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Ovalbumin particles are reduced to nano size using heat treatment techniques. Their structural patterns in their native state and in their pH denatured state were attempted. Denaturation is also a part of conformation and hence conformations due to pH and glucose were analyzed using FTIR spectroscopy. The interactions behind these conformations are unraveled and the role of glucose as cosolvent in restricting the denaturation is also revealed from the observed secondary structures of ovalbumin. Further, the characterization of these synthesized nano particles reveals the extent of their applications. The obtained results indicate that consideration of ovalbumin nanoparticles seems to favor a very clear trend of protein denaturation and the observed structural modifications are the result of development of non-covalent interactions by the cosolvent molecules., (Copyright © 2021. Published by Elsevier Inc.)

Solution based protein conjugation of small molecules involves multiple steps of chemical syntheses, bio-conjugation and purifications which are labor intensive and time-consuming. Since many small molecules have limited water solubility, conjugation to a protein is also a relatively low efficiency process in aqueous solutions. In this study, a model in situ protein conjugation of small molecules was achieved onto SPR surfaces, using progesterone and ovalbumin as a model small-molecule-protein system. The in situ protein conjugation not only eliminated the requirement for wet chemistry, but also provided an easy way to optimize the assay performance and screen various molecular linkers., (Copyright © 2017. Published by Elsevier Inc.)

Development of a simple method for preparation of stable open tubular (OT) columns for proteins separation by capillary electrochromatography is still challenging. In this work, the titanium oxide (TiO2) nanoparticles coated OT column was successfully prepared for separation of proteins by capillary electrochromatography. The polydopamine (PDA) film was first formed in the inner surface of a fused-silica capillary by the self-polymerization of dopamine under alkaline conditions. Then the TiO2 coating was deposited onto the surface of pre-modified capillary with PDA by a liquid phase deposition process. The plentifully active hydroxyl groups in PDA coating can chelate with Ti(4+) to boost the nucleation and growth of TiO2 film. The as-prepared TiO2 coated OT column was characterized by scanning electron microscopy and measurement of electroosmotic flow. Furthermore, the influence of liquid phase deposition time on the TiO2 coating was investigated. The TiO2 coated OT column was used for successful separation of two variants of β-lactoglobulin and eight glycoisoforms of ovalbumin. The column demonstrated good repeatability and stability. The relative standard deviations of migration times of proteins representing run-to-run, day-to-day, and column-to-column were less than 3.7%. Moreover, the application of the column was verified by successful separation of acidic proteins in egg white., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Polycyclic aromatic hydrocarbons (PAHs) can form DNA-binding compounds that show genotoxicity and carcinogenicity. Pyrene, as a PAH, was covalently linked to carrier protein bovine serum albumin and ovalbumin. A monoclonal antibody (McAb) was produced that showed high cross-reactivity values with chrysene (169.73%), benzo[a]pyrene (693.34%), benzo[a]anthracene (16.36%), and indeno[1,2,3-cd]pyrene (40.96%) and showed no significant cross-reactivity values with other homologues (<0.1%). A competitive enzyme-linked immunosorbent assay (ELISA) was developed for detection of pyrene and some homologues in water samples. The detection limit of the assay was 65.08 pg ml(-1). The average recoveries of PAHs from tap water, lake water, and mineral water were 99.13, 99.74, and 99.19%, respectively, indicating that matrices of water samples do not interfere with the assay. The results demonstrated that the developed ELISA seems to be a potential method for monitoring of pyrene and some homologous PAHs in water samples., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Theoretical consideration is given to the effect of cosolutes (including buffer and electrolyte components) on the determination of second virial coefficients for proteins by small-angle X-ray scattering (SAXS)-a factor overlooked in current analyses in terms of expressions for a two-component system. A potential deficiency of existing practices is illustrated by reassessment of published results on the effect of polyethylene glycol concentration on the second virial coefficient for urate oxidase. This error reflects the substitution of I(0,c3,0), the scattering intensity in the limit of zero scattering angle and solute concentration, for I(0,0,0), the corresponding parameter in the limit of zero cosolute concentration (c3) as well. Published static light scattering results on the dependence of the apparent molecular weight of ovalbumin on buffer concentration are extrapolated to zero concentration to obtain the true value (M2) and thereby establish the feasibility of obtaining the analogous SAXS parameter, I(0,0,0), experimentally., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Among the components that make up a lateral-flow immunochromatographic assay (ICA), antibody is the key. In this paper, salbutamol (SAL) as a model analyte was meticulously designed to prepare immunogen and coating antigen in distinctly different ways. Four hybridoma cell lines were prepared and identified. Among them, C9 had highest affinity, best dose-response behavior, lowest limit of detection, and highest specificity and was chosen to be labeled with colloidal gold as the detector reagent and applied on the conjugate pad. Goat anti-mouse antibody and SAL-BSA conjugate were sprayed on a nitrocellulose membrane as test line and control line, respectively. Under the optimized conditions, the ICA strip was constructed based on a binding inhibition format. Color intensity on the test line was visually distinguishable from that of the negative sample within 5 min, with the visual detection limit of 1 ngml(-1) in phosphate-buffered saline. Cross-reactions with other β-agonists were not found (<1%). The results from ICA were in a good agreement with those obtained by enzyme-linked immunosorbent assay. The developed ICA has potential as a useful on-site screening tool for SAL in swine urine., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Proteins separated by SDS-polyacrylamide gel electrophoresis can be stained with organic dyes, the most popular being Coomassie brilliant blue R-250. Coomassie R-250 staining of ovalbumin in an SDS-PAGE gel increased linearly from 2.5 to 60 min. Direct red 81 and amido black staining approached saturation in 10 min. Scatchard analysis showed that the number of direct red 81 and amido black ligands bound to ovalbumin was fourfold higher than that of Coomassie R-250. Direct red 81 and amido black stain proteins in an SDS-polyacrylamide electrophoresis gel in 10 min., (2010 Elsevier Inc. All rights reserved.)

A method for rapid determination of free D-aspartic acid (D-Asp) in mammals has been established using a highly specific mouse monoclonal antibody against D-Asp for the first time. An anti-D-Asp monoclonal antibody was obtained by the immunization of bovine-serum-albumin-conjugated D-Asp to BALB/c mice. The obtained antibody has a high specificity toward D-Asp but shows a slight cross-reactivity to all other D- and L- amino acids including L-Asp. The calibration range of the competitive enzyme linked immunosorbent assay (ELISA) is 0.016-16 micromol/mL D-Asp in rat serum samples. The precisions of this method were evaluated by inter-plate and intraplate assays, and the relative standard deviation values were 4.8% and 4.5%, respectively. The values of D-Asp determined by the present ELISA have a good correlation to those determined by high-performance liquid chromatography with the correlation coefficient of 0.963. Using this ELISA, the time course of D-Asp in the rat serum after intravenous administration was successfully demonstrated. The present method provides a simple and high-throughput determination of D-Asp in mammals, and is a useful tool for clarifying the physiological roles and diagnostic values of this D-amino acid.

Size exclusion chromatography (SEC) coupled with online light scattering, viscometry, refractometry, and UV-visible spectroscopy provides a very powerful tool for studying protein size, shape, and aggregation. This technique can be used to determine the molecular weight of the component peaks independent of the retention times in the SEC column and simultaneously measure the hydrodynamic radius and polydispersity of the protein. We applied this technology by coupling an Agilent Chemstation high-performance liquid chromatography system with a diode array UV-visible detector and a Viscotek 300 EZ Pro triple detector (combination of a light scattering detector, refractometer, and differential pressure viscometer) to characterize and compare the molecular properties of a number of monoclonal antibodies. Our studies reveal that different monoclonal immunoglobulin Gs (IgGs) and chimeric IgGs show slightly different retention times and therefore different molecular weights in gel filtration analysis. However, when they are analyzed by light scattering, refractometry, and viscometry, different IgGs have comparable molecular weight, molecular homogeneity (polydispersity), and size. Gel filtration coupled with UV or refractive index detection suggests that antibodies purified and formulated for preclinical and clinical development are more than 95% monomer with little or no detectable soluble aggregates. Light scattering measurements showed the presence of trace amounts of soluble aggregate in all the IgG preparations. The different IgG molecules showed different susceptibility to heat and pH. One of the murine antibodies was considerably less stable than the others at 55 degrees C. The application of this powerful technology for the characterization of monoclonal antibodies of therapeutic potential is discussed.

An analytical centrifuge cell was modified to detect refraction of light transmitted through the cell caused by refractive index gradients formed by sedimenting solute during centrifugation. Sedimentation velocity and sedimentation equilibrium experiments were carried out in this cell on solutions containing high concentrations of protein and polysaccharide in a Beckman-Coulter XLA analytical ultracentrifuge. Analysis of the results indicates that in the absence of an optical artifact easily recognized as a "black band," the dependence of apparent absorbance upon radial position reported by the instrument may be considered a reliable measure of the solute concentration gradient., (Copyright 2003 Elsevier Science (USA))

Insertion of the reactive center loop in beta-sheet A in serpins has been typically inferred from the increased stability of the cleaved form to thermal- and urea-induced denaturation. We describe a convenient and rapid fluorescence-based method that differentiates the loop-inserted form from the loop-exposed form in ovalbumin, a prototypic noninhibitory serpin. Recombinant wild-type and R345A ovalbumins in the intact form bind ANS with equilibrium dissociation constants of 116 and 125 microM and a maximal fluorescence increase of 200 and 264%, respectively, in pH 6.8 buffer. Cleavage of the two proteins with porcine pancreatic elastase results in a 1.6- and 2.6-fold increase in the ANS-binding affinity. While cleavage of the reactive center loop in rR345A ovalbumin results in a approximately 200% increase in the ANS fluorescence, the rWT protein exhibits a approximately 50% decrease. Similar experiments with alpha(1)-proteinase inhibitor and antithrombin, two inhibitory serpins that exhibit reactive center loop insertion, show a decrease in ANS fluorescence on cleavage with porcine pancreatic elastase and thrombin, respectively. Denaturation studies in guanidinium hydrochloride indicate that the reactive center loop is inserted in the main body of the serpin in the cleaved form of rR345A mutant, while it is exposed in the cleaved form of rWT ovalbumin. These results demonstrate that ANS fluorescence change is an indicator of the loop-inserted or loop-exposed form in these recombinant ovalbumins, and thus could be advantageously used for probing reactive center loop insertion in ovalbumins. The major increase in fluorescence for the rR345A mutant on cleavage primarily arises from a change in ANS binding rather than from the generation of an additional ANS-binding site.

In the light scattering technique, glycosylation gives rise to excess light scattering for glycoproteins. Assuming additivity of refractive index and using an appropriate refractive index increment for carbohydrate, one can determine the degree of glycosylation from the excess light scattering. Here we have used size-exclusion chromatography in combination with online light scattering, UV absorbance, and refractive index. The results show that the technique accurately determines the carbohydrate content of recombinant stem cell factor., ((c)2001 Elsevier Science.)

We developed a convenient method for the synthesis of dextran-based multivalent probes containing N-linked oligosaccharides which is efficient even in a small scale. Oligosaccharides were derivatized with succinic dihydrazide and dimethylamine borane under a mild acidic condition. The derivatized oligosaccharides were then conjugated in a good yield to periodate-oxidized dextran (500 kDa). Thus, the conjugates containing 120 to 140 oligosaccharide chains per dextran molecule were successfully synthesized. Their practical advantage was shown by the example that the asialofetuin oligosaccharide-dextran conjugate has much higher affinity to Ricinus communis agglutinin (RCA-I) than asialofetuin oligosaccharide itself or asialofetuin. The conjugates were further labeled with fluorescent reagent or biotinylation reagent containing a hydrazino group by the use of the unreacted aldehyde groups of the oxidized dextran, yielding probes with similar densities of fluorophores or biotin groups. Direct binding of the biotinylated asialofetuin oligosaccharide-dextran probe to RCA-I coated on the titer plate at a concentration of 50 ng/50 microl was easily detected using 50 fmol (as oligosaccharides) of the probe. The method for the synthesis of dextran-based oligosaccharide probes will facilitate the investigation of carbohydrate-mediated molecular interactions based on the native oligosaccharide structures., (Copyright 2000 Academic Press.)

A simple and rapid enzyme-linked immunosorbent assay (ELISA) method for quantitation of acrolein and 4-hydroxy-2-nonenal (HNE)-modified proteins was developed. Microtiter plate wells were precoated and blocked simultaneously with epitope-bound bovine caseins as matrix proteins, and aldehyde-modified proteins were quantitated by a competition assay with a monoclonal antibody specific for acrolein-modified lysine or HNE-modified histidine epitopes. Minimal reaction times required for the coating/blocking; first monoclonal antibody and the peroxidase-conjugated second antibody binding steps were 3, 3, and 7 min, respectively, the former two steps being found to be or akin to diffusion-rate-limiting reactions. The convenient ELISA should find an application for analyses of the intricate processes involved in oxidative stress and carcinogenic insult. The epitope-attachment methodology may also be advantageous for the quantitation of various other biologically important haptenic molecules., (Copyright 1999 Academic Press.)

We describe the construction and use of a dodecyl sulfate-sensitive electrode cell to measure the activity of the detergent in biological samples. The electrode is based on the incorporation of a cetyltrimethylammonium/dodecyl sulfate complex in a siloxane polymer membrane. The cell records changes in the activity of SDS from 10(-6) to 10(-5) M SDS up to the critical micellar concentration. In aqueous solutions the cell follows Nernst' law with an electrometric response which is not affected by protein per se, but is modified by supporting electrolytes like NaCl. We demonstrate by comparison with equilibrium dialysis that the electrode can be used both to detect the high-affinity binding sites of serum albumin for SDS and to follow cooperative binding of the detergent to serum albumin, ovalbumin, and beta-lactoglobulin in the concentration interval 10(-4)-10(-3) M of unbound SDS. We conclude that the electrode has properties which should enable its use to monitor changes in SDS activity during interaction with biological material. The electrode may also be used to measure the activity of other detergents which, like SDS, form a sparingly soluble complex with cetyltrimethylammonium bromide., (Copyright 1998 Academic Press.)

The fluorophore 2-aminoacridone has been used to label a number of branched oligosaccharides previously released from various glycoproteins. Complex glycans were derivatized via their reducing end with this fluorophore by a Schiff's base mechanism followed by reduction to a secondary amine using sodium cyanoborohydride. This process of derivatization was carried out efficiently and in a nonselective manner over a period of 30 min at 90 degrees C. The resulting derivatives were separated with high resolution by capillary electrophoresis using borate buffer, containing taurodeoxycholate, as the separation buffer. The method described does not require the removal of sialic acid residues prior to derivatization, so that treatment of glycans with N-acetyl neuraminidase provided useful and additional structural information. Attempts have also been made to relate the electrophoretic mobility of branched oligosaccharides with their molecular volume.

An assay for protein-bound phosphate with a capacity to determine 100 pmol of phosphate is described. It is based on the combination of two well known methods: the alkaline hydrolysis of phosphate from seryl and threonyl residues in phosphoproteins and the quantification of the released phosphate by the use of malachite green and phosphomolybdate.

Synthetic peptides are increasingly being used as antigens to generate highly specific antisera. Screening the antipeptide sera by enzyme-linked immunosorbent assay (ELISA) can suffer from carrier crossreactivity or lack sensitivity due to poor adsorption or presentation of the peptides. In this work we describe a procedure utilizing a heterobifunctional crosslinker to effect the directional coupling of synthetic peptides to poly-L-lysine preadsorbed to microtiter plates. Plates prepared by this method conferred precision, sensitivity, and specificity to an ELISA for antipeptide antisera. In a competitive ELISA format this method permitted detection of specific peptides to 3.7 x 10(-10) M and provided an assay sensitive to protein structure in solution.

High-performance capillary electrophoresis (HPCE) is rapidly gaining acceptance as an analytical tool for the study of biological macromolecules. In the present study, the utility of HPCE for separation of glycoproteins is highlighted using a pure ovalbumin preparation. Ovalbumin, the 43-kDa glycoprotein of avian egg white, is known to be heterogeneous in nature with at least nine different carbohydrate structures having been identified on the single Asn residue. HPCE separation in an 87-cm capillary containing borate buffer and 1 mM putrescine resolves five major protein peaks in less than 30 min under nondenaturing conditions. This effect appears to be specific to glycoproteins since analysis of the nonglycosylated protein carbonic anhydrase under the same conditions showed no enhanced separation. The sodium borate buffer is proposed to play a key role in the separation by preferentially complexing with the diols of specific carbohydrate moieties on ovalbumin. Addition of putrescine enhances resolution by slowing bulk flow through the capillary and allowing electrophoretic separation of what is deduced to be closely related glycoforms of ovalbumin. Dephosphorylation of the ovalbumin with either calf intestinal alkaline phosphatase or potato acid phosphatase results in a shift of all peaks to a more rapid migration time and is consistent with a loss of negative charge. This suggests that all major ovalbumin isoforms are phosphorylated to the same degree and that heterology among ovalbumin isoforms resides solely in the carbohydrate structure. The enhanced resolution obtained with the employment of longer capillaries and modifiers of endo-osmotic flow was not restricted to ovalbumin since partial resolution of pepsin isoforms was observed under the same conditions.

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.

A method based on amino acid analysis has been developed for monitoring the covalent conjugation of synthetic peptide haptens to carrier proteins. The marker amino acid, alpha-aminobutyric acid, is included in the sequence during peptide synthesis. Following reaction, the carrier protein-conjugate is freed of excess peptide by two successive rounds of gel filtration chromatography. Amino acid analysis of a hydrolysate of the conjugate allows the calculation of the coupling ratio of the peptide to the carrier protein. Two typical procedures for conjugation, carbodiimide cross-linking and cysteine-thiol reaction with maleimidyl-proteins, have been evaluated.

A general method by polyacrylamide gel electrophoresis for the determination of sulfhydryls and disulfides in a protein was developed. The method included a two-step alkylation procedure: the first step consisted of alkylation of the sulfhydryl groups with iodoacetic acid in the presence and absence of 8 M urea; the second step consisted of alkylation of the disulfide groups with iodoacetamide after reduction with a thiol. By high-pH urea gel electrophoresis, all the half-cystine residues in a protein could be categorized into three states: reactive sulfhydryls, nonreactive sulfhydryls, and disulfide bonded. The particular advantage of the method is that the states of half-cystines in different protein species can be analyzed independently both in isolated protein and in biological translation systems.