Oxidative modification of amino acids in porcine myofibrillar protein isolates exposed to three oxidizing systems

Susceptibility of amino acids in myofibrillar protein isolate (MPI) exposed to three oxidizing matrixes commonly encountered in muscle foods was compared. MPI suspensions (20 mg protein/mL) in 15 mM piperazine-N,N bis(2-ethane sulphonic acid) buffer (pH 6.0) were oxidized with an iron-catalyzed oxidizing system (IOS, 0.01 mM FeCl₃, 0.1 mM ascorbic acid, 0.0–10.0 mM H₂O₂), a lipid-oxidizing system (LOS, 0.0–10.0 mM linoleic acid, 3750 units of lipoxidase/mL), or a metmyoglobin (MetMb) oxidizing system (MOS, 0.0–0.5 mM H₂O₂/MetMb) for 24 h at 4 °C. Changes were quantitatively analyzed by determining amino acids on a reverse-phase liquid chromatographic (LC) system. In IOS, the amount of cysteine, methionine and tyrosine decreased (P < 0.05) with increasing [H₂O₂]. In LOS, only cysteine and methionine were lowered at increasing linoleic acid concentrations. In MOS, the quantity of alanine, cysteine, glycine, histidine, leucine and lysine, as well as the total amount of amino acids were significantly reduced at high concentrations of MetMb/H₂O₂. The results suggest that under typical meat processing conditions, iron- and metmyoglobin-catalyzed reactions play a major role in the oxidation of amino acids in muscle proteins.     

Oxidation‐Initiated Myosin Subfragment Cross‐Linking and Structural Instability Differences between White and Red Muscle Fiber Types

Both white and red muscles are commonly used in meat processing, and protein cross‐linking, which may be affected by oxidants, is a key factor affecting the product quality. In this study, myofibrillar proteins (MPs) extracted from postrigor chicken Pectoralis major (PM, predominantly white) and Gastrocnemius (GN, predominantly red) muscles were subjected to a ?OH‐oxidizing system (10 μM FeCl₃, 0.1 mM ascorbic acid, with 0, 5, 10, or 20 mM H₂O₂) at pH 6.2, 4 °C for 18 h. The solubility of nonoxidized (control) PM MPs (63%) was higher than that of control GN MPs (41%). After oxidation with ?OH generated at 5 mM H₂O₂, protein solubility decreased by 46% and 21% for PM and GN, respectively, due to aggregation. Chemical and electrophoretic analyses indicated H₂O₂‐dose‐dependent losses of sulfhydryls and the concomitant formation of disulfides which were more pronounced in PM protein samples. Oxidation favored cross‐linking of myosin rod or tail in PM MPs compared to an equal susceptibility of myosin subfragment‐1 (s‐1) and rod to ?OH in GN MPs. Both Ca‐ and K‐ATPase activities in GN myosin were more sensitive to ?OH than their PM counterparts, indicating a less stable s‐1 region of GN myosin to oxidation. The uncoiling of rods from PM myosin was more rapid than that in GN myosin during heating. Oxidation induced cross‐linking via disulfide bonds hindered the unfolding of rod, particularly in PM myosin. These data revealed the molecular events that underscore the necessity of meat processing and formulation control based on muscle fiber types.     

羟自由基和高铁肌红蛋白氧化体系对牦牛肉肌原纤维蛋白氨基酸含量变化的影响

本研究比较分析了牦牛肉成熟过程中及羟自由基氧化体系(iron-catalyzed oxidizing system,IOS)和高铁肌红蛋白氧化体系(metmyoglobin oxidizing system,MOS)在特定氧化强度下牦牛肉肌原纤维蛋白氨基酸含量的变化.结果表明,在牦牛肉成熟过程中,苏氨酸、半胱氨酸、甘氨...     

Negative impacts of in-vitro oxidative stress on the quality of heat-induced myofibrillar protein gelation during refrigeration

To investigate the effects of pre-oxidized myofibrillar protein (MP) on stability of heat-induced MP gelation throughout 28 days of refrigeration, a hydroxyl radical generating system (10 μM FeCl₃, 0.1 mM ascorbic acid, with 10 mM H₂O₂) was employed. Results demonstrated that an increase of carbonyl content followed a similar trend in both pre-oxidized and non-oxidized protein gels. Unexpectedly, pre-oxidized protein gels yielded increased (P < 0.05) hardness at 14 days of storage compared to the initial day of storage. The water holding capacity (WHC) had a significant positive correlation with gel hardness, percentage of immobile water, and T₂₂, while having a negative correlation with carbonyl group content and T₂₃ (P < 0.01). These results suggest that the pre-oxidation treatment increases the susceptibility of MP gel matrices to undergo oxidation and thus provides a better comprehension of the consequences that in vitro pre-oxidation treatments have on protein matrix systems.     

Concentration effects of hydroxyl radical oxidizing systems on biochemical properties of porcine muscle myofibrillar protein

The objective of the study was to determine the dose-dependency of myofibrillar protein oxidation on oxidizing ferric ion. Pork myofibrillar protein isolates (MPI) were suspended in 15 mM piperazine-N,N bis(2-ethane sulfonic acid) (PIPES) buffer (pH 6.0) with 0.6 M NaCl, and incubated at 4 °C for 24 h with two levels of ferric ion (0.01 and 0.1 mM FeCl₃) at eight concentrations of hydrogen peroxide (0.00–10 mM H₂O₂). In both high and low [FeCl₃] oxidizing systems, the Ca-ATPase activity steadily increased with the H₂O₂ concentration. On the other hand, K-ATPase activity, protein carbonyl content, and 2-thiobarbituric acid-reactive substances increased with H₂O₂ up to 1.0 mM, and then gradually declined. Protein unfolding and loss of myosin heavy chain occurred continuously with increasing H₂O₂ concentrations. All changes, except for K-ATPase activity, were generally more rapid and extensive in the high [FeCl₃] oxidizing system. Overall, the biochemical changes in MPI exposed to ferric iron-oxidizing systems were more pronounced at high [FeCl₃] than at low [FeCl₃], but the pattern of the biochemical alterations appeared to be independent of the FeCl₃ concentration.     

Protein Oxidation at Different Salt Concentrations Affects the Cross‐Linking and Gelation of Pork Myofibrillar Protein Catalyzed by Microbial Transglutaminase

In a fabricated then restructured meat product, protein gelation plays an essential role in producing desirable binding and fat‐immobilization properties. In the present study, myofibrillar protein (MFP) suspended in 0.15, 0.45, and 0.6 M NaCl was subjected to hydroxyl radical stress for 2 or 24 h and then treated with microbial transglutaminase (MTGase) in 0.6 M NaCl (E : S = 1 : 20) at 4 and 15 °C for 2 h. Protein cross‐linking and dynamic rheological tests were performed to assess the efficacy of MTGase for mediating the gelation of oxidized MFP. MTGase treatments affected more remarkable polymerization of myosin in oxidized MFP than in nonoxidized, especially for samples oxidized at 0.6 M NaCl. Notably, the extent of MTGase‐induced myosin cross‐linking at 15 °C in oxidized MFP improved up to 46.8%, compared to 31.6% in nonoxidized MFP. MTGase treatment at 4 °C for MFP oxidized in 0.6 M NaCl, but not MFP oxidized in 0.15 M NaCl, produced stronger gels than nonoxidized MFP (P < 0.05). The final (75 °C) storage modulus (G′) of oxidized MFP gels was significantly greater than that of nonoxidized, although the G′ of the transient peak (～44.5 °C) showed the opposite trend. Overall, oxidation at high salt concentrations significantly improved MTGase‐mediated myosin cross‐linking and MFP gelation. This might be because under this condition, MTGase had an increased accessibility to glutamine and lysine residues to effectively initiate protein–protein interactions and gel network formation.     

Xanthan Enhances Water Binding and Gel Formation of Transglutaminase-Treated Porcine Myofibrillar Proteins

ABSTRACT: In this study, the effect of xanthan on dynamic rheological properties, textural profile, and water binding of transglutaminase (TG)-treated myofibrillar protein (MP) gels was investigated. In experiment 1, MP suspensions (40 mg/mL protein, 0.6 M NaCl) at pH 6.45 with or without 0.05% xanthan were treated with 0%, 0.1%, 0.2%, 0.3%, 0.4%, and 0.5% TG; in experiment 2, MP suspensions (40 mg/mL protein, 0.6 M NaCl) at pHs 6.13, 6.30, 6.45, 6.69 with or without 0.05% xanthan were treated with 0.3% TG. Treated samples were analyzed with differential scanning calorimetry for thermal stability and oscillatory rheometry and Instron penetration tests for gelation properties. The TG treatments lowered the transition temperature (T_m) of MP by as much as 6 °C (P < 0.05) but increased apparent enthalpy of denaturation. However, there was no detectable thermal stability difference between MP samples with or without xanthan. The shear storage modulus (G′) of MP gels increased markedly upon treatments with ≥0.3% TG, and the presence of xanthan further enhanced the gel strength (P < 0.05). The addition of 0.05% xanthan decreased cooking loss of TG-treated MP gels by 17% to 23% when compared with gels without xanthan at all pH levels evaluated (6.13 to 6.67). Thus, the combination of TG and xanthan offered a feasible means to promote cross-linking and gelation of MP while reducing cooking losses.     

Hydroxyl Radical-Stressed Whey Protein Isolate: Functional and Rheological Properties

The objective of the study was to examine the sensitivity of whey protein functionality to oxidizing radicals. Whey protein isolate (WPI) was oxidatively stressed by incubation at 20 °C for 3, 5, and 10 h in hydroxyl radical-generating media containing 0.1 mM ascorbic acid, 0.1 mM FeCl₃, and 1–10 mM H₂O₂. Protein solubility decreased (P?<?0.05) with increasing H₂O₂ concentrations and oxidation time. Surface properties of WPI, including both emulsifying and foaming activities, exhibited significant improvements (P?<?0.05) at H₂O₂ concentrations up to 5 mM and oxidation time up to 5 h. The longer oxidation time or higher H₂O₂ concentrations tended to diminish the surface functionality. However, the oxidative stress, though decreasing the onset gelling temperature, had a general detrimental effect on WPI gelation (hardness, springiness, and storage modulus). The results indicated opposing effects of oxidation on WPI: detrimental to hydrodynamic properties (solubility, gelation) but beneficial to surface properties (emulsification, foaming).     

Enzyme catalyzed oxidative gelation of sugar beet pectin: Kinetics and rheology

Sugar beet pectin (SBP) is a marginally utilized co-processing product from sugar production from sugar beets. In this study, the kinetics of oxidative gelation of SBP, taking place via enzyme catalyzed cross-linking of ferulic acid moieties (FA), was studied using small angle oscillatory measurements. The rates of gelation, catalyzed by horseradish peroxidase (HRP) (EC 1.11.1.7) and laccase (EC 1.10.3.2), respectively, were determined by measuring the slope of the increase of the elastic modulus (G′) with time at various enzyme dosages (0.125–2.0 U mL⁻¹). When evaluated at equal enzyme activity dosage levels, the two enzymes produced different gelation kinetics and the resulting gels had different rheological properties: HRP (with addition of H₂O₂) catalyzed a fast rate of gelation compared to laccase (no H₂O₂ addition), but laccase catalysis produced stronger gels (higher G′). The main effects and interactions between different factors on the gelation rates and gel properties were examined in response surface designs in which enzyme dosage (0.125–2.0 U mL⁻¹ for HRP; 0.125–10 U mL⁻¹ for laccase), substrate concentration (1.0–4.0%), temperature (25–55 °C), pH (3.5–5.5), and H₂O₂ (0.1–1.0 mM) (for HRP only) were varied. Gelation rates increased with temperature, substrate concentration, and enzyme dosage; for laccase catalyzed SBP gelation the gel strengths correlated positively with increased gelation rate, whereas no such correlation could be established for HRP catalyzed gelation and at the elevated gelation rates (>100 Pa min⁻¹) gels produced using laccase were stronger (higher G′) than HRP catalyzed gels at similar rates of gelation. Chemical analysis confirmed the formation of ferulic acid dehydrodimers (diFAs) by both enzymes supporting that the gelation was a result of oxidative cross-linking of FAs.     

Inhibition of Oxidant-Induced Biochemical Changes of Pork Myofibrillar Protein by Hydrolyzed Potato Protein

ABSTRACT:  The objective of the study was to investigate the role of hydrolyzed potato protein (HPP) in protecting myofibril protein isolate (MPI) from oxidative modification. MPI prepared from pork muscle was suspended (30 mg protein/mL) in 15 mM piperazine- N , N -bis(2-ethane sulfonic acid) buffer (pH 6.0) with 0, 0.3, 0.75, and 1.5 mg/mL of antioxidative HPP (1-h Alcalase hydrolysate). Oxidation was induced by incubating the protein suspensions at 4 °C for 24 h with (1) an iron-catalyzed oxidizing system (IOS: 0.01 mM FeCl₃, 0.1 mM ascorbic acid, and 1.0 mM H₂O₂) and (2) a metmyoglobin-oxidizing system (MOS: 0.1 mM metmyoglobin and 0.1 mM H₂O₂). Changes in oxidized MPI were measured as thiobarbituric acid-reactive substances (TBARS), protein carbonyl content, Ca- and K-ATPase activities, and ultraviolet (UV) spectra. Oxidation increased the production of TBARS and protein carbonyls by 2.9- and 0.24-fold in IOS and 5.6- and 2.2-fold in MOS, respectively. The 2 oxidizing systems altered the Ca- and K-ATPase activities and exposed hydrophobic groups buried in MPI. The presence of HPP reduced the extent of MPI oxidation in all physicochemical categories tested. Therefore, HPP may be used as a potential functional ingredient in meat products to enhance their oxidative stability.     

Chemical, Physical, and Gel-forming Properties of Oxidized Myofibrils and Whey- and Soy-protein Isolates

Myofibrils, oxidized with FeCl₃/H₂O₂/ascorbate, exhibited an increase in carbonyls and amines, SH→SS conversion, peptide scission, myosin polymerization, and a decrease in thermal stability and gel‐formation ability. Amino‐acid side chains of whey‐protein isolates (WPI) and soy‐protein isolates (SPI) were also modified during oxidation, but the thermal stability of WPI or SPI was not significantly altered. Oxidation increased elasticity of SPI gel but not that of WPI gel. Similarly, oxidation promoted interactions of myofibrils with SPI but not with WPI, resulting in > 30% increases in elasticity of the myofibril/SPI composite gel over its nonoxidized control. Hence, in processed meats where oxidation occurs, the presence of soy proteins may enhance the functionality of myofibrillar proteins.     

Effects of oxidative modification on gel properties of isolated porcine myofibrillar protein by peroxyl radicals

AAPH-derived (2,2′-azobis (2-amidinopropane) dihydrochloride) peroxyl radicals were selected as representative free radicals of lipid peroxidation to investigate the effects of oxidative modifications on isolated porcine myofibrillar protein structures as well as their rheological and gelling properties. Incubation of myofibrillar protein with increasing concentrations of AAPH resulted in a gradual increase (p < 0.05) in carbonyl content and SH → S–S conversion. Results from SDS-PAGE indicated that medium (~ 1 mM) and relatively high (> 3 mM) concentrations of AAPH induced aggregation of myosin and denaturation of myosin, troponin and tropomyosin, respectively. These structural changes resulted in changes on gelation of myofibrillar protein. Low level protein oxidation (AAPH ≤ 0.5 mM) had no remarkable effect (p > 0.05) on the viscoelastic pattern of myofibrillar protein gelation. Moderate oxidative modification (AAPH ~ 1 mM) enhanced the water-holding capacity (WHC) and texture properties of gels, while further oxidation (AAPH > 3 mM) significantly reduced the gel quality.     

Effects of chicken myofibrillar protein concentration on protein oxidation and water holding capacity of its heat-induced gels

The effects of chicken myofibrillar protein (MP) concentrations (20, 40 and 60 mg/mL) on MP oxidation and the subsequent effects on water holding capacity (WHC) of heat-induced gels were investigated. MP oxidation stressed by a hydroxyl radical-generating system (10 µM FeCl₃, 0.1 mM ascorbic acid, and 1 mM H₂O₂) was evaluated by carbonyl content. Water distribution, microstructure visualization, free sulfhydryl (FSH) groups and molecular forces of the gels were determined to illustrate the effects on WHC. Compared to the non-stressed group, the stressed proteins were highly oxidized where the carbonyl content (p?<?0.01) increased with decreasing MP concentrations (increased by 24.67% at 20 mg/mL). As the MP concentrations were decreased, the percentage of immobile water (decreased by 8.01% at 20 mg/mL, while increased by 5.98% at 60 mg/mL), ionic bonds, hydrogen bonds, and FSH groups (p?<?0.05) of the oxidized gels were decreased, the gel network of the oxidized groups was impaired which was with more cracks and protein aggregates, the hydrophobic interactions (increased by 24% at 20 mg/mL) and the percentage of free water (p?<?0.05) (increased by 7.80% at 20 mg/mL, but decreased by 6.13% at 60 mg/mL) of the oxidized gels were enhanced, and the resultant WHC was reduced (decreased by 17.75 and 8.07% at 20 and 40 mg/mL, respectively, but increased by 8.54% at 60 mg/mL). The results indicated that the MP concentration could be a potential influencing factor that affected the protein oxidation and subsequently affected the WHC of gels.     

肌原纤维蛋白氧化对谷氨酰胺转移酶介导的交联反应及凝胶性的影响

研究不同盐浓度下肌原纤维蛋白的氧化对后继的微生物谷氨酰胺转移酶(MTG)交联反应及凝胶能力的影响。分散在三种盐浓度(0.15、0.45和0.6mol/L NaCl)下的肌原纤维蛋白在4℃分别氧化2h和24h,然后在0.6mol/L NaCl条件下加入MTG于30℃交联2h。通过电泳和溶解度研究蛋白的氧化对MTG交联反应的影响,通过凝胶强度研究蛋白的氧化对MTG交联反应后凝胶性的影响。研究表明,三种盐浓度下的氧化都会促进MTG交联反应,特别是高盐条件下的氧化(提高量达47.8%)。所有样品经MTG交联后凝胶强度均显著增加(p<0.05),高盐条件下氧化的蛋白其凝胶强度明显高于低盐条件,然而在所有盐浓度下,氧化蛋白的凝胶强度增幅均小于未氧化的。结果表明过多的交联反应反而不利于蛋白成胶性。      

大豆分离蛋白对肌原纤维蛋白加热过程中结构及流变特性的影响

大豆分离蛋白（soybean protein isolate,SPI）作为优质的植物蛋白常被用于肉制品加工中,以提高产品产量和质地。研究添加SPI对肌原纤维蛋白（myofibrillar protein,MP）凝胶特性及MP加热过程中结构和流变特性的影响。结果表明：添加10%、20% SPI能提升混合凝胶的凝胶强度及保水性（P＜0.05）;加热过程中混合蛋白凝胶二级结构发生改变,但其变化规律尚不明确;添加SPI使混合凝胶储能模量及损耗模量下降;混合凝胶上清液十二烷基硫酸钠-聚丙烯酰胺凝胶电泳图谱显示,肌球蛋白重链、肌动蛋白、SPI部分亚基均是参与凝胶形成的蛋白质。     

Extreme pH treatments enhance the structure-reinforcement role of soy protein isolate and its emulsions in pork myofibrillar protein gels in the presence of microbial transglutaminase

Alkali (pH₁₂) and acid (pH_1.5) pH-treated soy protein isolates (SPI) were incorporated (0.25–0.75% protein) into sols of myofibrillar protein (MP, 3%, in 0.6 M NaCl at pH 6.25) with or without 0.1% microbial transglutaminase (TG) to investigate the potential as meat processing ingredients. Static and dynamic rheological measurements showed significant enhancements of MP gelling ability by the inclusion of pH_1.5-treated as well as preheated SPI (90 °C, 3 min). A 7-h incubation with TG accentuated the gel-strengthening effect by these SPI samples. The B subunit in 11S of SPI was the main component manifesting structure reinforcement in the mixed gels. The MP gelling properties were also greatly improved (P < 0.05) by the addition of 10% canola oil emulsions stabilized by pH-treated SPI. The principal force in the MP gels incorporated with pH-treated SPI was hydrophobic patches; in the presence of TG, cross-linking of previously dissociated A and B subunits of 11S was also intimately involved.     

Effect of Partial Substitutes of NaCl on the Cold-Set Gelation of Grass Carp Myofibrillar Protein Mediated by Microbial Transglutaminase

The objectives of this study were to investigate the changes and the relationship between structure and physiochemical properties of low sodium salt substitutes (NaCl partially replaced by KCl, CaCl₂, and MgCl₂) on grass carp myofibrillar protein gels mediated by microbial transglutaminase during cold-set gels and to provide more information about the gel characteristics. The gel strength, water holding capacity, whiteness, rheological characteristics, differential scanning calorimeter (DSC), and Raman spectra of cold-set gels were determined. The Raman spectra data were fitted to four secondary structures (α-helix, β-sheet, β-turn, and random coil). The gel properties of cold-set gels varied both with the low sodium salt types and incubation time. Myofibrillar protein (MP) gels added with NaCl and KCl had significantly higher water holding capacity than the MgCl₂, CaCl₂, and control groups. Additionally, the results showed that the gel strength and G’ value increased with the incubating time. No significant difference was detected in whiteness between the NaCl group and partial substituted groups. Cold-set gels added with the same molar amount of NaCl and KCl had fairly similar gel properties. There is a strong correlation between structural properties and gel properties of MP gels determined by DSC during the cold-set gelation process.     

Curtailing Oxidation‐Induced Loss of Myosin Gelling Potential by Pyrophosphate Through Shielding the S1 Subfragment

In muscle food processing, where oxidation is inevitable, phosphates are usually added to improve water binding. This present study attempted to investigate the interactive roles of protein oxidation and pyrophosphate (PP) during thermal gelation of myosin. Myosin isolated from pork muscle was solubilized in 0.5 M NaCl at pH 6.2 then oxidatively stressed with an iron‐redox cycling system that produces hydroxyl radicals with or without 1 mM PP and 2 mM MgCl₂ at 4 °C for 12 or 24 h then heated to 50 °C at 1.3 °C/min. Protein conformational stability was measured by differential scanning calorimetry, and covalent cross‐linking was examined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis following chymotrypsin digestion. The binding of PP to myosin suppressed disulfide bond formation in myosin subfragments 1 and 2 and partially inhibited oxidation‐initiated cross‐linking of heavy meromyosin during myosin gelation with a lesser effect on light meromyosin. In the presence of PP, myosin exhibited less loss of conformational integrity upon oxidation than myosin without PP. Rheological analysis from 20 to 75 °C indicated up to 32% decreases (P < 0.05) in elastic modulus (G′) of myosin gels due to oxidation. However, the presence of 1 mM PP, which also lowered the gelling capacity of myosin, inhibited the oxidation‐induced G′ by nearly half (P < 0.05). These results suggest that the protection of myosin head from oxidative modification by PP can be a significant factor for the minimization of gelling property losses during cooking of comminuted meats.     

Effects of malondialdehyde-induced protein modification on water functionality and physicochemical state of fish myofibrillar protein gel

Effects of malondialdehyde (MDA)-induced modification on water distribution in fish myofibrillar proteins (MP) gels were investigated using nuclear magnetic resonance (NMR) and magnetic resonance imaging (MRI). The oxidative modifications of MP gels were evaluated by surface hydrophobicity, gel strength, water holding capacity (WHC), scanning electron microscopy (SEM) and SDS-PAGE. Influence of heating procedure on water distribution and functional properties of modified MP gels was also investigated. Results from NMR and MRI indicated that the water functionality was strongly affected by the modification level, which corresponded to the changes of water holding capacity of MP upon MDA modification. Compared with unmodified MP gels, the T₂ relaxation times of modified sample increased significantly, indicating an alteration of water-protein interaction upon MDA-induced modification. The fraction of P₂₃ declined from 7.66% to 0.15% as the MDA addition increased from 0 to 50 mM. Moreover, the relaxation components T_2b disappeared with the addition of MDA mainly due to enhanced protein flexibility and surface hydrophobicity. Besides, the P₂₃ (free water) of heated MP samples increased by 5.41 times compared with that of unheated MP samples.     

The effect of Fenton’s reactants and aldehydes on the changes of myoglobin from Eastern little tuna (<Emphasis Type="Italic">Euthynnus affinis</Emphasis>) dark muscle

The influences of Fenton’s reactants (H₂O₂ and FeCl₂) and aldehydes (hexanal and hexenal) on changes of oxymyoglobin and metmyoglobin from Eastern little tuna (Euthynnus affinis) dark muscle were studied. In the presence of H₂O₂, both oxymyoglobin and metmyoglobin were rapidly oxidized into ferrylmyoglobin based on spectra patterns. In the presence of Fe²⁺ and/or H₂O₂, the changes in fluorescent intensity of myoglobin were noticeable, but there were no changes in aggregation ratio. Release of non-heme iron from myoglobin was mainly governed by H₂O₂. When aldehydes were incorporated, the oxidation of oxymyoglobin and conformational changes of globin were more pronounced. No release of non-heme iron was noticeable, suggesting the stability of heme moiety toward aldehydes. Hexenal had a great impact on cross-linking of oxymyoglobin and metmyoglobin via covalent modification. Alteration of myoglobin redox state might be enhanced by conformational changes of globin induced by both Fenton’s reactants and aldehydes.