosomes and occasionally in the sinusoidal spaces. No significant radioactivity was found associated with other cytoplasmic organelles or the nucleus. It is concluded that lysosomes of the non-parenchymal cells are primarily responsible for the degradation of denatured extracellular protein that enters the liver.     

Preferential interaction of albumin-binding proteins, gp30 and gp18, with conformationally modified albumins. Presence in many cells and tissues with a possible role in catabolism.

Albumin binding to the endothelial surface apparently initiates its transcytosis via plasmalemmal vesicles and also increases capillary permselectivity. Several albumin-binding proteins, which, we call gp60, gp30, and gp18, have been identified; however, their functional relationship to each other is unclear. In this study, we show that gp30 and gp18 are both variably expressed by cultured rat fibroblasts, smooth muscle cells, and endothelial cells and are present in all tissues examined (heart, lung, skeletal muscle, diaphragm, duodenum, kidney, fat, brain, adrenal, pancreas, and liver). The binding of albumin-gold complexes (A-Au) to gp30 and gp18 was compared with that of native and modified albumins. Monomeric native bovine serum albumin (BSA) interacted much less avidly than A-Au and BSA that was chemically modified by formaldehyde (Fm-BSA) or maleic anhydride (Mal-BSA). Mal-BSA and A-Au have similar affinity constants for gp30 and gp18 (KD approximately 3-7 micrograms/ml (50-100 nM)), which is 1000-fold greater than BSA. These interactions were Ca(2+)-independent but sensitive to pH (< 6.0) and high salt concentrations (> or = 1.0 M). Comparative biochemical characterization provided evidence of conformational changes for Mal-BSA, Fm-BSA, and A-Au. Anti-native BSA serum recognizes BSA much more avidly than modified BSA. Mal-BSA, Fm-BSA, and A-Au are much more sensitive to trypsin digestion than BSA. Cellular processing was also examined. A-Au and Mal-BSA bound at the endothelial cell surface were degraded, whereas BSA was not. Our results indicate that: (i) gp30 and gp18, unlike gp60, are expressed in all tissues tested regardless of the type of endothelia lining the microvasculature and the local mechanism of transendothelial albumin transport; (ii) BSA conformationally modified by either surface adsorption or chemical means not only interacts more avidly with gp30 and gp18 than native albumin but also is preferentially degraded by the cells; (iii) A-Au and native albumin are not equivalent probes for detecting albumin interaction sites; and (iv) gp30 and gp18 exhibit binding behavior resembling scavenger receptors. The possible roles of gp30 and gp18 in albumin binding, transcytosis, endocytosis, and even protein catabolism are discussed.     

A correlation between changes in conformation and molecular properties of bovine serum albumin upon succinylation

Using succinic anhydride, six succinylated derivatives of bovine serum albumin having percent modification in the range of 23-87% were prepared and their physicochemical and immunological properties were studied. Measurements of Stokes radius, frictional ratio, UV spectra, solvent perturbation, solubility, and immunological cross-reactivity against anti-bovine serum albumin antiserum revealed that the protein undergoes gradual changes in its native conformation with increase in the degree of succinylation. These changes were less marked below 50% modification but became pronounced above 50% modification. However, even the maximally modified preparation (87%) contained a significant amount of folded structure. Interestingly, though the measurements of various molecular properties revealed significant changes in 23-49% modified preparations, the solubility parameters for these preparations which were obtained at high ionic strength were indistinguishable from those of the native protein. The various results taken together suggest that at lower degrees of chemical modification, the conformational changes were produced mainly because of an increase in electrostatic free energy, whereas at higher degrees of modification, steric hindrance in addition to the electrostatic factor seems to make a substantial contribution to the conformational changes in the modified proteins.