An integrated process for purification of lysozyme, ovalbumin, and ovomucoid from hen egg white

This article describes an integrated process for simultaneous purification of lysozyme, ovalbumin, and ovomucoid from hen egg white. The crude egg white extract was passed through a cation exchanger Streamline trade mark SP and the bound lysozyme was eluted with 5% ammonium carbonate, pH 9.0, containing 1 M NaCl after elution of avidin. This partially purified lysozyme was further purified 639-fold on dye-linked cellulose beads. Ovalbumin and ovomucoid did not bind to Streamline SP. Ovalbumin could be precipitated from this unbound fraction by 5% trichloroacetic acid, and ovomucoid was removed from the supernatant by precipitation with ethanol. The yields of lysozyme, ovomucoid, and ovalbumin were 77, 94, and 98%, respectively. All the purified proteins showed single bands on sodium dodecyl sulfate polyacrylamide gel electrophoresis. All the steps are easily scalable, and the process described here can be used for large-scale simultaneous purification of these proteins in the pure form.     

Gelatin-Immobilised Poly(hydroxyethyl methacrylate) Cryogel for Affinity Purification of Fibronectin

In this work, fibronectin purification from human plasma with the gelatin-immobilised poly(hydroxyethyl methacrylate) (PHEMA) cryogel has been evaluated. The PHEMA cryogel was prepared by cryo-polymerisation which proceeds in an aqueous solution of monomer frozen inside a plastic syringe. The PHEMA cryogel contained interconnected macrochannels of 10–200 μm in diameter. Gelatin molecules were covalently immobilised onto the PHEMA cryogel via carbodiimide activation. The gelatin-immobilised PHEMA cryogel was used to purify fibronectin from human plasma. Fibronectin adsorption from human plasma on the PHEMA cryogel was 0.30 mg/ml, while much higher adsorption values, up to 38 mg/ml, was obtained with the gelatin-immobilised PHEMA cryogel. The fibronectin adsorption capacity of the gelatin-immobilised PHEMA cryogel did not change with an increase in the flow rate of plasma. Up to 92 % of the adsorbed fibronectin was eluted using 2 M urea containing 1 M NaCl as elution agent. The adsorption–elution cycle was repeated ten times using the same PHEMA cryogel. No remarkable decrease was detected in the adsorption capacity of the gelatin-immobilised PHEMA cryogel.     

Purification,Partial Characterization,and CNBr-Sepharose Immobilization of a Vasorelaxant Glucose/Mannose Lectin from Canavalia virosa Seeds

A novel mannose/glucose-binding lectin from Canavalia virosa (designated as ConV) has been purified from seeds of C. virosa by affinity chromatography on a mannose-Sepharose 4B column. ConV strongly agglutinates rabbit erythrocytes and was inhibited by monosaccharides (D-mannose, D-glucose, and α-methyl-D-mannoside) and glycoproteins (ovalbumin and fetuin). SDS-PAGE revealed three bands corresponding to three subunits (α, β, and γ) confirmed by ESI mass spectrometry with exact mass of 25,480?±?2 Da, 12,864?±?1 Da, and 12,633?±?1 Da, respectively. The purified lectin was more stable in pH ranging from 7.0 to 9.0, supported up to 80?ºC without any loss in activity and unaffected by EDTA. ConV showed no toxicity against Artemia sp. nauplii and relaxed endothelized rat aorta, with the participation of the lectin domain. In our tests, the lectin immobilized on CNBr-Sepharose was capable of binding 0.8 mg of ovalbumin per chromatography, allowing the use of ConV as a tool for capture and purification of glycoproteins.     

Preconcentration of Trace Cadmium (II) and Copper (II) in Environmental Water Using a Column Packed with Modified Silica Gel-Chitosan Prior to Flame Atomic Absorption Spectrometry Determination

A new column loaded with modified silica gel-chitosan is proposed as a preconcentration system for adsorption of trace cadmium (II) and copper (II). The optimization steps were performed under dynamic conditions, involving pH, sample flow rate, eluent selection, concentration, volume, and flow rate. Trace Cd(II) and Cu(II) were quantitatively adsorbed by the modified silica gel-chitosan. The metal ions adsorbed on the separation column were eluted with 0.1 M HNO₃ and determined by flame atomic absorption spectrometry. Under the optimum conditions, this method allowed the determination of cadmium and copper with limits of detection (LOD) of 20 ng L^?1 and 38 ng L^?1, respectively. The relative standard deviation values (RSDs) for 1.0 mg L^?1 of cadmium and 1.0 mg L^?1 of copper were 2.62% and 2.85%, respectively.     

Anacardium occidentale Bark Lectin: Purification, Immobilization as an Affinity Model and Influence in the Uptake of Technetium-99M by Rat Adipocytes

Lectins, proteins that recognize carbohydrates, have been immobilized on inert supports and used in the screening or purification of glycoproteins. Anacardium occidentale bark infusion has been used as a hypoglycemic agent in Brazil. The toxicity of natural products may be evaluated determining their capability to alter the biodistribution of technetium-99M (^99mTc). This work reports the isolation and characterization of a lectin from A. occidentale bark (AnocBL), its evaluation as an affinity support for glycoprotein isolation and lectin effect on the uptake of ^99mTc by rat adipocytes. AnocBL was isolated from 80?% ammonium sulphate supernatant by affinity chromatography on fetuin?Cagarose. SDS?CPAGE showed a single protein band of 47?kDa. The monossacharide l-arabinose and the glycoproteins fetuin, asialofetuin, ovomucoid, casein, thyroglobulin, peroxidase, fetal bovine serum and IgG inhibited the activity. The lectin activity was stable until 70?°C and at a pH range of 3.0?C7.5. AnocBL?CSepharose column bound fetuin indicating that the lectin matrix may be used to obtain glycoconjugates of biotechnological interest. In vitro assay revealed that glucose and insulin increase ^99mTc uptake by rat adipocytes. AnocBL decreases ^99mTc uptake, and this effect was not detected in the presence of glucose. Fetuin inhibited AnocBL effect in all insulin concentrations.     

Synthesis of boronate-silica hybrid affinity monolith via a one-pot process for specific capture of glycoproteins at neutral conditions

In this study, a boronate-silica hybrid affinity monolith was prepared for specific capture of glycoproteins at neutral pH condition. The monolith was synthesized via a facile one-pot procedure in a stainless steel column by concurrently mixing hydrolyzed alkoxysilanes tetramethoxysilane and vinyltrimethoxysilane, organic monomer 3-acrylamidophenylboronic acid and initiator 2,2′-azobisisobutyronitrile together. The polycondensation of alkoxysilanes and copolymerization of organic monomer and vinyl-silica monolith were carried out successively by reacting at different temperatures. After optimizing the preparation conditions, the resulting hybrid affinity monolith was systematically characterized and exhibited excellent affinity to both cis-diol-containing small molecules and glycoproteins at neutral and physiological pH, including adenosine, horseradish peroxidase, transferrin and ovalbumin. The binding capacity of ovalbumin on monolith was measured to be 2.5 mg g^?1 at pH 7.0. Furthermore, the hybrid affinity monolith was applied to the separation of transferrin from bovine serum sample at a physiological condition. Good repeatability was obtained and the relative standard deviations of retention time were 1.15 and 4.77 % (n?=?5) for run-to-run and column-to-column, respectively.     

Ionization and fragmentation of N-linked glycans as silver adducts by electrospray mass spectrometry

[M+Ag]+ ions were produced by electrospray from neutral high-mannose, hybrid and complex N-linked glycans obtained from bovine ribonuclease, chicken egg glycoproteins, bovine fetuin and porcine thyroglobulin by the addition of silver nitrate to the electrospray solvent. Both singly and doubly charged ions were produced but, as the signals were split between the two silver isotopes, sensitivity was not as high as with the sodium adducts reported earlier. Collision-induced dissociation (CID) spectra were dominated by ions produced by glycosidic cleavages, mainly of the B- and Y-type. Internal cleavage ions involving both B and Y cleavages were very prominent but cross-ring fragments were generally of very low abundance or absent. Silver was very efficient at cleaving the glycosidic bonds, so much so that spectra tended to contain glycosidic ions at most possible combinations of the constituent monosaccharides.     

Simultaneous production and separation of no-carrier-added 111In, 109Cd from alpha particle induced silver target

A natural silver foil was bombarded by 30 MeV α-particles which produced ¹¹¹In, ¹⁰⁹Cd and ^106mAg in the target matrix. ¹¹¹In and ¹⁰⁹Cd were separated from the Ag target matrix employing ion-exchange chromatography and liquid–liquid extraction (LLX). In the chromatographic separation, the active solution containing the NCA products were adsorbed in the column containing Dowex 50 and were eluted with HNO₃. Bulk silver and ¹⁰⁹Cd were sequentially eluted with 1 M HNO₃. After complete elution of ¹⁰⁹Cd and the bulk, ¹¹¹In was eluted with 1.5 M HNO₃. In the LLX, the NCA ¹¹¹In was extracted to 1 % HDEHP (di-2(ethylhexyl)phosphoric acid) from 10^?2 M HNO₃ solution, leaving cadmium and bulk silver quantitatively in the aqueous phase. The NCA ¹⁰⁹Cd was separated from the bulk Ag by precipitating Ag as AgCl. NCA ¹¹¹In was stripped back quantitatively from HDEHP phase using 8 M HNO₃.     

Measurement of neutral sugars in glycoproteins as dansyl derivatives by automated high-performance liquid chromatography

Automated high-performance liquid chromatography was used to analyse dansylhydrazine derivatives of neutral sugars in unfractionated acid hydrolysates of four well-characterized glycoproteins: fetuin, ovalbumin, alpha-1-acid glycoprotein and bovine submaxillary mucin. After a simple single-step derivatization at 65 degrees C the sugar derivatives in protein hydrolysates chromatographed as single peaks on reversed-phase C18 columns. The isocratic solvent consisted of 20% (v/v) aqueous acetonitrile containing 0.01 M formic acid, 0.04 M acetic acid and 0.001 M triethylamine. The triethylamine significantly increases the sugar peak height at 254 nm. Repeated automatic sample injection without deterioration of column performance or interference from dansyl hydrazine is not possible with published methods, but was achieved by cleaning the column between each analysis with a solvent of 20% (v/v) acetonitrile and 80% (v/v) methanol. Hydrolysis with 2 M trifluoroacetic acid is superior to 2 M hydrochloric acid for both sugar recovery and convenience but must continue for 6-8 h at 105 degrees C to ensure complete sugar release. We confirmed that mannose is present in most preparations of human high-molecular-weight salivary glycoproteins, and also examined purified bovine skin proteodermatan sulphate. p-Nitrophenylhydrazine derivatives of neutral sugars are readily produced, but do not chromatograph as successfully as the dansyl derivatives while phenylhydrazine derivatives are not easily produced at 65 degrees C. Further development of the method should be possible by producing other hydrazine derivatives of neutral sugars.     

Chromatography of beta-glucuronidase from bovine liver. A study of the enzyme binding sites of prepared adsorbents.

beta-Glucuronidase from bovine liver was adsorbed to the adsorbents prepared with CH-Sepharose 4B and either the competitive inhibitor or its analogs such as p-aminophenyl 1-thio-beta-D-glucuronic acid, -glucoside, -galactoside, and N-acetyl glucosaminide. The adsorbed enzyme was eluted at 0.1 or 0.5 M NaCl by a stepwise gradient. Chromatography of the enzyme was also performed by using the adsorbents prepared with Epoxy-activated Sepharose 6B and amine compounds or other compounds. In order to see whether the hydroxyl groups of the sugar parts in the ligand are necessary for the adsorption of the enzyme, chromatography was performed by using the adsorbents prepared with sugar derivatives as the ligand. As a result, it was found that beta-glucuronidase had an affinity for adsorbents prepared with either acetyl derivatives or methoxy derivatives of glycosides and CH-Sepharose 4B. From the results of elution of the enzyme with NaCl from adsorbents having amide bonding, it was clarified that the affinity of the enzyme for adsorbents without glycosides in the ligands correlated with acidity of the amide in the adsorbents. Hydrogen bond chromatography was performed with the prepared adsorbents. The enzyme was adsorbed under a high concentration of ammonium sulfate, and the elution of the adsorbed enzyme from adsorbents was examined by the degradation of salt. The enzyme was most easily eluted from aminoethyl 1-thio-beta-D-glucuronic acid-CH Sepharose 4B at 0.9 M ammonium sulfate and at 0.5 M concentration of the salt with p-aminophenyl 1-thio-beta-D-glucuronic acid-CH Sepharose 4B.(ABSTRACT TRUNCATED AT 250 WORDS)     

Super-Paramagnetic Nanoparticles by Surface Imprinting on Graphene Oxide Modified Iron (II,III) with Application for the Determination of Ovalbumin by Absorption Spectroscopy

Protein surface imprinting produces materials capable of selective recognition and capture of proteins. Herein, a protein surface imprinted polymer on graphene oxide modified super-paramagnetic Fe₃O₄ nanoparticles is reported. The molecularly imprinted polymer was synthesized by ultrasound-assisted suspension polymerization, using ovalbumin as the template molecule, 3-aminophenylboronie acid as the functional monomer, and methylene-bis-acrylamide as the cross-linking agent. The nanoparticles were approximately 40 nanometers in size and super-paramagnetic. Moreover, these particles demonstrated considerably high adsorption capacity, fast adsorption kinetics, and selective binding affinities toward the template protein ovalbumin. The calibration curve of ovalbumin was linear from 5.0 × 10^?11 to 1.0 × 10^?10 molar. The limit of detection of ovalbumin was 2.0 × 10^?11 M. These results show that this super-paramagnetic material has potential for biological macromolecule separation and determination.     

Preparation of a boronate-functionalized affinity hybrid monolith for specific capture of glycoproteins

A novel strategy for preparation of a boronate affinity hybrid monolith was developed using a Cu(I)-catalyzed 1,3-dipolar azide–alkyne cycloaddition (CuAAC) reaction of an alkyne–boronate ligand with an azide-functionalized monolithic intermediate. An azide-functionalized hybrid monolith was first synthesized via a single-step procedure to provide reactive sites for click chemistry; then the alkyne–boronate ligands were covalently immobilized on the azide-functionalized hybrid monolith via an in-column CuAAC reaction to form a boronate affinity hybrid monolith under mild conditions. The boronate affinity monolith was characterized and evaluated by means of elemental analysis, Fourier transform infrared spectroscopy, and scanning electron microscopy. The boronate affinity hybrid monolith exhibited excellent specificity toward nucleosides and glycoproteins, which were chosen as test cis-diol-containing compounds under neutral conditions. The binding capacity of the monolith for the glycoprotein ovalbumin was 2.36 mg?·?g^-1 at pH 7.0. The practicability of the boronate affinity hybrid monolithic material was demonstrated by specific capture of the glycoproteins ovalbumin and ovotransferrin from an egg sample.

Kinetics of protein adsorption by nanoporous carbons with different pore size

Kinetics of bovine serum albumin and ovalbumin adsorption by nanoporous carbons with different main pore sizes (1.6, 5, 7.8 and 28 nm) was studied. Experimental kinetics curves were well described by multi-exponential equation with different number of exponents (from 1 to 4). Protein adsorption kinetics showed significant dependence on pore size of carbonaceous adsorbent. Correlation between pore size distribution and amount of protein adsorbed revealed threshold pore size 7.3 nm for BSA and 6.8 nm for OVA, which are close to hydrodynamic diameter of protein molecules. The fastest and the highest adsorption of proteins were observed in carbons having developed porosity with pore sizes larger than 15 nm.     

Calorimetric and volumetric data of salting of hen egg lysozyme using NaCl solution at pH 8.8

Heat effects and densities of hen egg lysozyme in the phosphate buffer at pH 8.8 and various NaCl concentrations were determined at 25°C by LKB 10700-2 microcalorimeter and an Anton Paar 60/602 densimeter. The relation between the changes of the enthalpy and apparent molar volumes vs. molality of NaCl were determined. The data are discussed together with the data obtained previously for hen egg lysozyme solutions with NaCl salt in Na-acetate buffer pH 4.2.     

Highly efficient adsorbing noble metal ions by 3,4-dihydroxycinnamic acid-modified activated carbon

A method was established for the preconcentration of trace Au(III), Pd(II) and Pt(IV) by activated carbon modified with 3,4-dihydroxycinnamic acid. The separation and preconcentration conditions of analytes were investigated, such as effects of pH, the contacting time, the sample ?ow rate and volume, the elution condition and the interfering ions. At a pH of 1.0, the maximum static sorption capacity of the sorbent was found to be 374.8, 96.6 and 137.5 mg g^?1 for Au(III), Pd(II) and Pt(IV), respectively. The adsorbed metal ions were effectively eluted with 2.0 mL of 4% thiourea in 0.5 M HCl solution and determined by inductively coupled plasma optical emission spectrometry. The detection limit (3σ) of this method defined by IUPAC was found to be 0.12, 0.18 and 0.32 ?g L^?1 for Au(III), Pd(II) and Pt(IV), respectively. The relative standard deviation (RSD) was lower than 3.0% (n = 8) towards standard solutions. The method has been validated by analysing certified reference materials and successfully applied to the determination of trace Au(III), Pd(II) and Pt(IV) in road sediments samples.     

Isolation of N-linked glycopeptides by hydrazine-functionalized magnetic particles

We introduce a novel combination of magnetic particles with hydrazine chemistry, dubbed as hydrazine-functionalized magnetic particles (HFMP) for isolation of glycopeptides. Four methods have been developed and compared for the production of HFMP by hydrazine modification of the surface of the carboxyl and epoxy-silanized magnetic particles, respectively. The evaluation of the capability and specificity of HFMP as well as the optimization of the coupling condition for capturing of glycoproteins were systematically investigated. The results showed that HFMP prepared by adipic dihydrazide functionalization from carboxyl-silanized magnetic particles (HFCA) displayed the maximum capture capacity and isolated efficiency for glycoprotein. When measured with glycoproteins, the capacity of the HFCA (1 g) for coupling bovine fetuin was 130?±?5.3 mg. The capability of this method was also confirmed by successful isolation of all formerly glycosylated peptides from standard glycoproteins and identification of their glycosylation sites, which demonstrated the feasibility of the HFCA as an alternative solid support for isolation of glycoproteins/glycopeptides.

Rapid analysis of N‐linked oligosaccharides in glycoproteins (ovalbumin,ribonuclease B and fetuin) by reversed‐phase ultra‐performance liquid chromatography with fluorescence detection and electrospray ionization time‐of‐flight mass spectrometry

Rapid, selective and sensitive determination of N‐linked oligosaccharides in glycoproteins (ovalbumin, ribonuclease B and fetuin) was performed by ultra‐performance liquid chromatography (UPLC) with fluorescence (FL) and electrospray ionization time‐of‐flight mass spectrometry (ESI‐TOF‐MS). The asparaginyl‐oligosaccharide moiety was first liberated from each glycoprotein by pronase E (a proteolitic enzyme). The oligosaccharide fractions separated by gel‐permeation chromatography were labeled with 1‐pyrenesulfonyl chloride (PSC, a fluorescence reagent), separated by UPLC in a short run time, and then detected by FL and TOF‐MS. The PSC‐labeled oligosaccharides were selectively identified from the FL detection and then sensitively determined by ESI‐TOF‐MS. As the results, 15, eight and four kinds of N‐linked oligosaccharides were detected from ovalbumin, ribonuclease B and fetuin, respectively. Because the present method is rapid (within 9 min), selective and sensitive (approximate 60 fmol, S/N = 5), the determination of N‐linked oligosaccharides in various glycoproteins seems to be possible. Copyright © 2008 John Wiley & Sons, Ltd.     

Relative quantitation of glycosylation variants by stable isotope labeling of enzymatically released N-glycans using [12C]/[13C] aniline and ZIC-HILIC-ESI-TOF-MS

Glycan reductive isotope labeling (GRIL) using [¹²C]- and [¹³C]-coded aniline was used for relative quantitation of N-glycans. In a first step, the labeling method by reductive amination was optimized for this reagent. It could be demonstrated that selecting aniline as limiting reactant and using the reductant in excess is critical for achieving high derivatization yields (over 95 %) and good reproducibility (relative standard deviations ～1–5 % for major and ～5–10 % for minor N-glycans). In a second step, zwitterionic–hydrophilic interaction liquid chromatography in capillary columns coupled to electrospray mass spectrometry with time-of-flight analyzer (μZIC-HILIC-ESI-TOF-MS) was applied for the analysis of labeled N-glycans released from intact glycoproteins. Ovalbumin, bovine α₁-acid-glycoprotein and bovine fetuin were used as test glycoproteins to establish and evaluate the methodology. Excellent separation of isomeric N-glycans and reproducible quantitation via the extracted ion chromatograms indicate a great potential of the proposed methodology for glycoproteomic analysis and for reliable relative quantitation of glycosylation variants in biological samples.     

Separation of hafnium from tungsten by extraction chromatography with TOA in HCl–H<Subscript>2</Subscript>O<Subscript>2</Subscript> mixture

In order to measure ¹⁸²Hf by accelerator mass spectrometry (AMS), a chemical procedure for separation of hafnium from tungsten has been developed by extraction chromatography. The extraction chromatographic behavior of hafnium and tungsten has been studied using tri-n-octylamine (TOA) as the stationary phase, HCl–H₂O₂ mixture and NH₃·H₂O as the mobile phase. The effects of H₂O₂ concentration, column loading and column dimensions are investigated. Hf and W with microgram amounts are successfully separated on a chromatographic column (Ø5 × 196 mm), on which Hf is hardly retained after completely eluted with 6 M HCl–1% H₂O₂ and W strongly adsorbed is then eluted with 3 M NH₃·H₂O. The decontamination factor for tungsten is 3.0 × 10⁵ and the recovery of hafnium is better than 99% using a single column separation.     

3-Aminobenzamide and 3-aminobenzoic acid, tags for capillary electrophoresis of complex carbohydrates with laser-induced fluorescent detection

The efficiencies in derivatization of reducing carbohydrates were compared by capillary electrophoresis using maltose as a model with nine monoaminobenzene derivatives by reductive amination in the presence of sodium cyanoborohydride. We found that aminobenzene derivatives substituted at the 3-position showed good reactivity with reducing carbohydrates as expected from the reaction mechanism, although the fluorescence intensities and molar absorptivities of these derivatives were not as high as those of 2- and 4-aminobenzene derivatives. The reagents, 3-aminobenzamide and 3-aminobenzoic acid, which showed the highest reactivity, were applied to the labeling of carbohydrate chains obtained from some sialic acid-containing glycoprotein samples, and also high-mannose and hybrid-type oligosaccharides. Capillary electrophoresis of these labeled carbohydrate chains in an inner surface-modified capillary with (50% phenyl)methylpolysiloxane allowed excellent separation of sialic acid-containing carbohydrate chains derived from fetuin and thyroglobulin as well as high mannose-type and hybrid-type carbohydrates derived from bovine pancreas ribonuclease B, soybean agglutinin and hen ovalbumin. The lower limit of calibration was as low as the 10(-16) mol (injected amount) with helium-cadmium laser induced detection.