Immune complex transfer enzyme immunoassays for anti-angiotensin I IgG in serum using angiotensin I conjugates prepared by two different methods

Anti-angiotensin I IgG in serum was measured by immune complex transfer enzyme immunoassays using angiotensin I conjugates prepared by two different methods. In the first method, angiotensin I was conjugated to dinitrophenyl bovine serum albumin and beta-D-galactosidase through covalent links. Anti-angiotensin I IgG in rabbit serum was reacted simultaneously with dinitrophenyl bovine serum albumin-angiotensin I conjugate and beta-D-galactosidase-angiotensin I conjugate, and the complex formed of the three components was trapped onto (anti-dinitrophenyl group) IgG-coated polystyrene balls. After washing, the complex was eluted from the polystyrene balls with dinitrophenyl-L-lysine and transferred to goat (anti-rabbit IgG) IgG-coated polystyrene balls. Beta-D-Galactosidase activity bound to (anti-rabbit IgG) IgG-coated polystyrene balls was assayed by fluorometry. In the second method, biotinylated angiotensin I was coupled with dinitrophenyl bovine serum albumin-avidin conjugate and Beta-D-galactosidase-avidin conjugate and substituted for the two conjugates in the first method. The detection limits of anti-angiotensin I IgG in serum were 10-30 ng/liter (0.2-0.6 pg/assay). These methods were 330 to 1,000-fold more sensitive and much less affected by serum effect than the conventional enzyme immunoassay, in which an angiotensin I-bovine serum albumin-coated polystyrene ball was incubated with anti-angiotensin I IgG in serum and, after washing, with (anti-rabbit IgG) Fab'-peroxidase conjugate. The first method was more sensitive than the second method, but the second method may be superior in applicability to the first method.     

Avidin- or streptavidin-biotin as a highly sensitive method to stain total protein on membranes

A sensitive method for staining proteins after transfer from polyacrylamide gels to nitrocellulose paper is described. Transferred proteins are first derivatized by reaction of the nitrocellulose replica with sulfosuccinimidobiotin and are then reacted sequentially with streptavidin, rabbit anti-streptavidin, and horseradish peroxidase-conjugated goat anti-rabbit IgG antibody. Incubation with the enzyme substrate α-chloronaphthol, produces dark protein bands against a white background. The binding of streptavidin to the proteins is dependent on biotin derivatization as demonstrated by competition with biotinylated bovine serum albumin or 10 nM biotin. The procedure detects less than 5ng of transferred protein in a single band and is thus 5–10 times more sensitive than horseradish peroxidase-conjugated avidin alone. For bovine serum albumin, the method is comparable in sensitivity to silver staining of protein in polyacrylamide gels.     

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。     

Protein-cationic detergent interaction. Fourier transform infrared and laser Raman spectroscopic studies on the interaction between proteins and dodecylpyridinium bromide.

Fourier transform infrared and laser Raman spectroscopies were used to study the effects of dodecylpyridinium bromide on the conformation of haemoglobin, myoglobin, bovine serum albumin, ribonuclease, ovalbumin, lysozyme, trypsin and beta-lactoglobulin in aqueous solution. Addition of the cationic detergent caused a decrease in alpha-helix conformation in highly helical proteins. At low detergent concentrations stabilization of beta-sheet conformation was observed.     

Tryptophanyl fluorescence of sodium dodecyl sulfate treated and 2-mercaptoethanol reduced proteins: a simple assay for tryptophan

Relative tryptophanyl fluorescence intensities of eleven different proteins (bovine liver glutamate dehydrogenase, bovine pancreas trypsin and α-chymotrypsinogen, egg white lysozyme, ovalbumin, bovine serum albumin and γ-globulin, bovine heart and rabbit muscle lactate dehydrogenases, rabbit muscle glyceraldehyde-3-phosphate dehydrogenase, and yeast alcohol dehydrogenase) were evaluated as a function of the physical state of the protein, i.e., native, denatured with intact disulfide bonds, and denatured with reduced disulfide bonds.     

Novel thymine-functionalized polystyrenes for applications in biotechnology. 2. Adsorption of model proteins

This paper investigates the adsorption of bovine serum albumin (BSA) and bovine hemoglobin (BHb) model proteins onto novel thymine-functionalized polystyrene (PS-VBT) microspheres, in comparison with polystyrene (PS) microspheres. Maximum adsorption was obtained for both proteins near their corresponding isoelectric points (pI at pH = 4.7 for BSA and 7.1 for BHb). FTIR and adsorption isotherm analysis demonstrated that, although both proteins were physisorbed onto PS through nonspecific hydrophobic interactions, adsorption onto the functionalized copolymers occurred by both physisorption and chemisorption via hydrogen bonding. FTIR analysis also indicated conformational changes in the secondary structure of BSA and BHb adsorbed onto PS, whereas little or no conformation change was seen in the case of adsorption onto PS-VBT. Atomic force microscopy (AFM), consistent with the isotherm results, also demonstrated monolayer adsorption for both proteins. AFM images of BSA adsorbed onto copolymers with 20 mol % surface VBT loading showed exclusively end-on orientation. Adsorption onto copolymers with lower functionality showed mixed end-on and side-on orientation modes of BSA, and only the side-on orientation was observed on PS. The AFM results agreed well with theoretically calculated and experimentally obtained adsorption capacities. AFM together with calculated and observed adsorption capacity data for BHb indicated that this protein might be highly compressed on the copolymer surface. Adsorption from a binary mixture of BSA and BHb onto PS-VBT showed good separation at pH=7.0; approximately 90% of the adsorbed protein was BHb. The novel copolymers have potential applications in biotechnology.     

Fourier Transform Infrared Reflection Absorption Spectroscopy (FT-IRAS) of fibrinogen adsorbed on metal and metal oxide surfaces

We have investigated the possibilities of using Infrared Reflection Absorption Spectroscopy in the study of the interaction of proteins with metal surfaces. Structural information can be obtained since the infrared radiation at the metal surface interacts only with dipole transition moments perpendicular to the metal surface. Fibrinogen spontaneously adsorbed from solution onto gold, titanium and aluminum was used as model systems. The infrared studies were carried out on dried protein films. The amide I bands of fibrinogen adsorbed on the metal surfaces shift towards higher frequencies (ca. 20 cm-1) relative to the same band in buffer solution. The magnitude of these shifts indicates that conformational change of the protein occurs upon adsorption on metal surfaces. The change in conformation of the fibrinogen also can partly be due to one week of drying at room temperature. The amide I and amide II bands show a slightly different behaviour in terms of frequency and intensity for each metal-protein system studied. The side chains appeared to be more substrate sensitive than the peptide group. Orientational effects were observed for a number of side-chain related groups.     

Thickness and density of protein films by optical mixing spectroscopy.

The adsorption of protein films on polystyrene latex spheres was studied by optical mixing spectroscopy. With this technique, we show that both the hydrodynamic thickness of protein films and their optical density can be measured. Thus, we found that films of the glycoproteins isolated from the human erythrocyte membrane were four times as thick as films of either human serum albumin or bovine serum albumin for about the same surface coverage. This result suggests an end-on orientation for the adsorbed glycoprotein molecules, which is consistent with the model proposed by others for the orientation of these molecules at the surface of the red blood cell itself.     

Raman and surface enhanced Raman spectroscopy of 2,2,5,5-tetramethyl-3-pyrrolin-1-yloxy-3-carboxamide labeled proteins: bovine serum albumin and cytochrome c

2,2,5,5-Tetramethyl-3-pyrrolin-1-yloxy-3-carboxamide (tempyo) labeled bovine serum albumin and cytochrome c at different pH values were prepared and investigated using Raman-resonance Raman (RR) spectroscopy and surface enhanced Raman scattering (SERS) spectroscopy. The Raman spectra of tempyo labeled proteins in the pH 6.7-11 range were compared to those of the corresponding free species. The SERS spectra were interpreted in terms of the structural changes of the tempyo labeled proteins adsorbed on the silver colloidal surface. The tempyo spin label was found to be inactive in the Raman-RR and SERS spectra of the proteins. The alpha-helix conformation was concluded to be more favorable as the SERS binding site of bovine serum albumin. In the cytochrome c the enhancement of the bands assigned to the porphyrin macrocycle stretching mode allowed the supposition of the N-adsorption onto the colloidal surface.     

N-tris-(beta-D-galactopyranosyloxymethyl)glycine methylamide as an antigenic determinant of neoglycoproteins]

A chemical synthesis of N-tris (beta-D-galactopyranosyloxymethyl) glycine methylamide (trisgalactosylglycine) has been carried out. Trisgalactosylglicine derivatives of bovine serum albumin, ovalbumin and amyloglucosidase from Aspergillus have been obtained. The binding of trisgalactosylglycine residues to bovine serum albumin and ovalbumin was performed by the carbodiimide method; amyloglucosidase galactosylation was performed by using the reductive amination method. The latter technique seems to be the most mild one because it does not interfere with the peptide structure of the protein being analyzed. The antiserum specifically raised against the trisgalactosylglycine derivative of bovine serum albumin as well as the monospecific antibodies isolated from it can interact with both the antigen and the trisgalactosylglycine derivatives of ovalbumin and amyloglucosidase. Native proteins are not precipitated with this antiserum. This suggests that the trigalactosylglycine residues (bovine serum albumin, ovalbumin, amyloglucosidase) covalently bound to various proteins act as immunologic determinants regardless of the mode of their binding.     

Non-covalent binding of azo compound to peptide chain: interactions of biebrich scarlet and naphthochrome green with four model proteins

We studied the non-specific interactions of two azo compounds: biebrich scarlet (BS) and naphthochrome green (NG), with four model proteins: bovine serum albumin, ovalbumin, poly-l-lysine and hemoglobin by UV-VIS spectrometry, fluorophotometry and circular dichroism melting technique. The optimal acidities of NG and BS for binding to proteins correspond to the physiological pHs of skin and gastro tissues. The saturation binding numbers of BS and NG on peptide chains were determined and the effects of electrolytes and temperature were investigated. These interactions were fitted by the Temkin absorption model and their thermodynamic parameters were calculated. The different bindings of BS and NG to proteins were compared from their molecular structures. We inferred that an ion-pair electrostatic interaction first fixes azo compounds to basic amino acid residues and subsequent binding involves the collective action of other non-covalent bonds: hydrogen bond, van der Waals force, and hydrophobic interaction. This combination of bonds caused a change of secondary conformation of protein from β-sheet to helix and the possible process was illustrated. The potential protein toxicity resulting from such a non-specific binding was analyzed. Besides, the interaction of BS with peptide chains was applied to protein assay.     

Versatile polymer microspheres for injection therapy: aspects of fluoroscopic traceability and biofunctionalization

Synthesis and characterization of a series of novel microspheres featuring (i) radiopacity (i.e., clear fluoroscopic traceability) and (ii) an outer surface exposing aldehyde groups are reported. The aldehydes allowed us to tether proteins onto the particles' surface under mild conditions, under which the protein conformation and, hence, structural motifs for biorecognition are preserved. Essential monomer building blocks were (i) 4-iodobenzoyl-2-oxo-ethylmethacrylate (4-IEMA) for radiopacity and (ii) propenal for surface tethering of proteins. The particles demonstrated good X-ray visibility and cytocompatibility. Procedures to couple proteins onto the surface were optimized using fluorescent bovine serum albumin (FITC-BSA) or collagen (FITC-collagen). Furthermore, radiopaque microparticles with unlabeled bovine collagen type I were produced. The presence of immobilized collagen was verified with narrow-scan X-ray photoelectron spectroscopy. Fibroblasts readily adhere to and grow on the collagen-modified surfaces, whereas this was much less the case for the unmodified controls. The results led us to suggest that immobilized nondenatured collagen may transform filler particles from passive space-occupying objects to particles that cross-talk with surrounding tissues.     

Conformational changes and orientation of Humicola lanuginosa lipase on a solid hydrophobic surface: an in situ interface Fourier transform infrared-attenuated total reflection study

This study was done to better understand how lipases are activated at an interface. We investigated the conformational and solvation changes occurring during the adsorption of Humicola lanuginosa lipase (HLL) onto a hydrophobic surface using Fourier transform infrared-attenuated total reflection spectroscopy. The hydrophobic surfaces were obtained by coating silicon attenuated total reflection crystal with octadecyltrichlorosilane. Analysis of vibrational spectra was used to compare the conformation of HLL adsorbed at the aqueous-solid interface with its conformation in solution. X-ray crystallography has shown that HLL exists in two conformations, the closed and open forms. The conformational changes in HLL caused by adsorption onto the surface were compared with those occurring in three reference proteins, bovine serum albumin, lysozyme, and alpha-chymotrypsin. Adsorbed protein layers were prepared using proteins solutions of 0.005 to 0.5 mg/mL. The adsorptions of bovine serum albumin, lysozyme, and alpha-chymotrypsin to the hydrophobic support were accompanied by large unfoldings of ordered structures. In contrast, HLL underwent no secondary structure changes at first stage of adsorption, but there was a slight folding of beta-structures as the lipase monolayer became complete. Solvation studies using deuterated buffer showed an unusual hydrogen/deuterium exchange of the peptide CONH groups of the adsorbed HLL molecules. This exchange is consistent with the lipase being in the native open conformation at the water/hydrophobic interface.     

Nascent chicken ovalbumin contains the functional equivalent of a signal sequence

Highly purified mRNA for chicken ovalbumin has been translated in a cell-free protein synthesizing system from rabbit reticulocytes in the presence or absence of EDTA-stripped microsomal membranes from dog pancreas. Nascent--but not completed--ovalbumin was transferred across the microsomal membrane, as demonstrated by cotranslational core glycosylation of ovalbumin nascent chains, by resistance to posttranslational proteolysis of only the glycosylated ovalbumin chains, and by cosedimentation with the membrane of exclusively the glycosylated form. Furthermore, nascent chains of bovine prolactin were observed to compete with nascent ovalbumin for transfer across the microsomal membrane. However, no competition for membrane sites was observed between nascent chains of rabbit globin and either nascent ovalbumin or prolactin. We interpret these results to suggest that nascent ovalbumin contains the functional equivalent of a signal sequence for transfer across membranes, and that membrane components involved in the segregation of secretory proteins with cleaved signal sequences also function in the segregation of ovalbumin.     

Effects of adsorption to aluminum salt adjuvants on the structure and stability of model protein antigens

The effect of adsorption onto aluminum salt adjuvants on the structure and stability of three model protein antigens was studied using fluorescence and Fourier transform infrared spectroscopies, as well as isothermal titration and differential scanning calorimetric techniques. Lysozyme was preferentially adsorbed to aluminum phosphate (Adju-Phos), whereas ovalbumin and bovine serum albumin were better adsorbed to aluminum hydroxide (Alhydrogel). A linearized Langmuir adsorption isotherm was used to obtain information regarding the binding interactions between proteins and adjuvants. Binding energetics and stoichiometry data obtained from isothermal titration calorimetry measurements were complex. Based on the spectroscopic and differential scanning calorimetry studies, the structure of all three proteins, when adsorbed to the surface of an aluminum salt, was altered in such a way as to render the proteins less thermally stable. Besides the pharmaceutical significance of this destabilization, we consider the possibility that this phenomenon may facilitate the presentation of antigens and thus contribute to the adjuvant activity of the aluminum salts.     

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.     

Antibodies against p-aminophenyl-beta-D-galactopyranoside-containing proteins

The antibodies were prepared from antisera of rabbits immunized with bovine serum albumin containing covalently bound p-aminophenyl-beta-D-galactopyranoside (APG) and purified by affinity chromatography on APG-containing ovalbumin immobilized by BrCN-activated Sepharose 4B. The antibodies possessed a selective specificity for APG and interacted with different APG-containing proteins, including APG-containing lysosomal alpha-glucosidase. The purified antibodies are immunoglobulins of G type as was determined from the molecular weights of native and dissociated antibodies and from the immunochemical assays with antibodies against rabbit IgG.     

Synthesis of N-glycoproteins with a native type of carbohydrate-peptide bond

Reaction of beta-oligoglycosylamines obtained from carbohydrate chains of N-glycoproteins (ovalbumin, ovomucoid, riboflavin-binding glycoprotein from hen egg white, and asialofetuin) with bovine serum albumin, lysozyme, and poly(L-Asp) in presence of water-soluble carbodiimide gave rise to a series of glycoconjugates, modelling natural N-glycoproteins. Carbohydrate-peptide bond was shown to be of N-glycosylamide type with participation of Asp and Glu residues. The method allows one to obtain synthetic N-glycoproteins from oligomannoside, complex and hybrid oligosaccharide chains, and may find application both in biochemistry and biotechnology for improvement of physico-chemical properties of unglycosylated proteins.     

Three pure chaperone proteins of Escherichia coli--SecB, trigger factor and GroEL--form soluble complexes with precursor proteins in vitro.

Diverse studies of three cytoplasmic proteins of Escherichia coli--SecB, trigger factor and GroEL--have suggested that they can maintain precursor proteins in a conformation which is competent for membrane translocation. These proteins have been termed 'chaperones'. Using purified chaperone proteins and precursor protein substrates, we find that each of these chaperones can stabilize proOmpA for translocation and for the translocation-ATPase. These chaperones bind to proOmpA to form isolable complexes. SecB and GroEL will also form complexes with another exported protein, prePhoE. In contrast, these chaperones do not form stable complexes with a variety of soluble proteins such as SecA protein, bovine serum albumin, ovalbumin or ribonuclease A. While chaperones may transiently interact with soluble proteins to catalyze their folding, the stable interaction between chaperones and presecretory proteins, maintaining an open conformation which is essential for translocation, may commit these proteins to the secretion pathway.     

Thermodynamic study of forces involved in bovine serum albumin and ovalbumin partitioning in aqueous two-phase systems

The partitioning of bovine serum albumin and ovalbumin in different two-phase aqueous polymer systems is investigated using a thermodynamic approach. Systems used were polyethylene glycols (PEGs) of molecular weights 1000 to 10,000 Da and Dextran T500 (500,000 Da). Ovalbumin transfer to the top phase is exothermic, which suggests an electrostatic interaction between the hydroxyl groups of PEG and the hydrophilic side chain of the protein, whereas the bovine serum albumin partition is an endothermic process that is entropically driven, which coincides with its high surface hydrophobicity. The effect of PEG molecular weight on enthalpy and heat capacity changes, associated with the partition of both proteins, is examined on the basis of a preferential interaction of low-molecular-weight PEG with the protein surface.