Degree Of Deamidation Research Articles

Effects of Enzymatic Deamidation by Protein-Glutaminase on Structure and Functional Properties of Wheat Gluten

Protein-glutaminase (PG) purified from Chryseobacterium proteolyticum was used to investigate its deamidation effects on wheat gluten. Water-insoluble gluten was able to be deamidated to the extent of deamidation degree (DD) 72% in 200 mM sodium phosphate buffer (pH 7) at 40 degrees C for 30 h. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis exhibited an upper shift of gluten bands with only deamidation for 1.5-2.0 h (DD 35-45%) compared to the bands of nondeamidated gluten. Results of Fourier transform infrared analysis revealed alterations in secondary structure of gluten by PG deamidation. The assignment within amide I region showed decreases in both inter- (around 1695 cm(-1)) and intramolecular beta-sheets (around 1680 cm(-1)) by deamidation suggesting the deterioration of the aggregation ability of gluten molecules. Solubility and emulsification properties of gluten at pH 7 were improved by deamidation, while both properties at pH 3 were deteriorated by deamidation. Enzyme-linked immunosorbent assay identified that allergenicity of deamidated gluten as compared to the nondeamidated cohorts was decreased remarkably as the deamidation time was prolonged.

Deamidation as a Consequence of β-Elimination of Phosphopeptides

Beta-elimination procedures often precede mass spectrometric analyses of phosphorylated peptides. Unfortunately, the commonly employed reaction conditions facilitate the deamidation of amide-containing residues. In addition to being 1 Da heavier than their amide counterparts, the newly created acidic residues greatly influence peptide tandem mass spectra. The effects of deamidation are investigated for five different amide-containing synthetic peptides exposed to beta-elimination conditions. MALDI-generated ions are analyzed with a tandem TOF mass analyzer. Methodologies for estimating the degree of deamidation from peptide mass spectra are presented, the influence that adjacent residues exert on the rate of deamidation is catalogued, and the impact that deamidation can have on peptide tandem mass spectra is demonstrated. The complications this side reaction can cause for automated data interpretation are also noted.

Effects of Enzymatic Deamidation by Protein-Glutaminase on Structure and Functional Properties of α-Zein

The performance of novel protein-glutaminase (PG) purified from Chryseobacterium proteolyticumon alpha-zein was investigated. Highly insoluble alpha-zein was able to be deamidated to the extent of deamidation degree 62% by using 50 mM potassium phosphate (pH 8) containing 11.7% ethanol, at 40 degrees C for 137 h. Analysis by sodium dodecyl sulfate polyacrylamide-gel electrophoresis showed that deamidated and non-deamidated zeins have different mobilities. Results of circular dichroism spectra revealed the decline in alpha-helix contents of alpha-zein by deamidation. Besides, Fourier transform infrared spectroscopy analysis demonstrated alterations in the secondary structure of alpha-zein by deamidation. The assignment of the amide I region showed a remarkable decrease in antiparallel intermolecular beta-sheets (around 1690 cm(-1)) as an indication of the weakening aggregation ability of the deamidated molecules. Solubility and emulsification properties of alpha-zein, particularly at pH 7, were remarkably improved after the deamidation by PG. Gas chromatography and peroxide value studies pointed out that deamidated alpha-zein in powder form exhibited an inferior antioxidative property as compared with the non-deamidated one.

Characterization of glutamine deamidation in a long, repetitive protein polymer via bioconjugate capillary electrophoresis.

We describe a novel method for the determination of glutamine deamidation in a long protein polymer via bioconjugate capillary electrophoresis. Since the current best technique for detection of glutamine (or asparagine) deamidation is mass spectrometry, it is practically impossible to precisely detect the degree of deamidation (i.e., how many residues are deamidated in a polypeptide) in a large protein containing a significant number of glutamine (or asparagine) residues, because the mass difference between native and deamidated residues is just 1 atomic mass unit. However, by covalently attaching polydisperse protein polymers (337 residues) to a monodisperse DNA oligomer (22 bases), the degree of glutamine deamidation, which could not be determined accurately by mass spectrometry, was resolved by free-solution capillary electrophoresis. Electrophoretic separations were carried out after different durations of exposure of the protein to a cyanogen bromide cleavage reaction mixture, which is a general treatment for the purpose of removing an oligopeptide affinity purification tag from fusion proteins. For protein polymers with increasing extents of deamidation, the electromotive force of DNA + polypeptide conjugate molecules increases due to the introduced negative charge of deamidated glutamic acid residues, and consequently CE analysis reveals increasing differences in the electrophoretic mobilities of conjugate molecules, which qualitatively shows the degree of deamidation. Peak analysis of the electropherograms enables quantitative determination of the first four deamidations in a protein polymer. A first-order rate constant of 0.018 h(-1) was determined for the deamidation of a single glutamine residue in the protein polymer during the cyanogen bromide cleavage reaction.

Generation of fine powders of recombinant human deoxyribonuclease using the aerosol solvent extraction system.

To investigate the feasibility of using the Aerosol Solvent Extraction System (ASES) to produce fine powders of recombinant human deoxyribonuclease (rhDNase), lysozyme-lactose and rhDNase-lactose powders from aqueous based solutions. The ASES technique using high pressure carbon dioxide modified with ethanol or ethanol and triethylamine was used for the generation of rhDNase powders and protein-lactose powders from aqueous based solutions. Particle size, morphology, size distributions, crystallinity, and powder aerosol performance were measured. The biochemical integrity of the processed rhDNase was assessed by testing the monomer content and the degree of deamidation. RhDNase precipitated as spherical particles in the size range between 50 and 500 nm. The primary nano-sized particles were agglomerated to micron-sized clumps of particles during the precipitation process. The median particle size and the fine particle fraction were functions of the operating temperature and the nozzle system used. RhDNase was substantially denatured in the ASES process using carbon dioxide modified with ethanol as anti-solvent. However almost complete recovery of the monomer was achieved using carbon dioxide modified with ethanol-triethylamine as an anti-solvent. Lysozyme-lactose and rhDNase-lactose powders were also precipitated as agglomerated spheres using the ASES process. The powders were amorphous except for those with lactose content higher than 45%. Micron-sized particles of rhDNase suitable for inhalation delivery were generated from aqueous based solutions using the modified ASES technique. The biochemical integrity of the rhDNase powder is a function of the antisolvent and the operating temperature.

Action of protein-glutaminase on alpha-lactalbumin in the native and molten globule states.

The action of a novel protein-glutaminase from microorganisms on alpha-lactalbumin was investigated. When alpha-lactalbumin in the native state was incubated with protein-glutaminase, the deamidation proceeded gradually, i.e., the deamidation degree increased to 20% and 55% after 4 and 24 h, respectively. The transformation of alpha-lactalbumin from the native state to the molten globule state caused an increase in the rate of the enzyme-catalyzed deamidation, particularly in the early stage. The deamidation degree for the molten globule state reached 61% after 4 h, followed by a gradual increase to 66% after 24 h. CD spectral analyses of deamidated alpha-lactablumin revealed that the stability of the tertiary structure of alpha-lactablumin was closely related to the degree of deamidation, whereas the secondary structure was not affected by deamidation. Glutamine residues in alpha-lactalbumin to be modified by protein-glutaminase were identified as Gln[39], [43], [54], and [65]. Conformational characteristics of the amino acid sequence around these glutamine residues are discussed.

Some structural properties of ovalbumin heated at 80°C in the dry state

Dry heating of ovalbumin at 80°C improved the gel properties, including the gel strengthening and transparency upon the subsequent heating for gelation [Matsudomi, N., Ishimura, Y., & Kato, A., (1991). Improvement of gelling properties of ovalbumin by heating in dry state. Agricultural and Biological Chemistry, 55, 879–881.]. Some structural properties of the dry-heated ovalbumin responsible for such gel properties were studied. The electrophoretic patterns revealed the formation of ovalbumin polymer through hydrophobic and disulfide protein-protein interaction during heating in the dry state. The slight changes in circular dichroism spectrum and surface hydrophobicity indicated mild conformational changes in ovalbumin molecules. Differential scanning calorimetry themograms showed that enthalpy of denaturation of ovalbumin was markedly decreased with an increase of dry-heating time. The deamidation of ovalbumin was found to occur during the dry-heating process, and the degree of deamidation reached 10% after dry heating at 80°C for 7 days. It was assumed that the partially unfolded and deamidated ovalbumin produced by the dry heating forms specific soluble aggregates during subsequent heating for gelation, resulting in the formation of an ordered gel matrix.

Surface functional properties of native, acid-treated, and reduced soy glycinin. 2. Emulsifying properties.

Emulsifying properties of native and chemically modified soy glycinins were studied. The influence of ionic strength, protein sample composition and concentration, and assay conditions on the flocculation-creaming process and coalescence resistance was analyzed. Differences in these emulsifying properties were exhibited by native glycinins, which have a variable content of 4S, 11S, and 15S forms. Structure and functionality of native glycinin were modified by means of combined treatments: mild acidic treatments without heating or with heating at variable time and with or without disulfide bonds reduction. Modified glycinins presented different degrees of deamidation, surface hydrophobicity, and molecular mass. A slight enhancement of emulsifying stability at moderated deamidation degrees was observed. In different protein samples, a positive relationship between the flocculation-creaming rate constant and equilibrium oil volume fraction of emulsions with surface hydrophobicity was detected. A remarkable difference was observed between reduced and nonreduced samples, mainly with respect to behavior at low or high ionic strength.

Acid modification of proteins from soymilk residue (okara)

Protein was extracted from soy residue (okara) at alkaline pH, and was modified by mild acid treatments. The degree of deamidation and peptide bond hydrolysis ranged from 10 to 70% and 6 to 15%, respectively. Size exclusion gel filtration chromatography revealed that there was a progressive degradation of the okara protein. Solubility was increased markedly by the modification, while other functional properties such as emulsifying and foaming properties were also improved. The okara protein products have good essential amino acid profiles, and acid modification also increased the in vitro digestability and available lysine content of okara protein. The results indicate that changes in functional properties of okara protein by acid modification were attributed to changes in physicochemical properties such as decreases in molecular size, higher net titratable charge and surface hydrophobicity. The low solubility of okara protein makes it difficult to be incorporated into many food systems. Improved solubility and other functional properties by acid modification will enhance the utilization okara protein as food ingredient.

Decreased IgE-binding with Wheat Gluten by Deamidation.

The major wheat allergens contain a number of glutamine residues, suggesting that deamidation would be a promising method to produce hypoallergenic wheat proteins. Gluten was deamidated by acid under various conditions. The 30%-, 50%- and 90%-deamidated glutens were reacted with sera of patients allergic to wheat proteins. The results indicate that the reactivity was dramatically decreased according to the degree of deamidation.

Deamidation-induced fragmentation of maize zein, and its linked reduction in fatty acid-binding capacity as well as antioxidative effect

Zein lost its antioxidative effect in proportion to the degree of deamidation. Changes in molecular weight distribution of the protein fraction by deamidation were examined by means of gel filtration with Sephacry S-200 and SDS-electrophoresis in 12% polyacrylamide gel. The deamidation reaction (mild acid-hydrolysis by 0.05N HCl in 70% ethanol) was accompanied by fragmentation of zein subunits. Concomitantly, the surface hydrophobicity as well as fatty acid-binding capacity decreased with progressed deamidation. There was a close correlation ( r = 0.98) between the antioxidative effect and the fatty acid-binding capacity, but not the surface hydrophobicity. A large part of the antioxidative effect of zein is directly or indirectly attributable to its capacity of burying unsaturated lipid in the inter- or intra-molecular hydrophobic spaces in which the amide groups are intimately involved.

The association of deamidation of Bcl-xL and translocation of Bax to the mitochondria through activation of JNK in the induction of apoptosis by treatment with GSH-conjugated DXR

We investigated the induction of apoptosis via deamidation of Bcl-xL and translocation of Bax to the mitochondria by treatment with GSH-DXR. GSH-DXR treatment of HepG2 cells, which did not express GST P1-1, exhibited deamidation of Bcl-xL, and the degree of deamidation was related to the activation of caspase-3. Overexpression of GST P1-1 in HepG2 cells decreased both the Bcl-xL deamidation and caspase-3 activation induced by treatment with GSH-DXR. Bcl-xL deamidation and caspase-3 activation were also suppressed by co-treatment with SP600125, a specific inhibitor of JNK activity. Overexpression of wild-type Bcl-xL in HepG2 decreased GSH-DXR-induced apoptosis although deamidation was observed. However, expression of the deamidated mutant of Bcl-xL, in which aspartic acid was substituted for both arginine 52 and 66 (N52,66D-Bcl-xL), exhibited high sensitivity for the induction of apoptosis. Expression of the Bcl-xL mutant, in which alanine was substituted for both arginine 52 and 66 (N52,66A-Bcl-xL), suppressed deamidation and showed resistance to the induction of apoptosis by treatment with GSH-DXR. On the other hand, endogenous Bax and overexpressed Flag-Bax were localized in the cytosolic fraction of HepG2 cells. Treatment of the cells with GSH-DXR caused translocation of Flag-Bax to the mitochondrial fraction following the induction of apoptosis. The induced apoptosis was enhanced by the expression of Flag-Bax. Moreover, Flag-Bax was partly located in the mitochondrial fraction in N52,66D-Bcl-xL-expressed cells without the induction of apoptosis. Therefore, the induction of apoptosis by treatment of HepG2 with GSH-DXR was enhanced, thereby facilitating the release of cytochrome c by both deamidated inactivation of Bcl-xL and functional translocation of Bax to the mitochondria via JNK activation. Deamidation of Bcl-xL might be induced in order to translocate Bax to the mitochondria.

Deamidation of lysozyme during the storage of egg white.

Deamidation of lysozyme was observed during storage in a buffer solution and in egg white. The peak corresponding to native lysozyme from Bio-Rex 70 column chromatography was gradually decreased, while the peaks corresponding to deamidated lysozyme were increased during storage in 0.1 m carbonate buffer at pH 9.5. A similar change was observed during storage in egg white, but the change in egg white was larger than that in the buffer solution. A detailed analysis of the elution peaks from the Bio-Rex 70 column suggested that one to three residues of amide in lysozyme were mainly deamidated during storage in the buffer solution, and that more than three residues in lysozyme were deamidated during storage in egg white. There were significant differences in lysozyme activity between native and deamidated lysozyme, the activity being decreased in proportion to the degree of deamidation.

Functional properties of deamidated gluten obtained by treating with chymotrypsin at alkali pH.

The functional properties of gluten obtained by treating with chymotrypsin at alkali pH were investigated. The gluten was treated by chymotrypsin at pH 10.0 and 20°C, and was found to be deamidated to a state that was scarcely subject to proteolysis by chymotrypsin. The degree of deamidation of the gluten reached about 25% by this treatment for 2 hr. The functional properties of the gluten thus obtained were investigated in regard to deamidation. The enzymatically deamidated gluten greatly improved such functional properties as solubility and emulsifying ability. In particular, the solubility of the treated gluten was remarkably high in the pH range of 5 to 8, in which native gluten is insoluble. It was apparent that the improvement in functional properties of gluten was mainly due to the deamidation induced by treating with chymotrypsin at pH 10.0 and 20°C.

Increase in vesicle permeability mediated by myelin basic protein: effect of phosphorylation of basic protein

The two most basic charge isomers of myelin basic protein (BP), components 1 and 2 (C1 and C2), which presumably differ in the degree of deamidation, were purified from bovine BP by cation-exchange chromatography. Two additional specific types of posttranslational modifications were introduced into the purified isomers: (1) C-terminal arginine deficient derivatives of C1 and C2 were prepared by incubating the isomers with a carboxypeptidase, and (2) phosphorylated derivatives of C1 (1.6 and 1.7 mol of phosphate/mol of protein) were prepared by incubating C1 with the protein kinase from rabbit muscle. The ability of these charge isomers to increase the permeability of multilamellar vesicles composed of phosphatidylserine/phosphatidylcholine (1:11.5 w/w) and sphingomyelin/cholesterol/phosphatidic acid (1:1:0.2 w/w/w) was measured by monitoring the release of a water-soluble spin-label (tempocholine chloride) from the vesicles. The increase in vesicle permeability caused by BP was taken as a measure of the degree of perturbation of the bilayer by the protein, most likely by penetration partly into the bilayer. All classes of charge isomers (naturally occurring or generated in vitro) were more effective at increasing vesicle permeability than was poly(L-lysine), a polycation that only interacts electrostatically with the bilayer. Although C1 and C2 and their C-terminal-deficient derivatives did not differ in the amount of marker released, the phosphorylated derivative of C1 caused a smaller increase in vesicle permeability than did the other isomers, suggesting that phosphorylation had altered the ability of the protein to perturb the bilayer.(ABSTRACT TRUNCATED AT 250 WORDS)

Conformation and surface properties of deamidated gluten.

The surface properties of deamidated gluten were investigated with respect to their conformational changes. The helix content of gluten decreased curvilinearly with its decrease of deamidation. The surface tension decreased in proportion to the degree of deamidation. On the other hand, the surface hydrophobicity of gluten increased remarkably in proportion to the degree of deamidation. The emulsifying properties of gluten were improved greatly by deamidation, correlating linearly with the surface hydrophobicity. From these results, the relationships between the conformational changes and functional properties of deamidated gluten are discussed.