Structural modification of soy protein by the lipid peroxidation product acrolein

ArticleinLWT- Food Science and Technology 43(1):133-140 · January 2010with 22 Reads
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Abstract
Acrolein was selected as a representative secondary byproduct of lipid peroxidation to investigate the effect of oxidative modification of reactive aldehyde on soy protein structure. Acrolein reacted with histidine, lysine and cysteine residues in soy protein to form covalent adducts, leading to protein carbonylation and degradation of sulfhydryl groups. Circular dichroism spectra showed that soy protein modification by acrolein was related to loss of α-helix and increase of β-sheet structure. The decrease in solubility, surface hydrophobicity and intrinsic fluorescence indirectly implied that acrolein induced soy protein aggregation, and results obtained by size-exclusion chromatography directly showed that gradual aggregation of soy protein was induced by increasing concentration of acrolein. Results of sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that acrolein caused soy protein cross-linking which non-disulphide covalent bonds were involved in the formation of cross-linking, and subunits of β-conglycinin were more vulnerable to acrolein than that of glycinin.

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    In order to investigate the effects of small molecular aldehydes (among secondary lipid oxidation products) on the muscle structure and structural properties of myofibrillar proteins in large yellow croaker (Pseudosciaena crocea), the fish muscle was treated with different concentrations (0~10 mmol/L) of acrolein in an ex vivo oxidation experiment. The results showed that treatment with acrolein could lead to increased gaps between myofibers in large yellow croaker. Muscle fiber fracture and fragmentation occurred when the acrolein concentration reached 10 mmol/L, and the water-holding capacity of the fish muscle decreased when the acrolein concentration was greater than 0.1 mmol/L. The carbonyl content, bityrosine content, and surface hydrophobicity of the myofibrillar proteins increased significantly with an increase in the acrolein concentration above 0.1 mmol/L, whereas the contents of total sulfhydryl groups decreased significantly. Active sulfhydryl groups were sensitive to acrolein, and their contents decreased by 40.8% compared with the control at an acrolein concentration of 0.01 mmol/L. The SDS-PAGE results showed that acrolein oxidation could cause conjunction of some protein subunits, which formed aggregates in the area above 200 ku. In addition, the oxidation caused damage of the gel properties of the myofibrillar proteins, including decreases in the gel strength, water-holding capacity, and gel whiteness. All these results indicate that acrolein can damage the muscle structure of large yellow croaker and cause oxidation of the myofibrillar protein, thus damaging the structure and functional properties of the protein. ©, 2014, South China University of Technology. All right reserved.
  • Article
    The effects of radio frequency (RF) heating treatments with different final temperatures (70, 80, and 90 °C) and electrode gaps (120, 160, and 200 mm) on the structural characteristics of soy protein isolate (SPI) dispersion were investigated. The results showed that RF heating significantly influenced free sulfhydryl groups and surface hydrophobicity of SPI. Free sulfhydryl groups increased with the increase of final temperature. The hydrophobicity of the RF-heated sample was higher than the original SPI without RF treatment. The highest hydrophobicity of the RF-heated SPI was found with electrode gap of 200 mm at 90 °C. RF heating treatment resulted in the reduction of ultraviolet absorption of SPI indicating the change of three-dimensional positions of soy protein but did not modify the protein primary structure of SPI. The Fourier transform infrared spectroscopy showed that hydration of SPI was decreased by RF heating. The self-reassembly from random coil structure to β-sheet structure suggested that RF heating treatment can change the secondary structure of soy protein to be more orderly.
  • Article
    1. The soybean meal (SBM) was heated at 100°C for 1, 2, 4 and 8 h, respectively, and their resultant oxidative status was evaluated. 2. A total of 400 one-day-old Arbor Acres broilers were randomly divided into 5 treatments with 8 replicates of 10 birds each, and fed with diets containing non-heated SBM (NHSBM) or 1 of 4 heated SBMs (HSBMs, SBMs heated at 100°C for 1, 2, 4 and 8 h, respectively) for 42 days. 3. The contents of carbonyl in the SBMs were both linearly and quadratically increased, whereas the nitrogen solubility index, and in vitro digestibility of crude protein (CP) and dry matter (DM) in the SBMs were both linearly and quadratically decreased as heating time increased (P < 0.05). The concentrations of sulfhydryl and total sulfhydryl in the SBMs were linearly decreased as heating time increased (P < 0.05). 4. The average daily gain was linearly decreased while the feed conversion ratio (FCR) was linearly increased in broilers as heating time of dietary HSBMs increased during both d 22-42 and d 0-42 of study (P < 0.05), though FCR of broilers during d 22-42 study were unaffected when the heating time of dietary HSBMs was 1 h (P > 0.05). The serum glucose concentration and the activity of trypsin at d 42, and the apparent total digestibility of CP and DM were all linearly reduced in broilers when heating time of dietary HSBMs increased (P < 0.05). However, 1 h HSBM has a numerical higher CP and DM digestibility than NHSBM. The serum urea nitrogen contents were both linearly and quadratically increased at both d 21 and 42 (P < 0.05), and relative pancreas weight was linearly increased at d 42 in broilers as heating time of dietary HSBMs increased (P < 0.05).
  • Article
    Flavour binding or release behaviour from the meat matrix is very important for its sensory properties. The interaction between flavour substance (nonanal) and myofibrillar proteins (MPs) was investigated using protein fluorescence quenching at different temperatures. The results suggested that nonanal caused the fluorescence quenching mechanism of MPs combining dynamic and static quenching mode, and dynamic quenching played a dominant role. Nonanal mainly combined with tryptophan residues rather than tyrosine residues. The results of synchronous fluorescence spectra and circular dichroism (CD) revealed that the interaction between nonanal and MPs induced no significant conformational changes in MPs. The binding constant (K) and number of binding sites (n) (1.45–2.03) increased with temperature. The negative value of ∆G (−383.16 kJ mol⁻¹ to −397.30 kJ mol⁻¹) showed that the interaction of nonanal and MPs was spontaneous. The positive ∆H (180.18 kJ mol⁻¹, 181.48 kJ mol⁻¹) and ∆S (696.17 J mol⁻¹ K⁻¹, 688.32 J mol⁻¹ K⁻¹) indicated that the binding of nonanal to MPs driven by hydrophobic force.
  • Article
    BACKGROUND Soy protein isolate (SPI) has promising applications in various food products because of its excellent functional properties and nutritional quality. The structural and emulsifying properties of covalently modified SPI by (–)‐epigallocatechin‐3‐gallate (EGCG) were investigated. RESULTS SPI was covalently modified by EGCG under alkaline conditions. SDS‐PAGE analysis revealed that EGCG modification caused cross‐linking of SPI proteins. Circular dichroism spectra demonstrated that the secondary structure of SPI proteins was changed by EGCG modification. In addition, the modifications resulted in the perturbation of the tertiary structure of SPI as evidenced by intrinsic fluorescence spectra and surface hydrophobicity measurements. Oil‐in‐water emulsions of modified SPI had smaller droplet sizes and better creaming stability compared to those from unmodified SPI. CONCLUSION The covalent modification by EGCG improved the emulsifying property of SPI. This study provided an innovative approach for improving the emulsifying properties of proteins.
  • Article
    The objective of this study was to determine structure and functional properties changes of oxidised egg white protein derived from hydrogen peroxide (H2O2)/ferric chloride (FeCl3) /ascorbic acid hydroxyl radical‐generating systems at room temperature, including carbonyls, sulfhydryl and total sulfhydryl groups, dityrosine, free amino, surface hydrophobicity, particle size distribution, intermolecular forces, and foamability and emulsibility. Protein carbonyl and dityrosine content of egg white protein were increased (P < 0.05) with increasing the concentrations of H2O2. Oxidation decreased free sulfhydryl, total sulfhydryl groups and surface of hydrophobicity comparing with nonoxidised egg white protein. Oxidation also reduced its free amino content (P < 0.05). The low concentration of H2O2 contributing the average particle size of egg white protein was smaller than the control group. However, the high concentrations of H2O2 caused that egg white protein aggregated and the average particle size became larger. To sum up, oxidation made EWP denaturation and aggregation. The objective of this study was to determine structure and functional properties changes of oxidized egg white protein derived from Hydrogen peroxide (H2O2)/Ferric chlorid (FeCl3) /Ascorbic acid hydroxyl radical‐generating systems at room temperature, including carbonyls, sulfhydryl and total sulfhydryl groups, dityrosine, free amino, surface hydrophobicity, particle size distribution, intermolecular forces; and foamability and emulsibility.
  • Article
    We investigated the effect of acrolein, a byproduct of lipid oxidation, on the structure and gel properties of myofibrillar proteins (MPs) isolated from rabbit meat. As the acrolein concentration increased, the protein carbonyl compounds significantly accumulated (p < 0.05), and the total sulfhydryl content was significantly lost (p < 0.05). The results of circular dichroism spectra, surface hydrophobicity, UV absorption spectra and intrinsic fluorescence spectra evidenced that acrolein caused the disruption of α-helix structure, the exposure of hydrophobic sites and the unfolding of MPs. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis suggested that medium (0–1 mM) and high (5–10 mM) concentrations of acrolein could induce protein cross-linkage and protein aggregation, respectively. These structural changes could affect gelling properties of MPs involving gel strength and water holding capacity (WHC). The results of Raman spectroscopy indicated that moderate oxidative modification caused protein unfolding as well as the decline of α-helix structure and the increase of β-sheets structure in gels, thereby influencing the gel properties. Moderate oxidative modification (0–1 mM) improved gel strength and WHC, while excessive oxidative modification (5–10 mM) resulted in decreased gel properties. © 2018, Springer Science+Business Media, LLC, part of Springer Nature.
  • Article
    The aim of this study was to analyze the effect of AAPH on the conformational structure and allergenicity of shrimp tropomyosin (TM). The structure of AAPH-TM was evaluated by SDS-PAGE, fluorescence, circular dichroism (CD) and ultraviolet light (UV), and the allergenicity was evaluated by in vivo and in vitro methods. Results showed that AAPH can induce structural changes through TM aggregations. These aggregations can decrease the IgG/IgE binding capacity on immunoblot analysis. Further competitive inhibition ELISA (ciELISA) results showed the IC50 of AAPH-TM (AAPH 0–25 mmol/l) changed from 0.086 to 46.2 μg/ml, which correlated with TM structural changes. An RBL-2H3 cell assay showed that release rate of β-hexosaminidase and histamine decreased by 51.6% and 68.1% with AAPH (5 mmol/l) treatment, respectively. A mouse model showed AAPH-TM could decrease levels of IgE/IgG1, release of histamine and mMCP-1 in sera. In conclusion, AAPH induced TM aggregation can cause structural changes and decrease the allergenicity.
  • Article
    Surface activity and molecular changes of cuttlefish skin gelatin modified with oxidized linoleic acid (OLA) prepared at 60, 70 and 80 °C at different times were investigated. Modification of gelatin with OLA could improve surface activity of resulting gelatin as evidenced by the decreased surface tension and the increased foaming and emulsifying properties. Interaction between OLA and gelatin led to the generation of carbonyl groups, loss of free amino content and the increase in particle size of resulting gelatin. Emulsion stabilized by modified gelatin had the smaller mean particle diameter with higher stability, compared with that stabilized by gelatin without modification.
  • Article
    Full-text available
    The ability of unsaturated fatty acid methyl esters to modify amino acid residues in bovine serum albumin (BSA), glutamine synthetase, and insulin in the presence of a metal-catalyzed oxidation system [ascorbate/Fe(III)/O(2)] depends on the degree of unsaturation of the fatty acid. The fatty acid-dependent generation of carbonyl groups and loss of lysine residues increased in the order methyl linoleate < methyl linolenate < methyl arachidonate. The amounts of alkyl hydroperoxides, malondialdehyde, and a number of other aldehydes that accumulated when polyunsaturated fatty acids were oxidized in the presence of BSA were significantly lower than that observed in the absence of BSA. Direct treatment of proteins with various lipid hydroperoxides led to a slight increase in the formation of protein carbonyl derivatives, whereas treatment with the hydroperoxides together with Fe(II) led to a substantial increase in the formation of protein carbonyls. These results are consistent with the proposition that metal-catalyzed oxidation of polyunsaturated fatty acids can contribute to the generation of protein carbonyls by direct interaction of lipid oxidation products (alpha,beta-unsaturated aldehydes) with lysine residues (Michael addition reactions) and also by interactions with alkoxyl radicals obtained by Fe(II) cleavage of lipid hydroperoxides that are formed. In addition, saturated aldehydes derived from the polyunsaturated fatty acids likely react with lysine residues to form Schiff base adducts.
  • Article
    Soy proteins with soybean oil (9:1,w/w) were stored at 60°C to investigate the changes in intrinsic fluorescence during oxidation. The front-surface fluorescence of the oxidized samples showed excitation and emission maxima at 355 and 440 nm respectively. The fluorescent compounds were soluble in the organic layer of the chloroform–methanol (2:1,v/v). The solution fluorescence showed an excitation maximum at 365 nm and an emission maximum at 450 nm, and the intensity increased during storage. The interactions of oxidizing soybean oil and soy proteins also resulted in decreases of protein solubility, soluble protein hydrophobicity, and free amino groups of proteins. With an antioxidant (BHT) addition, the changes in fluorescence and in protein properties were inhibited. The intensity of the solution fluorescence showed high correlation with TBA value (r=0.968) and protein solubility (r=−0.979), which could serve as an indicator for oxidative deterioration of soy proteins and soybean oil systems.
  • Article
    Oxidative modification of soy protein by peroxyl radicals generated in a solution containing 2,2’-azobis (2-amidinopropane) dihydrochloride (AAPH) under aerobic condition was investigated. Incubation of soy protein with increasing concentration of AAPH resulted in gradual generation of protein carbonyl derivatives and loss of protein sulphydryl groups. Circular dichroism spectra indicated that exposure of soy protein to AAPH led to loss of α-helix structure. Effect of oxidation on tertiary structure was demonstrated by surface hydrophobicity and tryptophan fluorescence. Surface hydrophobicity steadily decreased, accompanied by loss and burial of some tryptophan residues, indicating that soy protein gradually aggregated. The results of the size exclusion chromatogram (SEC) implied that incubation caused an AAPH-dose-dependent increase of fragmentation and aggregation of oxidised soy protein. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) indicated that non-disulphide linkages were involved in aggregate formation, and β-conglycinin was more vulnerable to peroxyl radicals than glycinin.
  • Article
    Addition of tert-butylhydroquinone or a mixture of butylated hydroxyanisole and tert-butylhydroquinone (200 ppm on a lipid basis) during SPI processing gave increased protein solibility over that of the control (55%. 56% and 34%. respectively). These increased solubilities correspond to 32% and 18% decrease in oxidation of free sulfhydryls and 20% and 12% reduction in protein oxidation, as determined by protein carbonyl content. Increased protein solubilities, due to added antioxidants, were accompanied by higher total protein surface hydrophobicity, as determined by the sodium dodecyl sulfate (SDS) binding method, and soluble protein hydrophobicity, as determined by the fluorescence probe 8-anilino-1-naphthalene sulfonate (ANS).
  • Article
    BACKGROUND: Protein oxidation results in covalent modification of structure and deterioration of functional properties of target protein. Oxidation extent of soy protein was affected by the content and type of lipid peroxidation (LPO) products in defatted soybean flours during storage and processing. Malondialdehyde (MDA) was selected as a secondary byproduct of LPO to investigate the effects of oxidative modification of LPO-derived reactive aldehyde on soy protein structure.RESULTS: MDA reacted with ε-amino and sulfhydryl groups of soy protein, and resulted in an increase in protein carbonyl groups but a decrease in sulfhydryl/disulfide, free amines and lysine. The decrease in solubility, surface hydrophobicity and intrinsic fluorescence indirectly indicated that MDA induced soy protein aggregation, and results of high-performance size-exclusion chromatography directly showed that gradual aggregation of soy protein was induced by increasing concentration of MDA. Results of electrophoresis indicated that MDA caused soy protein aggregation, and non-disulfide covalent bonds were involved in aggregate formation.CONCLUSION: The results showed that sensitivity of soy protein was related to MDA concentration. Soy protein gradually aggregated with increase of MDA concentration; β-conglycinin was more sensitive to MDA modification than glycinin. Copyright © 2009 Society of Chemical Industry
  • Article
    Proteins are a major target for oxidants as a result of their abundance in biological systems, and their high rate constants for reaction. Kinetic data for a number of radicals and non-radical oxidants (e.g. singlet oxygen and hypochlorous acid) are consistent with proteins consuming the majority of these species generated within cells. Oxidation can occur at both the protein backbone and on the amino acid side-chains, with the ratio of attack dependent on a number of factors. With some oxidants, damage is limited and specific to certain residues, whereas other species, such as the hydroxyl radical, give rise to widespread, relatively non-specific damage. Some of the major oxidation pathways, and products formed, are reviewed. The latter include reactive species, such as peroxides, which can induce further oxidation and chain reactions (within proteins, and via damage transfer to other molecules) and stable products. Particular emphasis is given to the oxidation of methionine residues, as this species is readily oxidised by a wide range of oxidants. Some side-chain oxidation products, including methionine sulfoxide, can be employed as sensitive, specific, markers of oxidative damage. The product profile can, in some cases, provide valuable information on the species involved; selected examples of this approach are discussed. Most protein damage is non-repairable, and has deleterious consequences on protein structure and function; methionine sulfoxide formation can however be reversed in some circumstances. The major fate of oxidised proteins is catabolism by proteosomal and lysosomal pathways, but some materials appear to be poorly degraded and accumulate within cells. The accumulation of such damaged material may contribute to a range of human pathologies.
  • Article
    Aggregation of lipid-reduced soybean proteins (LRSP) was investigated by chemical analysis, spectroscopy, electrophoresis, SEC-HPLC and light scattering. Soybean proteins obtained from the model systems consisting of LRSP and different levels of linoleic acid and lipoxygenase (RSP 4 and 5) showed increased turbidity, protein oxidation, surface hydrophobicity but decreased sulfhydryl and disulfide contents. SDS-PAGE of RSP 4 and 5 revealed remarkable difference of electrophoretic bands for 7S subunits, comparing with those samples without linoleic acid and lipoxygenase. Fluorescence spectroscopy suggested other covalent linkages than disulfide bonds formed during the formation of aggregates. SEC-HPLC and laser light scattering indicated that aggregates with high molecular weight and large particle size existed in samples of RSP 4 and 5. The experimental evidences suggest that the aggregates were formed via non-covalent interactions, but covalent bonds were also involved.
  • Article
    Full-text available
    Lipid peroxidation often occurs in response to oxidative stress, and a great diversity of aldehydes are formed when lipid hydroperoxides break down in biological systems. Some of these aldehydes are highly reactive and may be considered as second toxic messengers which disseminate and augment initial free radical events. The aldehydes most intensively studied so far are 4-hydroxynonenal, 4-hydroxyhexenal, and malonaldehyde. The purpose of this review is to provide a comprehensive summary on the chemical properties of these aldehydes, the mechanisms of their formation and their occurrence in biological systems and methods for their determination. We will also review the reactions of 4-hydroxyalkenals and malonaldehyde with biomolecules (amino acids, proteins, nucleic acid bases), their metabolism in isolated cells and excretion in whole animals, as well as the many types of biological activities described so far, including cytotoxicity, genotoxicity, chemotactic, and effects on cell proliferation and gene expression. Structurally related compounds, such as acrolein, crotonaldehyde, and other 2-alkenals are also briefly discussed, since they have some properties in common with 4-hydroxyalkenals.
  • Article
    Effects of pulsed electric fields (PEF) treatment (0–547 μs and 0–40 kV/cm) on physicochemical properties of soybean protein isolates (SPI) were studied. Solubility, surface free sulfhydryls (SHF) and hydrophobicity of SPI dispersions (20 mg/ml) increased with the increment of the PEF strength and treatment time at constant pulse width 2 μs, pulse frequency 500 pulse per second (pps) and sample flow rate (1 ml/s). When the PEF strength and treatment time were above 30 kV/cm and 288 μs, solubility, surface SHF, and hydrophobicity of SPI decreased due to denaturation and aggregation of SPI by hydrophobic interactions and disulfide bonds. Size-exclusion chromatography and laser light scattering analyses demonstrated further that stronger PEF treatment-induced dissociation, denaturation and reaggregation of SPI. Circular dichroism analysis showed that PEF treatment did not produce significant secondary structure changes of SPI. These results suggested that controlled PEF could be applied to process liquid food including soybean protein ingredient and to modify their structure and function in order to get desired products.
  • Article
    Soybean lipoxygenase was inactivated to different degrees by dry heating of defatted soybean flour for 0, 5, 10, 15, 20 and 25 min and soy protein isolates were prepared thereof by isoelectric precipitation of the water extract of the defatted soybean flour. The fluorescence emission intensity at 420 nm of the chloroform–methanol extract of soy protein isolates, which was indicator of the existence of peroxidized lipid, varied in parallel with the lipoxygenase residual activity in defatted soybean flours. The dispersion of soy protein isolate showed an increasing turbidity with the increase of lipoxygenase residual activity in the starting defatted soybean flour, suggesting an elevated tendency to form insoluble aggregates during the preparation of soy protein isolate. Small deformation rheological test revealed that the gelling times were shorter for those soy protein isolates derived from low lipoxygenase activity defatted soybean flours than that of high lipoxygenase activity. Frequency sweep showed that G′ of soy protein isolate derived from low lipoxygenase defatted soybean flour was independent of oscillation frequency in contrast to that of derived from non dry-heated defatted soybean flour, the latter showed a marked frequency dependence. Large deformation test revealed that the gel hardness increased about 10 times after dry heating of defatted soybean flour for 20 min. As the increase of the lipoxygenase residual activity, the gel permeability increased markedly, suggesting that soy protein isolate from high lipoxygenase defatted soybean flour produced coarser textured gel, which corresponded well with the results of scanning electron microscopy.
  • Article
    The aggregation and structural properties of soy protein isolate (SPI), induced by high pressure (HP) treatment at 200–600 MPa were investigated by size-exclusion chromatography combined with multi-angle laser scattering (SEC-MALLS) and fourier transform infrared (FTIR) spectroscopy. HP treatment at lower pressure level (e.g., 200 MPa) resulted in formation of marked insoluble aggregate of SPI, while the treatment at higher pressure level (e.g., 600 MPa) led to transformation of insoluble aggregate to soluble one. The soluble aggregate formed at 400 or 600 MPa had much less mean molecular weight (MW) (about 5.2 × 106 g/mol) than that at 200 MPa (about 1.6 × 107 g/mol), and was also much more homogenous in MW distribution. FTIR analyses confirmed changes in secondary and tertiary structures, induced by HP treatment. These results can provide direct evidence or explanation for HP-induced modification of soy proteins.
  • Article
    Bicinchoninic acid, sodium salt, is a stable, water-soluble compound capable of forming an intense purple complex with cuprous ion (Cu1+) in an alkaline environment. This reagent forms the basis of an analytical method capable of monitoring cuprous ion produced in the reaction of protein with alkaline Cu2+ (biuret reaction). The color produced from this reaction is stable and increases in a proportional fashion over a broad range of increasing protein concentrations. When compared to the method of Lowry et al., the results reported here demonstrate a greater tolerance of the bicinchoninate reagent toward such commonly encountered interferences as nonionic detergents and simple buffer salts. The stability of the reagent and resulting chromophore also allows for a simplified, one-step analysis and an enhanced flexibility in protocol selection. This new method maintains the high sensitivity and low protein-to-protein variation associated with the Lowry technique.
  • Article
    The determination of available lysine in proteins by their reactions with 2,4,6-trinitrobenzenesulphonic acid has been examined in relation to the preparation of the sample, the conditions of the reaction, the hydrolysis of the trinitrophenylated protein and the effects of other amino-acids and related compounds. From these observations a revised method is proposed for the rapid routine screening of animal protein concentrates used in animal feeds. With the use of DL-lysine as a standard instead of ε-trinitrophenyllysine and with a simplified hydrolysis procedure, the technique is suitable for examining comparatively large numbers of samples. Interference by other amino compounds is slight, except that by cadaverine, hydroxylysine and ornithine. Results are given for the levels of available lysine in a range of animal proteins, which compare closely with values obtained by the Carpenter procedure in which 1-fluoro-2,4-dinitrobenzene is used.
  • Article
    Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products. Four major components of the head are cleaved during the process of assembly, apparently after the precursor proteins have assembled into some large intermediate structure.
  • Article
    Full-text available
    Acrolein (CH2=CH—CHO) is known as a ubiquitous pollutant in the environment. Here we show that this notorious aldehyde is not just a pollutant, but also a lipid peroxidation product that could be ubiquitously generated in biological systems. Upon incubation with BSA, acrolein was rapidly incorporated into the protein and generated the protein-linked carbonyl derivative, a putative marker of oxidatively modified proteins under oxidative stress. To verify the presence of protein-bound acrolein in vivo, the mAb (mAb5F6) against the acrolein-modified keyhole limpet hemocyanin was raised. It was found that the acrolein-lysine adduct, N ɛ-(3-formyl-3,4-dehydropiperidino)lysine, constitutes an epitope of the antibody. Immunohistochemical analysis of atherosclerotic lesions from a human aorta demonstrated that antigenic materials recognized by mAb5F6 indeed constituted the lesions, in which intense positivity was associated primarily with macrophage-derived foam cells and the thickening neointima of arterial walls. The observations that (i) oxidative modification of low-density lipoprotein with Cu²⁺ generated the acrolein-low-density lipoprotein adducts and (ii) the iron-catalyzed oxidation of arachidonate in the presence of protein resulted in the formation of antigenic materials suggested that polyunsaturated fatty acids are sources of acrolein that cause the production of protein-bound acrolein. These data suggest that the protein-bound acrolein represents potential markers of oxidative stress and long-term damage to protein in aging, atherosclerosis, and diabetes. • covalent modification of protein • antibody • atherosclerosis
  • Article
    Three soy protein isolates were prepared from two commercial defatted soybean flours (SG and ADM) and flour indigenously prepared from soybeans grown in Ontario (ON). Denaturation degree and protein composition of three isolates as measured by DSC and SDS–PAGE were not different significantly, but remarkable differences in ANS surface hydrophobicity and turbidity were noticed. Heat induced gelling properties of these soy protein isolates were examined by small deformation oscillatory rheological test. The critical gelling concentrations of the two isolates from commercial soy flours (SG and ADM) in distilled water were 90 mg/ml, much lower than 110 mg/ml for ON. The effect of type and concentration of salts on the gelling properties of the three protein isolates were also examined. In 1 mol/L NaCl solution, ADM had the higher gel strength than SG. It was found that the increase in gel strength of ON by addition of NaSCN was much greater than that in NaCl solution. N-Ethylmaleimide (NEM) was observed to increased the gel strength for all soy protein isolates examined despite its sulfhydryl groups blocking function. It is suggested that a non-heat aggregation of protein which led to turbidity development was responsible for the inferior gelling property of ON while the different gelling behavior between SG and ADM could be attributed to their difference in ANS surface hydrophobicity.
  • Article
    There is increasing evidence that aldehydes generated endogenously during lipid peroxidation contribute to the pathophysiologic effects associated with oxidative stress in cells and tissues. A number of reactive lipid aldehydes, such as 4-hydroxy-2-alkenals and malondialdehyde, have been implicated as causative agents in cytotoxic processes initiated by the exposure of biologic systems to oxidizing agents. Recently, acrolein (CH2 = CH-CHO), a ubiquitous pollutant in the environment, was identified as a product of lipid peroxidation reactions. The basis for this finding is an experimental approach that provides a measure of acrolein bound to lysine residues of protein. The identification of acrolein as an endogenous lipid-derived product suggests an examination of the possible role of this aldehyde as a mediator of oxidative damage in a variety of human diseases.
  • Article
    Protein oxidation is defined here as the covalent modification of a protein induced either directly by reactive oxygen species or indirectly by reaction with secondary by-products of oxidative stress. Oxidative modification of proteins can be induced experimentally by a wide array of prooxidant agents and occurs in vivo during aging and in certain disease conditions. Oxidative changes to proteins can lead to diverse functional consequences, such as inhibition of enzymatic and binding activities, increased susceptibility to aggregation and proteolysis, increased or decreased uptake by cells, and altered immunogenicity. There are numerous types of protein oxidative modification and these can be measured with a variety of methods. Protein oxidation serves as a useful marker for assessing oxidative stress in vivo. There are both advantages and disadvantages to using proteins for this purpose compared to lipids and DNA. Finally, it is important to monitor the degree of oxidative modification of therapeutic proteins manufactured for commercial use. This review will examine various aspects of protein oxidation, with emphasis on using proteins as markers of oxidative stress in biological samples.
  • Article
    Oxidative stress may be a hallmark of several neurodegenerative disorders, including Alzheimer's disease (AD) Huntington's, and Parkinson's diseases as well as amyotrophic lateral sclerosis. Acrolein is a highly reactive product of lipid peroxidation that is elevated in the brains of persons with AD. This alkenal potentially can react with proteins by Michael addition to alter their structure and function. In the present study, we used electron paramagnetic resonance in conjunction with a protein-specific spin label to monitor synaptosomal membrane protein conformational alterations induced by acrolein. A dose-dependent increased conformational alteration was observed. Consistent with this finding, protein carbonyl levels from protein-bound acrolein were significantly elevated. However, pretreatment of synaptosomes with glutathione ethyl ester (GEE) significantly ameliorated both the conformational alterations and protein carbonyls induced by acrolein. Based on this success, we tested the hypothesis that elevated levels of endogenous glutathione (GSH) would offer protection against acrolein-induced oxidative stress. In-vivo elevation of GSH (215% over control, P<0.04) was produced by i.p. injection of N-acetylcysteine (NAC), a known precursor of GSH. Synaptosomes were treated with vehicle or 2 nM acrolein, the level of this alkenal found in AD brain. In contrast to synaptosomes from control animals, which had significantly increased protein carbonyl levels following addition of 2 nM acrolein, synaptosomes that were isolated from NAC-treated rodents and treated with 2 nM acrolein showed no increased carbonyl levels compared to untreated controls. These results demonstrate protection by increased in-vivo GSH levels against acrolein-induced oxidative stress at levels found in AD brain and are consistent with the notion that methods to increase endogenous GSH levels in neurodegenerative diseases associated with oxidative stress may be promising.
  • Article
    Acrolein, a representative carcinogenic aldehyde that could be ubiquitously generated in biological systems under oxidative stress, shows facile reactivity with the epsilon-amino group of lysine to form N(epsilon)-(3-formyl-3,4-dehydropiperidino)lysine (FDP-lysine) as the major product (Uchida, K., Kanematsu, M., Morimitsu, Y., Osawa, T., Noguchi, N., and Niki, E. (1998) J. Biol. Chem. 273, 16058-16066). In the present study, we determined the electrophilic potential of FDP-lysine and established a novel mechanism of protein thiolation in which the FDP-lysine generated in the acrolein-modified protein reacts with sulfhydryl groups to form thioether adducts. When a sulfhydryl enzyme, glyceraldehyde-3-phosphate dehydrogenase, was incubated with acrolein-modified bovine serum albumin in sodium phosphate buffer (pH 7.2) at 37 degrees C, a significant loss of sulfhydryl groups, which was accompanied by the loss of enzyme activity and the formation of high molecular mass protein species (>200 kDa), was observed. The FDP-lysine adduct generated in the acrolein-modified protein was suggested to represent a thiol-reactive electrophile based on the following observations. (i) N(alpha)-acetyl-FDP-lysine, prepared from the reaction of N(alpha)-acetyl lysine with acrolein, was covalently bound to glyceraldehyde-3-phosphate dehydrogenase. (ii) The FDP-lysine derivative reacted with glutathione to form a GSH conjugate. (iii) The acrolein-modified bovine serum albumin significantly reacted with GSH to form a glutathiolated protein. Furthermore, the observation that the glutathiolated acrolein-modified protein showed decreased immunoreactivity with an anti-FDP-lysine monoclonal antibody suggested that the FDP-lysine residues in the acrolein-modified protein served as the binding site of GSH. These data suggest that thiolation of the protein-bound acrolein may be involved in redox alteration under oxidative stress, whereby oxidative stress generates the increased production of acrolein and its protein adducts that further potentiate oxidative stress via the depletion of GSH in the cells.
  • Article
    Acrolein is a reactive lipid peroxidation byproduct, which is found in ischemic tissue. We examined the effects of acrolein on cytosolic aspartate aminotransferase (cAAT), which is an enzyme that was previously shown to be inhibited by glycating agents. cAAT is thought to protect against ischemic injury. We observed that acrolein cross-linked cAAT subunits as evidenced by the presence of high molecular weight bands following SDS-PAGE. Acrolein-modified cAAT resisted thermal denaturation when compared with native cAAT. We also observed a decrease in intrinsic fluorescence (290 nm, ex; 380 nm, em). These observations are consistent with an acrolein-induced change in conformation that is more rigid and compact than native cAAT, suggesting that intramolecular cross-links occurred. Acrolein also inhibited activity, and the inhibition of enzyme activity correlated with the acrolein-induced formation of cAAT cross-links.
  • Article
    Acrolein, a representative carcinogenic aldehyde, that could be ubiquitously generated in biological systems under oxidative stress shows facile reactivity with a nucleophile such as a protein. In this study, to gain a better understanding of the molecular basis of acrolein modification of protein, we characterized the acrolein modification of a model peptide (the oxidized B chain of insulin) by electrospray ionization-liquid chromatography/mass spectrometry method and established a novel acrolein-lysine condensation reaction. In addition, we found that this condensation adduct represented the major antigenic adduct generated in acrolein-modified protein. To identify the modification site and structures of adducts generated in the acrolein-modified insulin B chain, both the acrolein-pretreated and untreated peptides were digested with V8 protease and the resulting peptides were subjected to electrospray ionization-liquid chromatography/mass spectrometry. This technique identified nine peptides, which contained the acrolein adducts at Lys-29 and the N terminus, and revealed that the reaction of the insulin B chain with acrolein gave multiple adducts, including an unknown adduct containing two molecules of acrolein per lysine. To identify this adduct, we incubated N(alpha)-acetyllysine with acrolein and isolated a product having the same molecular mass as the unknown acrolein-lysine adduct. On the basis of the chemical and spectroscopic evidence, the adduct was determined to be a novel pyridinium-type lysine adduct, N(epsilon)-(3-methylpyridinium)lysine (MP-lysine). The formation of MP-lysine was confirmed by amino acid analysis of proteins treated with acrolein. More notably, this condensation adduct appeared to be an intrinsic epitope of a monoclonal antibody 5F6 that had been raised against acrolein-modified protein.
  • Article
    A proteomic approach was applied to mitochondrial protein isolated from the livers of rats fed a combination high-fat and ethanol diet to identify proteins modified by 4-hydroxynonenal (4-HNE). Using this approach, the endoplasmic reticulum chaperone, protein disulfide isomerase (PDI), which participates in the maturation of newly synthesized proteins through promoting correct disulfide formation, was consistently found to be modified by 4-HNE. Further mass spectral analysis of PDI isolated from the animals revealed modification of an active site Cys residue thought to be involved in client protein binding. To test the hypothesis that 4-HNE inhibits the chaperone, purified bovine PDI was treated with concentrations of 4-HNE ranging from 20 to 200 microM (10-100-fold molar excess aldehyde), resulting in 14-56% inhibition, respectively. Similar treatments with the lipid peroxidation products acrolein (ACR) and 4-oxononenal (4-ONE) resulted in 60 and 100% inhibition, respectively, suggesting inactivation of the chaperone via Cys modification. Thiol sensitivity was confirmed through concentration-dependent inhibition of PDI by the Cys modifier N-ethylmaleimide (NEM). While some degree of sensitivity to these lipid aldehydes is suggested by the data, when compared to inactivation of other proteins by 4-HNE, PDI has demonstrated a relative resistance. It was also observed that physiologic (e.g., 4 mM) concentrations of GSH were capable of removing the 4-HNE adducts, likely serving as a protective mechanism against inactivation by 4-HNE and other lipid peroxidation products. However, because an active site Cys was found to be modified by 4-HNE on PDI in vivo, it is possible that the protective effect of GSH on the chaperone decreases under conditions of sustained oxidative stress, such as during chronic alcohol consumption, as GSH is depleted. The data presented here thus suggest potential impairment of an important molecular chaperone during oxidative stress.
  • Article
    Oxidised lipids are reported to interact with proteins causing undesirable changes in nutritional and functional properties including a loss of amino acids, cross-linking and damage to proteins and DNA. ESR spectroscopy with spin trapping was used to study the type of radical species generated in methyl linoleate and the transfer of the radical to protein beta-lactoglobulin. Antioxidants vitamins C and E reduced lipid oxidation and subsequent transfer of the radical to the protein as shown by the shape and size of the radical adduct. Changes to protein molecular structure due to oxidation were investigated by multidimensional NMR spectroscopy and liquid chromatography. NMR spectra indicated that as a result of oxidation and protein denaturation, there was an increase in structural flexibility and some initially protected backbone amide groups were exposed as they become sharper and easily identifiable. Dityrosine was detected in all samples tested which is indicative of oxidative damage to proteins. Monitoring tyrosyl radicals and formation of dityrosine is of practical value in order to enhance the acceptability, nutritional and safety aspects of proteins.
  • Article
    Carbonylation of proteins is an irreversible oxidative damage, often leading to a loss of protein function, which is considered a widespread indicator of severe oxidative damage and disease-derived protein dysfunction. Whereas moderately carbonylated proteins are degraded by the proteasomal system, heavily carbonylated proteins tend to form high-molecular-weight aggregates that are resistant to degradation and accumulate as damaged or unfolded proteins. Such aggregates of carbonylated proteins can inhibit proteasome activity. Alarge number of neurodegenerative diseases are directly associated with the accumulation of proteolysis-resistant aggregates of carbonylated proteins in tissues. Identification of specific carbonylated protein(s) functionally impaired and development of selective carbonyl blockers should lead to the definitive assessment of the causative, correlative or consequential role of protein carbonylation in disease onset and/or progression, possibly providing new therapeutic approaches.
  • Article
    Effects of lipoxygenase I-B (LOX)-catalyzed oxidation of linoleic acid on soybean proteins was evaluated by electron spin resonance (ESR) and fluorescence spectroscopy in different model systems in the presence or absence of antioxidants. A strong central singlet signal was detected by ESR spectroscopy and identified as the carbon radical (g value range 2.0041-2.0054). A downfield shoulder attributed to the sulfur radical (g value 2.019-2.028) was also observed. The changes in soybean proteins were accompanied by an increase in fluorescence, indicating the formation of cross-links. Natural antioxidants such as ascorbic acid and alpha-tocopherol as well as synthetic antioxidants butyl hydroxytoluene (BHT) inhibited the development of both the free radical signal and the fluorescence when added to soybean proteins prior to incubation with linoleic acid and lipoxygenase I-B; the central singlet signal attributed to the carbon radical was reduced by 35-65%. This paper clearly indicates direct free radical transfer from oxidizing linoleic acid catalyzed by LOX to soybean proteins.
  • Article
    Acrolein, the most reactive of the alpha,beta-unsaturated aldehydes, is endogenously produced by lipid peroxidation, and has been found increased in the brain of patients with Alzheimer's disease. Although it is known that acrolein increases total protein carbonylation and impairs the function of selected proteins, no study has addressed which proteins are selectively carbonylated by this aldehyde. In this study we investigated the effect of increasing concentrations of acrolein (0, 0.005, 0.05, 0.5, 5, 50 microM) on protein carbonylation in gerbil synaptosomes. In addition, we applied proteomics to identify synaptosomal proteins that were selectively carbonylated by 0.5 microM acrolein. Acrolein increased total protein carbonylation in a dose-dependent manner. Proteomic analysis (two-dimensional electrophoresis followed by mass spectrometry) revealed that tropomyosin-3-gamma isoform 2, tropomyosin-5, beta-actin, mitochondrial Tu translation elongation factor (EF-Tu(mt)) and voltage-dependent anion channel (VDAC) were significantly carbonylated by acrolein. Consistent with the proteomics studies that have identified specifically oxidized proteins in Alzheimer's disease (AD) brain, the proteins identified in this study are involved in a wide variety of cellular functions including energy metabolism, neurotransmission, protein synthesis, and cytoskeletal integrity. Our results suggest that acrolein may significantly contribute to oxidative damage in AD brain.
  • Article
    Oxidative damage to proteins such as apolipoprotein B-100 increases the atherogenicity of low-density lipoproteins (LDL). However, little is known about the potential oxidative damage to apolipoprotein E (apoE), an exchangeable antiatherogenic apolipoprotein. ApoE plays an integral role in lipoprotein metabolism by regulating the plasma cholesterol and triglyceride levels. Hepatic uptake of lipoproteins is facilitated by apoE's ability to bind with cell surface heparan sulfate proteoglycans and to lipoprotein receptors via basic residues in its 22 kDa N-terminal domain (NT). We investigated the effect of acrolein, an aldehydic product of endogenous lipid peroxidation and a tobacco smoke component, on the conformation and function of recombinant human apoE3-NT. Acrolein caused oxidative modification of apoE3-NT as detected by Western blot with acrolein-lysine-specific antibodies, and tertiary conformational alterations. Acrolein modification impairs the ability of apoE3-NT to interact with heparin and the LDL receptor. Furthermore, acrolein-modified apoE3-NT displayed a 5-fold decrease in its ability to interact with lipid surfaces. Our data indicate that acrolein disrupts the functional integrity of apoE3, which likely interferes with its role in regulating plasma cholesterol homeostasis. These observations have implications regarding the role of apoE in the pathogenesis of smoking- and oxidative stress-mediated cardiovascular and cerebrovascular diseases.
  • Article
    Acrolein is a very reactive aldehyde present in cigarette smoke and endogenously generated by pathways such as lipid peroxidation and threonine metabolism by phagocytes. Acrolein has been shown to affect uptake of cholesterol by HDL. We hypothesized that acrolein could also have deleterious effects on paraoxonase 1 (PON-1) activity. We also determined whether free serum acrolein levels are higher in renal failure, and assessed whether they decrease after hemodialysis (HD) and whether this change correlates with increases in PON-1 activity. We incubated human HDL with 0-10 mmol/l acrolein for 2 h and measured PON-1 activity and structural changes. Acrolein was also measured in 40 end stage renal disease (ESRD) patients (before and after a hemodialysis session), and 40 control subjects. We found that acrolein inhibits PON-1 activity in HDL in a time and concentration dependent fashion. Inhibition occurred at 40% at 0.5 mmol/l and was cancelled by cysteine but not by aminoguanidine or carnosine. We confirm that free serum acrolein levels are higher in chronic renal failure patients and demonstrate that they are partially removed by HD. Decrease in acrolein levels after dialysis correlate with increases in PON-1 activity (r=0.32, p 0.01). Acrolein inactivates paraoxonase 1 in HDL, a process that is inhibited by N-acetylcysteine. We confirm that acrolein levels are higher in ESRD and show for the first time, data supporting that acrolein is partially removed by hemodialysis. Decrease in acrolein levels after dialysis correlates with increase in PON-1 activity. This could offer new insights to explain low PON-1 activities in smokers and renal failure subjects as well as pointing at thiol-conserving reducing compounds such as N-acetylcysteine, as putative therapeutic palliatives.
  • Article
    Full-text available
    Conjugated alpha,beta-unsaturated carbonyl derivatives such acrylamide, acrolein, and 4-hydroxy-2-nonenal (HNE) are members of a large class of chemicals known as the type-2 alkenes. Human exposure through diet, occupation, and pollution is pervasive and has been linked to toxicity in most major organs. Evidence suggests that these soft electrophiles produce toxicity by a common mechanism involving the formation of Michael-type adducts with nucleophilic sulfhydryl groups. In this commentary, the adduct chemistry of the alpha,beta-unsaturated carbonyls and possible protein targets will be reviewed. We also consider how differences in electrophilic reactivity among the type-2 alkenes impact corresponding toxicokinetics and toxicological expression. Whereas these concepts have mechanistic implications for the general toxicity of type-2 alkenes, this commentary will focus on the ability of these chemicals to produce presynaptic damage via protein adduct formation. Given the ubiquitous environmental presence of the conjugated alkenes, discussions of molecular mechanisms and possible neurotoxicological risks could be important. Understanding the neurotoxicodynamic of the type-2 alkenes might also provide mechanistic insight into neurodegenerative conditions where neuronal oxidative stress and presynaptic dysfunction are presumed initiating events. This is particularly germane to a recent proposal that lipid peroxidation and the subsequent liberation of acrolein and HNE in oxidatively stressed neurons mediate synaptotoxicity in brains of Alzheimer's disease patients. This endogenous neuropathogenic process could be accelerated by environmental type-2 alkene exposure because common nerve terminal proteins are targeted by alpha,beta-unsaturated carbonyl derivatives. Thus, the protein adduct chemistry of the conjugated type-2 alkenes offers a mechanistic explanation for the environmental toxicity induced by these chemicals and might provide insight into the pathogenesis of certain human neurodegenerative diseases.