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.     

The separation of proteins with heteropolyacids

The precipitation of proteins with heteropolyacids has been studied for the purpose of large scale primary purification. A precipitate will form if the pH of the reaction between purified ovalbumin, hemoglobin, trypsin, pepsin, bovine serum albumin, ovomucoid, gelatin or ribonuclease and tungstrophosphoric, tungstosilicic or molybdosilicic acid is close to the isoelectric point of the protein and does not cause the dissociation of the heteropolyacid. Below the isoelctric point, the percent precipitation depends on the conformational changes of the protein. The precipitation of ovalbumin with tungstophosphoric decreases as the ionic strength of the buffer increases and is independent, of the protein concentration. Mixtures of ovalbumin and bovine serum albumin, though having close isoelectric points, can be separated by varying the concentration of the precipitant. The electropositive groups which combine with the tungstophosphoric acid are guanidino, ε-amino and imidazole. No precipitation is given by the α-amino groups. Filtrates of microbial fermentations containing lactase, glucose aerode-hydrogenase, alkaline protease, amyloglucosidase, and transglucosylase have been purified by precipitation with heteropolyacids.     

磷酸酪氨酸蛋白专一抗体的制备和纯化

以偶联磷酸化酪氨酸的牛血清白蛋白（ＢＳＡ）作为免疫原免疫兔获得抗血清．自抗血清中分离获得抗体．自酪胺合成磷酸酪胺,并偶联到溴化氰活化的Ｓｅｐｈａｒｏｓｅ４Ｂ上．抗体经磷酸酪胺－Ｓｅｐｈａｒｏｓｅ４Ｂ亲和柱纯化,所得抗体专一性强,Ｄｏｔｂｌｏｔ显示：抗体仅对酪氨酸磷酸化的蛋白质包括酪氨酸磷酸化的血清白蛋白,溶菌酶,卵清蛋白起抗原抗体反应,而不识别非酪氨酸磷酸化的溶菌酶,卵清蛋白,也不识别作为免疫原的骨架成分ＢＳＡ,也不识别丝氨酸磷酸化的卵清蛋白和苏氨酸磷酸化的卵清蛋白．     

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.     

The antibody–antigen interaction at an aqueous–solid interface: A study by means of polarized infrared ATR spectroscopy

The determination of structural changes in antibodies due to their specific interaction with antigenic proteins is an important problem in understanding immunological responses. The method of polarized ATR infrared spectroscopy applied to protein films adsorbed on an appropriate solid surface can give information about the conformation of the polypeptide chains, as well as their orientation with respect to the surface. The adsorption of anti-rabbit serum albumin onto monomolecular films of rabbit serum albumin, bovine serum albumin, and ovalbumin, and of anti-ovalbumin onto films of rabbit serum albumin and ovalbumin at a Ge-aqueous interface have been studied by this technique. The intensity of the amide I absorption indicates that the strengths of binding of these three albumin proteins with anti-rabbit serum albumin is, under appropriate conditions, in the order rabbit > bovine ? ovalbumin; with anti-ovalbumin, it is ovalbumin ? rabbit. Since the frequencies of the amide I band appear near 1655 cm^?1 for all the proteins and protein complexes studied, the major contributions to their conformation comes from α-helix and random-coil structures. The average orientation of the transition moments of the amide I and A bands has been shown to be about 75° with respect to the surface normal. This indicates that the polypeptides chains are on the average approximately parallel to the surface for all the systems studied. Consequently, the effect of the specific antibody-antigen interaction on the conformation and orientation of the former seems negligible in these films.     

Enantioselective binding sites on bovine serum albumin to dansyl amino acids.

The enantioselective binding sites on bovine serum albumin were examined by HPLC using 19 racemic 5-N, N-dimethylamino-1-naphthalenesulfonyl derivatives of alpha-amino acids (dansyl amino acids) as chiral probes. On a bovine serum albumin bonded chiral stationary phase, seven L-forms eluted faster than their D-forms, while ten D-forms eluted before their L-forms. It was speculated that either two classes or two different binding sites exist on bovine serum albumin which can be distinguished by N-dansyl-L-proline and N-dansyl-D-norvaline. This was confirmed by fluorometric experiments where non-fluorescent 1-naphthalenesulfonyl derivatives were synthesized and competitive adsorption experiments were performed.     

Purification and various properties of asialoglycoprotein receptors from the mouse liver

The receptor for asialoglycoproteins was isolated from murine liver and was purified by means of biospecific chromatography on sepharose-Asialo-orosomucoid. The obtained receptor with an absorption maximum at 277 nm binds to the nonreducing terminal galactosyl residues of glycoproteins similar to the receptors from liver of other mammalians. The interaction between this receptor and desialylated glycoproteins requires the presence of calcium. The dependence of specific binding on the concentration of [125I]acialo-orosomucoid used as a ligand gives a saturating curve. The dissociation constant for the receptor-ligand complex is 0.4 X 10(-9) M. Similar to asialo-orosomucoid, the receptor binds the p-aminophenyl-beta-D-galactopyranoside derivatives of bovine serum albumin, ovalbumin and acid alpha-glucosidase synthesized by us earlier. Possible use of the asialoglycoprotein receptor as a highly specific carrier transporting the modified acid alpha-glucosidase to hepatocyte lysosomes is discussed.     

Spectral and fluorescent study of the interaction of squarylium dyes,derivatives of 3<Emphasis Type="Italic">H</Emphasis>-indolium,with albumins

Noncovalent interaction of intraionic squarylium dyes, derivatives of 3H-indolium, as well as the structurally analogous ionic indodicarbocyanine dye with serum albumins (human, bovine, rat) and, for comparison, with ovalbumin has been studied by spectral and fluorescent methods. The hydrophilic squarylium dye with sulfonate groups was found to interact with albumins more efficiently, which is probably due to the double negative charge on the dye molecule at the expense of the sulfonate groups and the ability to form hydrogen bonds with albumin. The hydrophilic indodicarbocyanine dye without the squarylium group in its structure binds to albumins much weaker than the structurally analogous squarylium dye. The dyes bind to ovalbumin less efficiently than to serum albumins. Along with the binding of monomeric dye molecules, the aggregation of the dyes on albumins is also observed. The hydrophobic squarylium dye without sulfonate groups tends to form aggregates in aqueous solutions, which partially decompose upon the introduction of albumin into the solution. The hydrophilic squarylium dye with sulfonate groups can be recommended for tests as a spectral-fluorescent probe for serum albumins in extracellular media of living organisms.     

Covalent linkage of ruthenium polypyridyl compounds to poly(L-lysine), albumins, and immunoglobulin G.

A series of ruthenium polypyridyl complexes has been covalently bound to poly(L-lysine), bovine serum albumin, human serum albumin, ovalbumin, and immunoglobulin G using different binding methods. The conjugation ratios and the luminescence properties of the bioconjugates are reported. All conjugates show nonsingle-exponential decay curves. Quenching of the emission by oxygen has been studied.     

Use of hexadecyl Fractosil as a hydrophobic carrier for adsorptive immobilization of proteins

Fractosil, a porous form of silica, has been used for the preparation of a hydrophobically derivatized carrier for protein immobilization. Interaction of a number of arbitrarily chosen proteins with hexadecyl-substituted Fractosil has been investigated. Binding of proteins was found to take place with retention of their native properties. Glutamate dehydrogenase, used as a model allosteric protein, was found to retain its catalytic and allosteric properties upon binding to the adsorbent in the form of suspension or column. Positive cooperative interactions for binding of bovine serum albumin and glutamate dehydrogenase to the matrix were observed. These findings are discussed in terms of hydrophobic interactions occurring between various residues of the protein molecules and the hydrophobic ligands in addition to those interactions which may occur with the unsubstituted gel. Results presented on immobilized glutamate dehydrogenase, trypsin, alpha-chymotrypsin, alpha-amylase, and amyloglucosidase clearly indicate possible potential of the support for continuous catalytic transformations.     

Determination of the concentration of protein by dry weight--a comparison with spectrophotometric methods

A protocol for dry weight determination of the concentration of protein, using 0.2-1.0 mg of protein per sample, has been presented and applied to nine proteins: bovine serum albumin, ovalbumin, bovine carbonic anhydrase B, galactoside binding protein (rabbit), lens calinaris lectin B, green pea lectin, soy bean agglutinin-m, wheat germ agglutinin, and antithrombin III. Dry weights, combined with spectrophotometry, have been used to calculate A1% 1 cm values at 280 nm for these proteins in dilute salt solutions and are compared with published values. From absorptivities at 288 and 280 nm in 6 M guanidine hydrochloride, the number of tryptophan residues per molecule has also been calculated and compared with literature values. These results demonstrate the utility of the present method of dry weight determination.     

Interaction between new neoglycoproteins and thed-Man/l-fuc receptor of rabbit alveolar macrophages

New types of neoglycoproteins, -caseins coupled with ovalbumin-derived asparagine oligosaccharides (AO), aspartate aminotransferase-phosphopyridoxylated AO complex (AAT-PG), and streptavidin-biotinylated AO complex (SA-BAO), were tested for their inhibitory effect on binding of bovine serum albumin derivatized with thiomannoside, Man-AI-BSA [Lee YC, Stowell CP, Krantz MJ (1976) Biochemistry 15:3956–63] by rabbit alveolar macrophages. The -casein derivatives and the AAT-PG complex increased binding affinity as the number of oligosaccharide chains attached was increased. Their inhibitory potencies were closely related to those of the Man-Al-BSA derivatives [Hoppe CA, Lee YC (1983) J Biol Chem 258:14193–99] on the basis of terminal mannose density. The SA-BAO complex containing three AO chains gave stronger inhibitory potency than the -casein derivative with three AO residues, suggesting that proper orientation of the oligosaccharides on the protein can affect the receptor-ligand interaction.Abbreviations BSA bovine serum albumin - Man₄₃-BSA BSA derivative containing on average 43 residues of Man linked through an amidino-linkage [7] - AO asparagine oligosaccharide (Man₅-GlcNAc₂-Asn) from ovalbumin - AAT aspartate aminotransferase - PG phosphopyridoxylated AO - SA streptavidin - BAO biotinylated AO     

The measurement of insoluble proteins using a modified Bradford assay

A technique for determining the amount of thermally denatured, insoluble protein is described. The assay has been validated using four globular proteins, bovine serum albumin, beta-lactoglobulin, lysozyme, and ovalbumin. It consists of a resolubilization protocol, using 8 M urea and 5% 2-mercaptoethanol, linked to the Bradford dye binding assay. The resolubilization protocol was carried out at 100 degrees C to enable complete recovery of all insoluble proteins. Beta-Lactoglobulin resolubilization was completed after heating for 1 min, whereas samples of bovine serum albumin, lysozyme, and ovalbumin required heating for 1.5 min. The assay can measure protein concentrations as small as 10 micrograms, typically with standard deviations of 3%, thus comparing favorably with the standard Bradford assay. Other types of denaturation, such as chemical denaturation causing subsequent insolubility, may be studied with this technique providing that there is no interference with the Bradford assay.     

Structural studies on metal-serum albumin. IV. The interaction of Zn(II), Cd(II) and Hg(II) with HSA and BSA.

There have been no detailed and reliable studies on the environment and configuration of Zn(II), Cd(II) and Hg(II) in the metal centers of human serum albumin and bovine serum albumin to date. In this paper the authentic evidence for the involvement of the cystinyl sulfur atoms in the ligation to the zinc group ions has been obtained from the X-ray photoelectron spectra. The belief that each of the zinc group ions possesses several similar binding sites in human- and bovine serum albumin and is bound to the deprotonated thiol group (-RS-) of the cysteinyl residues to form tetrahedral and linear metal centers has been further confirmed by the treatment of ligand to metal charge transfer data with Jorgensen's method. According to these results, we have inferred that these binding sites may be located at the seventeen disulfide bridges, most likely at the seven pairs of adjacent disulfide bridges between positions 75 and 567, in the serum albumins.     

Molecular characteristics of methylglyoxal-modified bovine and human serum albumins. Comparison with glucose-derived advanced glycation endproduct-modified serum albumins

The amino acid modification, gel filtration chromatographic, and electrophoretic characteristics of bovine and human serum albumins irreversibly modified by methylglyoxal (MG-SA) and by glucose-derived advanced glycation endproducts (AGE-SA) were investigated. Methylglyoxal selectively modified arginine residues at low concentration (1 mM); at high methylglyoxal concentration (100 mM), the extent of arginine modification increased and lysine residues were also modified. Both arginine and lysine residues were modified in AGE-SA. Analytical gel filtration HPLC of serum albumin derivatives suggested that the proportion of dimers and oligomers increased with modification in both low and highly modified MG-SA and AGE-SA derivatives relative to unmodified serum albumins. In SDS-PAGE analysis, dimers and oligomers of low-modified MG-SA were dissociated into monomers, but not in highly modified MG-SA. MG-SA had increased anodic electrophoretic mobility under nondenaturing conditions atpH 8.6, indicating an increased net negative charge, which increased with extent of modification; highly modified MG-SA and AGE-SA had similar high electrophoretic mobilities. MG-SA derivatives were fluorescent: the fluorescence was characteristic of the arginine-derived imidazoloneN -(5-methyl-4-imidazolon-2-yl)ornithine, but other fluorophores were also present. AGE-SA had similar fluorescence, attributed, in part, to glucose-derived imidazolones. AGE formed from glucose-modified proteins and AGE-like compounds formed from methylglyoxal-modified proteins may both be signals for recognition and degradation of senescent macromolecules.Abbreviations AGE advanced glycation endproduct - BSA bovine serum albumin - HSA human serum albumin - MG-SA methylglyoxal-modified serum albumin - MG-BSA methylglyoxal-modified bovine serum albumin - MG-HSA methylglyoxal-modified human serum albumin - AGE-SA AGE-modified serum albumin - AGE-BSA AGE-modified bovine serum albumin - AGE-HSA AGE-modified human serum albumin - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - HPLC high-performance liquid chromatography - FFI 2-(2-furoyl)-4(5)-(2-furanyl)-1H-imidazole     

牛血清蛋白酶联免疫检测试剂盒的研制及验证

为研制酶联免疫试剂盒以检测病毒性疫苗中残余牛血清蛋白(BSP)含量,制备高效价高纯度的兔抗BSP多克隆抗体作为包被抗体和酶标抗体,建立了ELISA双抗体夹心法并组建试剂盒,通过标准剂量曲线可对样品中所含BSP、BSA及B-IgG进行定量,经验证该方法标准曲线线性范围内r≥0.98,对BSP的检测限量为3ng/ml;分别检测5、10、20ng/ml含量的BSP时,试验内(n=12)和试验间(n=3)测定的变异系数在3.71%到7.29%之间,回收率在93.4%~106.3%,未见该方法与人血清白蛋白、卵清蛋白以及疫苗复合保护剂之间有交叉反应。该法敏感度高,准确性、重复性和稳定性好,可用于疫苗牛血清残余蛋白的质量控制。     

Precolumn labeling of reducing carbohydrates with 1-(p-methoxy)phenyl-3-methyl-5-pyrazolone: analysis of neutral and sialic acid-containing oligosaccharides found in glycoproteins.

A convenient precolumn labeling method was developed for the analysis of neutral and sialic acid-containing oligosaccharides in glycoproteins using 1-(p-methoxy)phenyl-3-methyl-5-pyrazolone (PMPMP). PMPMP reacts with a reducing oligosaccharide under slightly alkaline conditions (pH 8.3) to form a 2:1 adduct (bis-PMPMP derivative). Sialic acid residues in the oligosaccharides remain intact during the reaction. Tryptic glycopeptides digested with glycopeptidase A for oligosaccharide liberation can be directly derivatized with PMPMP without prior treatment. Separation of the labeled oligosaccharides was performed by reverse-phase high-performance liquid chromatography on a C-18 column with aqueous acetonitrile, and positional isomers such as isomeric triantennary tetradecasaccharides from bovine fetuin were completely resolved. The bis-PMPMP derivatives were labile in alkaline media to form mono-PMPMP derivatives; however, the mono-PMPMP derivatives could be easily reconverted to the original bis-PMPMP derivatives. The proposed method is simpler than the reductive pyridylamination method, and detection sensitivity could reach subnanomole range with a uv detector. Oligosaccharides from ribonuclease B (bovine pancreas), ovalbumin, thyroglobulin (porcine thyroid), fetuin (bovine), and transferrin (human) have been successfully analyzed to demonstrate the usefulness of this method as an alternative to the existing methods.     

Effect of molecular parameters on the binding of phenoxyacetic acid derivatives to albumins

The interaction of 12 phenoxyacetic acid derivatives with human and serum albumin as well as with egg albumin was studied by charge-transfer reversed-phase (RP) thin-layer chromatography (TLC) and the relative strength of interaction was calculated. Each phenoxyacetic acid derivative interacted with human and bovine serum albumins whereas no interaction was observed with egg albumin. Stepwise regression analysis proved that the lipophilicity of the derivatives exert a significant impact on their capacity to bind to serum albumins. This result supports the hypothesis that the binding of phenoxyacetic acid derivatives to albumins may involve hydrophobic forces occurring between the corresponding apolar substructures of these derivatives and the amino acid side chains.     

Interactions between 1-benzoyl-4-p-chlorophenyl thiosemicarbazide and serum albumin: investigation by fluorescence spectroscopy

The interactions between 1-benzoyl-4-p-chlorphenyl thiosemicarbazide (BCPT) and bovine serum albumin (BSA) or human serum albumin (HSA) have been studied by fluorescence spectroscopy. By the analysis of fluorescence spectrum and fluorescence intensity, it was showed that BCPT has a strong ability to quench the intrinsic fluorescence of both bovine serum albumin and human serum albumin through a static quenching procedure. The binding constants of BCPT with BSA or HSA were determined at different temperatures based on the fluorescence quenching results. The binding sites were obtained and the binding force were suggested to be mainly hydrophobic. The effect of common ions on the binding constants was also investigated. A new fluorescence spectroscopy assay of the proteins is presented. The linear range is 5.36-67.0 microg mL(-1) with recovery of 101.1% for BSA, and the linear range is 8.28-144.9 microg mL(-1) with recovery of 102.6% for HSA. Determination of the proteins in bovine serum or in human serum by this method gives results which are very close to those obtained by using Coomassie Brilliant Blue G-250 colorimetry. A practical method was proposed for the determination of BCPT in human serum samples.     

Carnosine binding: characterization of steric and charge requirements for ligand recognition.

The steric and charge requirements for binding of l-carnosine (β-alanyl-l-histidine) by bovine serum albumin were investigated with proton magnetic resonance (¹HMR) spectrometry. The histidinyl side chain of the dipeptide is responsible for primary recognition by the binding site. Furthermore, recognition is specific to a particular orientation of the histidinyl side chain that is determined by the other amino acid residue of the dipeptide. It was found that, although salts do not have a great effect on the binding of carnosine to bovine serum albumin, this binding cannot be measured by equilibrium dialysis in the presence of salt because of formation of a complex Donnan equilibrium. Carnosine, which has been postulated to have a role in olfaction, binds to the crude particulate fraction of nasal olfactory epithelium in the same steric orientation as it does to bovine serum albumin. Therefore, we have used the binding of carnosine to bovine serum albumin as a model system to test potential competitive inhibitors of carnosine binding that ultimately could be tested for activity in the olfactory pathway. It was found that the binding of carnosine to bovine serum albumin is a good model of nonspecific binding of carnosine to tissue preparations but not of the specific binding of carnosine to the nasal olfactory epithelium. In addition to requiring the proper conformation of the histidinyl residue, the binding to olfactory epithelium also appears to require recognition of the β-alanyl residue and of substituents on the imidazole ring. Evidence is provided that the carnosine binding by the nasal olfactory epithelium demonstrated by ¹HMR spectroscopy does not occur with the mature olfactory receptor neurons.