Previous studies have reported that myosin can be modified by oxidative stress and particularly by activated haem proteins. These reactions have been implicated in changes in the properties of this protein in food samples (changes in meat tenderness and palatability), in human physiology (alteration of myocyte function and force generation) and in disease (e.g. cardiomyopathy, chronic heart failure). The oxidant species, mechanisms of reaction and consequences of these reactions are incompletely characterized. In the present study, the nature of the transient species generated on myosin as a result of the reaction with activated haem proteins (horseradish peroxidase/H2O2 and met-myoglobin/H2O2) has been investigated by EPR spectroscopy and amino-acid consumption, product formation has been characterized by HPLC, and changes in protein integrity have been determined by SDS/PAGE. Multiple radical species have been detected by EPR in both the presence and the absence of spin traps. Evidence has been obtained for the presence of thiyl, tyrosyl and other unidentified radical species on myosin as a result of damage-transfer from oxidized myoglobin or horseradish peroxidase. The generation of thiyl and tyrosyl radicals is consistent with the observed consumption of cysteine and tyrosine residues, the detection of di-tyrosine by HPLC and the detection of both reducible (disulfide bond) and non-reducible cross-links between myosin molecules by SDS/PAGE. The time course of radical formation on myosin, product generation and cross-link induction are consistent with these processes being interlinked. These changes are consistent with the altered function and properties of myosin in muscle tissue exposed to oxidative stress arising from disease or from food processing.

INTRODUCTION

Oxidation of a protein by radicals in the presence of O2 can result in major alterations to the physical and chemical nature of the protein, including oxidation of side-chain groups, backbone fragmentation, cross-linking, unfolding, changes in hydrophobicity and conformation, altered susceptibility to proteolytic enzymes and formation of new reactive groups (DOPA, carbonyl and hydroperoxide groups). These alterations can result in loss of the structure and enzymatic activity of the protein, and hence cause biological perturbations [13].

Myosin is the most abundant protein in muscle tissue and is located in the myofibrillar part of the muscle. Several studies have shown that myosin is susceptible to oxidation, causing intermolecular cross-linking and aggregation of the protein [47]. The type of cross-linking seems to be highly dependent on how oxidation of myosin is initiated. Oxidation with myoglobin/H2O2 has been reported to result in non-disulfide bond formation, whereas oxidation with a metal-catalysed (Fenton-like) system results primarily in the formation of disulfide bonds. Evidence has been presented for the generation of thiyl radicals on myosin on reaction with the mild oxidant Ce(IV) in the presence of the spin trap PBN (N-t-butyl-α-phenylnitrone) [8]. However, the relevance of such radical formation to damage induced by haem proteins is unclear.

Initiation of oxidation of proteins by myoglobin/H2O2 and HRP (horseradish peroxidase)/H2O2 has attracted considerable attention, owing to the high abundance of these haem proteins in many cell types and tissues and the likely continuous formation of H2O2in vivo as a result of a plethora of sources of superoxide radicals within, and on the surface of, cells (e.g. via electron leakage from mitochondrial and endoplasmic reticulum electron transport chains, NADPH oxidases, lipoxygenases, xanthine oxidase [9]). Enzymatic dismutation (catalysed by the multiple forms of superoxide dismutase) and spontaneous dismutation of such radicals yields H2O2 and O2. H2O2 activation of peroxidases and Mb [myoglobin in the +3 (met) oxidation state] (a pseudo-peroxidase) is known to form radicals on these proteins as a result of electron transfer from the protein to the oxidized haem group [10]. A number of formation sites have been reported for these species (e.g. tyrosine, tryptophan and cysteine residues), with the sites being both species- and sequence-dependent [1115]. It has been demonstrated that such protein-derived radicals are able to damage other proteins via radical transfer reactions [1619]. Thus long-lived radicals have been detected on BSA and a number of other proteins incubated with H2O2-activated haem proteins [17]. The structural and functional consequences of these radical transfer reactions, and their role in disease, have yet to be fully elucidated.

It is well established that oxidation of skeletal muscle myosin occurs in a number of human diseases, including chronic heart failure [20] and animal models of diabetes [21], and as a result of muscle aging [21,22]. Evidence has also been presented for the modification of myosin light chains during myocardial ‘stunning’ as a result of short periods of ischaemia and subsequent reperfusion in rabbits [23,24]. Myosin oxidation has also been detected in intact myofibrils exposed to myoglobin and H2O2 [25] and in isolated myosin [6,20,26]. A number of other oxidizing systems, in addition to H2O2-activated haem proteins, have been proposed to play a role in myosin damage, including peroxynitrite [20,22], hydroxyl radicals [27] and reactive aldehyde groups [22].

Damage to skeletal muscle components is of considerable significance to the food industry, as oxidative modifications that occur during food processing and storage can lead to changes in the physical and functional properties of myosin. Alterations in protein conformation and the formation of protein aggregates through intermolecular cross-linking as a result of oxidation decrease gel-forming ability, protein solubility and water-holding capacity, which negatively influence sensory characteristics (e.g. juiciness and tenderness) and palatability [5,2830]. Biochemical properties, such as the ATPase activity of myosin, which plays a role in muscle contraction, have also been found to change as a result of oxidation of myofibrillar proteins [31].

In the present study, the mechanism of myosin oxidation by two enzymatic systems (myoglobin/H2O2 and HRP/H2O2) has been examined using EPR spectroscopy (both direct and spin-trap analysis), amino-acid consumption and product analysis by HPLC and SDS/PAGE. Comparative studies were also performed using a range of other oxidant systems. We demonstrated that a number of different radicals are generated on myosin (thiyl, tyrosyl and unidentified radical species) by H2O2-activated haem proteins, and that these reactions result in the depletion of cysteine and tyrosine residues, the formation of di-tyrosine and the generation of both reducible and non-reducible protein cross-links. These reactions may contribute to the loss of meat palatability and tenderness in food samples, and the loss of function of myosin in certain pathologies and aging.

EXPERIMENTAL

Materials

Myoglobin from horse heart (>90%), ATP, NEM (N-ethylmaleimide), DTT (dithiothreitol), mercaptoacetic acid, methanesulfonic acid (packed under Ar), o-phthaldialdehyde (with 2-mercaptoethanol added) and MNP (2-methyl-2-nitrosopropane) dimer were obtained from Sigma. H2O2 (30%), DTNB [5,5′-dithiobis-(2-nitrobenzoic acid)] and DTNP [2,2′-dithiobis-(5-nitropyridine)] were obtained from Merck. HRP was obtained from Roche Molecular Biochemicals. TEMPO (2,2,6,6-tetramethylpiperidine-N-oxyl) was obtained from Molecular Probes. EGTA was purchased from ACROS Organics (Morris Plains, NJ, U.S.A.). Nitrilotriacetic acid and (NH4)2Ce(SO4)2 were obtained from BDH. Buffer solutions were all of analytical grade. HPLC solvents were purchased from EMD Chemicals (Merck, Kilsyth, VIC, Australia).

Myosin preparation

Myosin was prepared from porcine longissimus dorsi muscle which had been frozen immediately in liquid nitrogen after slaughter, followed by storage at −18 °C. Myosin was isolated as described previously [32] with minor modifications. Thus 50 g of muscle, trimmed of adhering fat and connective tissue, was homogenized using a Ultra-Turrax apparatus in the presence of 3 volumes (v/w) of modified Guba–Straub solution [0.3 M KCl, 0.1 M KH2PO4, 0.05 M K2HPO4 and 1 mM EGTA (pH 6.4)]. The crude myofibrillar extract was diluted with 3 volumes of water (v/v) and filtered through a 400 μm plastic mesh. Water [6.5 volumes (v/v)] was added to the filtrate and the samples were allowed to precipitate on ice for >2 h. The clear supernatant was decanted and the remaining precipitate was centrifuged at 2000 g for 45 min at 4 °C. The precipitate was subsequently dissolved in 100 ml of 0.5 M KCl, 1 mM EGTA and 10 mM Tris/HCl (pH 7.5), treated with 2 mM ATP and 5 mM MgCl2, and centrifuged at 22500 rev./min for 30 min at 4 °C (Kontron TST28.38/17-28.000 rotor; Beckman Optima TM LE-80IC ultracentrifuge) in order to remove residual actomyosin. Myosin present in the supernatant was precipitated using ammonium sulfate. The fraction which precipitated between 38–50% (w/v) saturation was collected, re-suspended in the minimum possible volume of 0.5 M KCl, 1 mM EGTA and 10 mM Tris/HCl (pH 7.5) and dialysed overnight against three changes of 5 M KCl and 10 mM Tris/HCl (pH 7.5), using 6000–8000 Da molecular mass cut-off dialysis tubing (Spectra/Por; Spectrum, Rancho Dominguez, CA, U.S.A.). Purified myosin stocks were stored at −80 °C in small aliquots (1 ml). On the day of use, the myosin stock was thawed, diluted with 2 ml of 5 mM phosphate buffer (pH 7.0, I 1.0), and centrifuged at 2100 g for 5 min at 22 °C. The resulting solution was stored at 4 °C until used. The myosin concentration was determined by measuring the absorbance (A280) and comparing with the A280 of 1 g/l myosin (0.496).

Oxidation reactions

MNP (1 M) was prepared daily in 100% acetonitrile and protected from light. The final concentration of MNP in the reaction solutions did not cause precipitation of myosin. All other solutions were prepared in 5 mM phosphate buffer (pH 7.0, I 1.0) using water passed through a four-stage Milli Q system (Millipore) equipped with a 0.2 μm pore-size filter. Stock solutions of myoglobin, HRP and H2O2 were prepared daily. Myoglobin was purified using a PD-10 column (Pharmacia) and the concentration was determined from the optical absorbance at 525 nm using ϵ525 7700 M−1·cm−1 [33]. H2O2 concentrations were determined from their absorbance (A240) using ϵ240 39.4 M−1·cm−1 [34]. All concentrations stated in the text are final concentrations after mixing.

Blocking of thiol groups on myosin

Thiol groups on myosin were blocked by incubation of 15–18 μM myosin with 5 mM NEM for 90 min at 4 °C. The extent of blocking was measured using 1.5 mM DTNB [35], and under these conditions >95% of the thiol groups were blocked.

EPR spectroscopy

Samples for EPR spectroscopy were prepared by mixing 10–13 μM myosin with 300 μM Mb and 300 μM H2O2 or 100 μM HRP and 10 mM H2O2 in this order at 22 °C. Reaction times refer to the time after H2O2 addition. Additional oxidation systems were also used, consisting of: (i) 0.1 mM FeCl3, 1 mM sodium ascorbate and 20 mM H2O2, (ii) 0.5 mM Ce(IV) solution, prepared from 0.01 M nitrilotriacetic acid and 0.05 M (NH4)2Ce(SO4)2 [8], and (iii) 10 mM peroxynitrite, prepared as described in [36]. For experiments performed at 22 °C, the incubation mixture was subsequently transferred to an EPR flat cell (Wilmad, Buena, NJ, U.S.A.). For low-temperature EPR analysis, the reaction solution was frozen in liquid nitrogen in cylindrical EPR cells 40 s after addition of H2O2. EPR spectra were recorded on a Bruker EMX X-band spectrometer equipped with 100 kHz modulation and a standard 4103TM/9702 cavity. Typical spectrometer settings for analysis at 22 °C were: gain, 1.0×105; modulation amplitude, 2 G (Gauss; where 10 G=1 mT); time constant, 163.840 ms; sweep time, 83.886 s; centre field, 3480 G; field sweep width, 100 G; microwave power, 2.5 mW; and frequency, 9.7 GHz, with four acquisitions averaged. Typical spectrometer settings for analysis at 77 K were: gain, 1.0×105; modulation amplitude, 2 G; time constant, 163.840 ms; sweep time, 83.886 s; centre field, 3360 G; field sweep width, 160 G; microwave power, 2.5 mW; and frequency, 9.4 GHz, with eight acquisitions averaged. Spectral manipulations and signal integrations were performed using the WINEPR program (Bruker BioSpin).

Determination of thiol oxidation

Thiol concentrations in samples containing 13 μM myosin with varying concentrations (0, 25, 50, 75 and 100 μM) of Mb and H2O2 (1:1 ratio) or HRP and H2O2 (1:1 and 1:100 ratios) were determined at 22 °C after incubation for 2 min, using DTNP as described in [37]. Absorbance (A386) was measured 5 min after the addition of DTNP (0.1 mM final concentration) to the sample. Blanks where individual components were omitted were measured in parallel. The thiol concentration was calculated by using ϵ386 14 mM−1·cm−1.

Quantification of amino-acid consumption

Amino acid analysis of parent and oxidized protein samples was carried out on amino acid hydrolysates generated by methanesulfonic acid by HPLC with fluorescence detection after derivatization with o-phthaldialdehyde as described previously [35]. Results are expressed in mol of amino acid residues lost per mol of alanine residues to compensate for any loss of material during processing.

Quantification of tyrosine oxidized products

Myosin (13 μM) was incubated with 100 μM Mb and 100 μM H2O2 or 100 μM HRP and 10 mM H2O2 at 22 °C, with aliquots (100 μl) withdrawn after 0 min, 0.3 min, 10 min, 60 min and 24 h, and stored at −80 °C until analysis. Hydrolysis of the protein samples to free amino acids was performed as described in [38]. Samples (40 μl) were loaded on to a Shimadzu HPLC system equipped with a Zorbax ODS column with a Pelliguard guard column and eluted using a gradient solvent system [38]. The eluate was monitored in series by UV (set at 280 nm to quantify the parent tyrosine) and fluorescence detectors (λex 280 nm, λem 320 nm for DOPA and λex 280 nm, λem 410 nm for di-tyrosine) and compared with standard curves prepared using authentic materials (chemically synthesised parent tyrosine, DOPA and di-tyrosine). Results are expressed in mmol of oxidized product per mol of parent tyrosine to compensate for any loss of material during processing.

SDS/PAGE

Samples for SDS/PAGE were prepared and incubated as described above, with 2.5 μg of protein loaded directly on to the gel at the appropriate time points. NuPAGE® Novex Tris/acetate gels (3–8% gels; Invitrogen) were used following the manufacturer's protocol. DTT (0.1 M) was added directly to the samples when reducing conditions were required. Gels were stained with colloidal Coomassie Blue.

Statistical analysis

All experiments were performed in triplicate or greater, and results are means±S.D. except where otherwise indicated. Statistical analysis was performed using a 2-way ANOVA with Dunnett's post-hoc test. Statistical significance was assumed at P<0.05.

RESULTS

Detection of radical intermediates on myosin by direct EPR

Incubation (2, 30, 60 and 120 min) of myosin with either HRP and H2O2 (Figure 1A) or Mb and H2O2 (Figure 1B) at 22 °C, pH 7.0 and examination by direct EPR spectroscopy at 22 °C resulted in the detection of signals assigned to myosin-derived radicals (Figure 1). These signals were long-lived and could be detected for up to 120 min (Figure 1). The g value of these signals was determined for both oxidation systems to be approx. 2.005 by reference to TEMPO. The species generated with Mb and H2O2 decayed less rapidly than those generated by HRP and H2O2. Control experiments with the Mb/H2O2 system in the absence of myosin gave less intense signals (Figure 1B), which have been assigned to Mb-derived tyrosyl radicals as observed previously [11,16]. These features decayed more rapidly than those detected in the presence of myosin. No signals were observed with the HRP/H2O2 system in the absence of myosin (control, Figure 1A).

EPR spectra of myosin-derived radicals detected by direct EPR

Figure 1
EPR spectra of myosin-derived radicals detected by direct EPR

EPR spectra were recorded at 22 °C from reaction mixtures containing (A) 12 μM myosin, 100 μM HRP and 10 mM H2O2 and (B) 12 μM myosin, 300 μM Mb and 300 μM H2O2. Spectra were recorded at the indicated time points (2, 30, 60 and 120 min) after the addition of H2O2. The control spectra shown are those where myosin was excluded from the reaction mixture. Spectrometer settings are described in the Experimental section.

Figure 1
EPR spectra of myosin-derived radicals detected by direct EPR

EPR spectra were recorded at 22 °C from reaction mixtures containing (A) 12 μM myosin, 100 μM HRP and 10 mM H2O2 and (B) 12 μM myosin, 300 μM Mb and 300 μM H2O2. Spectra were recorded at the indicated time points (2, 30, 60 and 120 min) after the addition of H2O2. The control spectra shown are those where myosin was excluded from the reaction mixture. Spectrometer settings are described in the Experimental section.

The EPR signal detected with the HRP/H2O2/myosin system consisted of a single broad absorption (peak-to-peak line width 8.22 G), with poorly resolved fine structure, as shown by the presence of weak inflections on the signal envelope. No significant changes in line shape were detected over time. The long-lived nature of this signal, its g value, line shape and peak-to-peak line width are consistent with an assignment to (one or more) tyrosine-derived phenoxyl radicals present on myosin [18]. The initial EPR signal detected with the Mb/H2O2/myosin system was much broader than that from the HRP/H2O2/myosin system (peak-to-peak line width 11.10 G), with this becoming considerably sharper as the signal decayed (Figure 1B). These changes are consistent with the presence of multiple species. The broader component has been assigned to Mb-derived tyrosyl radicals (cf. [39,40]) and the narrower component to myosin-derived species (cf. the absence of this component in spectra from incubations performed in the absence of myosin and the similarity of this narrower signal to that detected with the HRP/H2O2/myosin system). This second myosin-derived species becomes more distinct on decay of the Mb-derived species (i.e. at longer reaction times; Figure 1B). The different line widths of the proposed Mb- and myosin-derived tyrosyl radicals are believed to arise from different conformations of the aromatic ring of these radicals relative to the methylene hydrogens through which the ring is attached to the backbone of the protein. It is well established [39,41,42] that the hyperfine coupling constants from these hydrogens is the major determinant of the overall line width and line shape of tyrosine phenoxyl radicals, with these values varying dramatically with the angle between the methylene C–H bonds and the plane of the tyrosine ring.

Analogous oxidation experiments carried out using other oxidizing systems [Fe2+, ascorbate and H2O2, Ce(IV) and peroxynitrite; see the Experimental section] in the presence of myosin did not result in the (direct) detection of any signals that could be assigned to myosin-derived radicals.

The role of thiol groups on myosin in the generation of the observed EPR signals (cf. previous evidence for the formation of disulfide bonds on oxidized myosin [5,2830]) was probed by pre-treating myosin with NEM (5 mM) to block these sites. Subsequent oxidation with Mb and H2O2 or HRP and H2O2 for 2 min resulted in a decrease in the intensity of the EPR signals when compared with the unmodified myosin (Figure 2). For the HRP system, the signal intensity decreased to 38% of that detected with the unmodified protein 2 min after initiation of oxidation (Figure 2, spectrum 1 compared with spectrum 2), and with the Mb system, the signal was reduced to 80% of the original intensity (Figure 2, spectrum 3 compared with spectrum 4). At longer incubation times, the intensity differences in the EPR spectra between the NEM-treated myosin and the native myosin was greater for both oxidizing systems (results not shown). The smaller decrease with the Mb system at 2 min is consistent with the presence of significant concentrations of Mb-derived radicals at this time point. These results are consistent with some of the observed signals requiring the presence of thiol groups for their formation. Whereas thiyl radicals have been detected previously on myosin {e.g. using Ce(IV) and spin-trapping [8]}, the observed EPR signals are not consistent with the direct detection of these species, as thiyl radicals are typically short-lived and yield highly anisotropic EPR signals [43]. Thus the effect of NEM is interpreted in terms of a requirement for thiol groups in the generation of some of the tyrosine phenoxyl radicals detected. The incomplete inhibition observed with the use of NEM (e.g. with the HRP/H2O2 system) is consistent with the additional occurrence of thiol-independent pathways of myosin radical formation.

Effect of blocking thiol groups on myosin on the nature of the myosin-derived radicals detected by direct EPR

Figure 2
Effect of blocking thiol groups on myosin on the nature of the myosin-derived radicals detected by direct EPR

EPR spectra were recorded at 22 °C from reaction mixtures containing: (1) 12 μM unmodified myosin, 100 μM HRP, 10 mM H2O2; (2) as spectrum (1), except with 12 μM thiol-blocked myosin; (3) 12 μM unmodified myosin, 300 μM Mb and 300 μM H2O2; and (4) as spectrum (3), except with 12 μM thiol-blocked myosin. All spectra were recorded 2 min after initiation of oxidation. The thiol groups on myosin were blocked by incubation with 5 mM NEM for 90 min at 4 °C prior to oxidation. Spectrometer settings are described in the Experimental section.

Figure 2
Effect of blocking thiol groups on myosin on the nature of the myosin-derived radicals detected by direct EPR

EPR spectra were recorded at 22 °C from reaction mixtures containing: (1) 12 μM unmodified myosin, 100 μM HRP, 10 mM H2O2; (2) as spectrum (1), except with 12 μM thiol-blocked myosin; (3) 12 μM unmodified myosin, 300 μM Mb and 300 μM H2O2; and (4) as spectrum (3), except with 12 μM thiol-blocked myosin. All spectra were recorded 2 min after initiation of oxidation. The thiol groups on myosin were blocked by incubation with 5 mM NEM for 90 min at 4 °C prior to oxidation. Spectrometer settings are described in the Experimental section.

Detection of radical intermediates on myosin by EPR spin-trapping

Inclusion of the nitroso spin trap MNP (100 mM) in both the HRP/H2O2/myosin and Mb/H2O2/myosin systems resulted in the detection of spin adduct signals. With the Mb/H2O2 system, identical EPR signals were detected at 22 °C in both the presence and absence of myosin, indicating that the majority of the radicals trapped were present on Mb (results not shown). The signals detected were similar to those reported previously for this system [14,44]. Addition of MNP 20 or 60 min after the addition of H2O2 resulted in similar EPR spectra, though the spectra were of lower intensity, indicating that some Mb radicals were present at these time points (results not shown); this is consistent with previous reports of long-lived radicals on H2O2-treated Mb [44]. Blocking of the thiol groups on myosin did not have any effect on the spin adduct signals, consistent with the observed species being Mb-derived.

Inclusion of MNP in the HRP/H2O2 system in the absence of myosin did not result in the detection of any protein-derived species under the conditions used. When myosin was present, multiple signals were detected, with the ratio of these species dependent on the reaction time and whether the thiol groups on myosin had been blocked. With unmodified myosin, a highly anisotropic spectrum was detected 2 min after the initiation of oxidation (Figure 3, spectrum 1) with the distance between the outermost features of this spectrum (2A′zz) being approx. 63.1 G. On the basis of the observed anisotropy, this signal is assigned to a slowly tumbling myosin-derived radical adduct. The signal from this species decayed rapidly, indicating that this adduct is short-lived (Figure 3, spectrum 2). In both the initial and subsequent spectra (e.g. at 45–60 min; Figure 3, spectrum 3) an isotropic triplet signal with a nitrogen hyperfine coupling [a(N)] of 15.35 G was also observed, with no discernable fine structure. This signal could be observed for many hours. These results are consistent with the presence of an adduct species with no β-hydrogen couplings. This signal has been assigned to a radical species arising from the trapping of a tyrosine-derived phenoxyl radical (through the ortho position) and the subsequent rearrangement of this species to the observed tertiary radical species as stated previously [45]. Treatment of this long-lived adduct species with the proteolytic enzyme pronase (from Streptomyces griseus, 0.1 mg/ml to 10 μM myosin) resulted in an increase in signal intensity consistent with the release of highly mobile radical adducts from initial myosin adduct species of more limited mobility (results not shown). A similar isotropic triplet signal [a(N) 15.6 G] has been detected previously in a HRP/H2O2/tyrosine/MNP system, with this signal assigned to a long-lived tyrosine-derived radical [46]. The signal detected in the current study is therefore assigned to a trapped tyrosine-derived radical present on myosin, consistent with the direct EPR results.

Detection of myosin-derived radicals by EPR spin-trapping

Figure 3
Detection of myosin-derived radicals by EPR spin-trapping

EPR spectra were recorded at 22 °C from reaction mixtures containing 12 μM myosin, 100 μM HRP and 10 mM H2O2 in the presence of 100 mM MNP at the following time points after the addition of H2O2: 2 min (spectra 1 and 4), 8 min (spectra 2 and 5) and 60 min (spectra 3 and 6). Spectra 1–3 were obtained from native myosin, and spectra 4–6 from thiol-blocked myosin. The thiol groups on myosin were blocked by incubation with 5 mM NEM for 90 min at 4 °C. Spectrometer settings are described in the Experimental section. The anisotropic features (indicated by 2A′zz in spectrum 1, and which are also present in the other spectra, although at lower concentrations) are assigned to a short-lived tryptophan or thiyl radical adduct. The asterisks (*) correspond to a long-lived tyrosine-derived species.

Figure 3
Detection of myosin-derived radicals by EPR spin-trapping

EPR spectra were recorded at 22 °C from reaction mixtures containing 12 μM myosin, 100 μM HRP and 10 mM H2O2 in the presence of 100 mM MNP at the following time points after the addition of H2O2: 2 min (spectra 1 and 4), 8 min (spectra 2 and 5) and 60 min (spectra 3 and 6). Spectra 1–3 were obtained from native myosin, and spectra 4–6 from thiol-blocked myosin. The thiol groups on myosin were blocked by incubation with 5 mM NEM for 90 min at 4 °C. Spectrometer settings are described in the Experimental section. The anisotropic features (indicated by 2A′zz in spectrum 1, and which are also present in the other spectra, although at lower concentrations) are assigned to a short-lived tryptophan or thiyl radical adduct. The asterisks (*) correspond to a long-lived tyrosine-derived species.

Repetition of the above experiments with thiol-blocked myosin resulted in the immediate detection of an isotropic triplet signal (Figure 3, spectrum 4) with an identical hyperfine coupling to the species described above. This adduct decayed rapidly (Figure 3, spectrum 5). At longer time points (e.g. 60 min; Figure 3, spectrum 6) a further persistent adduct species with a triplet spectrum [a(N) 15.6 G] identical with that detected for the native protein was observed. The identical signals detected at long time points with both unmodified and modified myosin are consistent with these being assigned to tyrosine-derived radical adducts generated by reactions that are thiol-independent. In contrast, the highly anisotropic short-lived adduct detected with native myosin, but not the thiol-blocked protein, is consistent with the trapping of either a tryptophan or a thiyl radical. The second short-lived triplet signal detected with the thiol-blocked protein may be due to a different population of tyrosine-derived species, or an additional trapped tertiary carbon-centred radical.

Detection of radical intermediates by low-temperature direct EPR

Previous studies have demonstrated that tryptophan-derived radicals are readily formed on proteins, owing to their low oxidation potentials [13]. Thus Trp14 peroxyl radicals have been detected on H2O2-treated Mb by low-temperature EPR [13,16,47]. The formation of analogous species on myosin as a result of damage-transfer from H2O2-activated HRP or Mb was therefore examined. Examination of rapidly frozen (in liquid nitrogen) samples of HRP/H2O2/myosin incubations by direct EPR at 77 K resulted in the detection of a similar signal to that detected at room temperature (22 °C) (cf. Figure 1A), consistent with the presence of identical radicals under both conditions; no evidence for tryptophan-derived peroxyl radicals was obtained. As with the room temperature studies, omission of myosin resulted in the loss of these spectral features.

Examination of the Mb/H2O2 system at 77 K in the absence of myosin resulted in the detection of anisotropic EPR signals which have been assigned, as previously, to a mixture of the Trp14 peroxyl radical and (one or more) tyrosine-derived phenoxyl radicals on Mb [16] (Figure 4). The inclusion of myosin in these incubations decreased the intensity of the Mb-derived signals in a concentration-dependent manner (Figure 4); no additional myosin-derived signals were detected. Blocking the thiol groups on myosin had no effect on either the nature or the intensity of the Mb-derived Trp14 peroxyl radical signal (results not shown). These results are consistent with the reaction of the Mb-derived Trp14 peroxyl radical with myosin, with the site(s) of reaction on myosin not including the thiol residues. The incomplete loss of these observed signals does not preclude the formation of either tyrosine-phenoxyl or tryptophan-derived species on myosin as a result of these reactions (i.e. the Mb species may be being replaced by species with similar EPR features on myosin).

Detection of radicals by low-temperature EPR

Figure 4
Detection of radicals by low-temperature EPR

EPR spectra of reaction mixtures containing 300 μM Mb and 300 μM H2O2 without (control) or with myosin (5 or 13 μM) were recorded at 77 K. Samples were frozen in liquid nitrogen 40 s after initiation of oxidation and subsequently examined by EPR. Features arising from the Trp14-peroxyl radical of myoglobin: gparallel (∼2.035) and gperpendicular (∼2.006) are indicated. Spectrometer settings are described in the Experimental section. Inset results are means±S.D. (n=3) of the EPR signal area in the absence or presence of 5 or 13 μM myosin.

Figure 4
Detection of radicals by low-temperature EPR

EPR spectra of reaction mixtures containing 300 μM Mb and 300 μM H2O2 without (control) or with myosin (5 or 13 μM) were recorded at 77 K. Samples were frozen in liquid nitrogen 40 s after initiation of oxidation and subsequently examined by EPR. Features arising from the Trp14-peroxyl radical of myoglobin: gparallel (∼2.035) and gperpendicular (∼2.006) are indicated. Spectrometer settings are described in the Experimental section. Inset results are means±S.D. (n=3) of the EPR signal area in the absence or presence of 5 or 13 μM myosin.

Quantification of amino acid side-chain oxidation

Oxidation of the thiol (cysteine) residues on myosin was quantified with DTNP. Both the Mb/H2O2 and HRP/H2O2 systems gave rise to rapid thiol loss; this was quantified after 2 min of oxidation at room temperature with a range of oxidant concentrations (0, 25, 50, 75 or 100 μM, Figure 5). Increasing the concentrations of either Mb and H2O2 or HRP and H2O2 (with fixed ratios of haem protein to H2O2) added to a fixed concentration of myosin (13 μM) resulted in a significant loss of myosin thiol groups. Use of the HRP/H2O2 system with a 1:100 ratio of HRP/H2O2 resulted in particularly rapid thiol loss (Figure 5A). Reduced thiol loss was observed at a 1:1 haem:H2O2 ratio for HRP/H2O2 and Mb/H2O2. Control experiments with Mb and HRP alone gave zero free thiol levels, as expected on the basis of their amino acid composition, indicating that all of the thiol groups consumed were on the myosin. Control experiments in the absence of haem protein, but with 25 μM–10 mM H2O2, did not result in significant loss of thiol groups on the myosin (results not shown).

Consumption of thiol and tyrosine residues on oxidation of myosin by activated haem proteins

Figure 5
Consumption of thiol and tyrosine residues on oxidation of myosin by activated haem proteins

(A) Myosin (13 μM) was incubated with the indicated concentrations of Mb and H2O2 (1:1 ratio; black bars), HRP and H2O2 (1:1 ratio; grey bars), or HRP and H2O2 (1:100 ratio; white bars) for 2 min at 22 °C before assessment of residual thiol groups on myosin using DTNP as described in the Experimental section. Results are means±S.D. (n=3) after incubation with the stated concentration of oxidant system. (B) Myosin (13 μM) was incubated with 100 μM HRP and 10 mM H2O2 for the indicated times before removal of 100 μl aliquots for quantification of the remaining amino acids after acid hydrolysis of the protein. Grey bars are myosin samples incubated with the oxidant system, white bars correspond to incubation controls of myosin in the absence of oxidant. Results for HRP and H2O2 alone have been subtracted from the reported values. Tyrosine levels are expressed per mol of alanine residues present in the protein (mol Tyr/mol Alanine) in order to compensate for any loss during processing. For further details see the Experimental section. Results are means±S.E.M. (n=3–6). No significant change in concentration was detected for any of the other amino acids examined (see text for details).

Figure 5
Consumption of thiol and tyrosine residues on oxidation of myosin by activated haem proteins

(A) Myosin (13 μM) was incubated with the indicated concentrations of Mb and H2O2 (1:1 ratio; black bars), HRP and H2O2 (1:1 ratio; grey bars), or HRP and H2O2 (1:100 ratio; white bars) for 2 min at 22 °C before assessment of residual thiol groups on myosin using DTNP as described in the Experimental section. Results are means±S.D. (n=3) after incubation with the stated concentration of oxidant system. (B) Myosin (13 μM) was incubated with 100 μM HRP and 10 mM H2O2 for the indicated times before removal of 100 μl aliquots for quantification of the remaining amino acids after acid hydrolysis of the protein. Grey bars are myosin samples incubated with the oxidant system, white bars correspond to incubation controls of myosin in the absence of oxidant. Results for HRP and H2O2 alone have been subtracted from the reported values. Tyrosine levels are expressed per mol of alanine residues present in the protein (mol Tyr/mol Alanine) in order to compensate for any loss during processing. For further details see the Experimental section. Results are means±S.E.M. (n=3–6). No significant change in concentration was detected for any of the other amino acids examined (see text for details).

Quantification of the possible loss of other amino acids on myosin was assessed by HPLC after hydrolysis to free amino acids. Only results for the HRP/H2O2/myosin system are considered, as the lower concentrations of HRP protein relative to myosin employed in this system allowed changes on the myosin to be discerned after subtraction of the levels seen with HRP/H2O2 alone. The higher concentration of Mb used in the Mb/H2O2/myosin system prevented the attribution of any loss to a particular protein. Of all the amino acids analysed (arginine, aspartic acid/asparagine, glutamic acid/glutamine, glycine, histidine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine residues), significant changes were only detected for tyrosine residues (Figure 5B), though there was a (non-significant) trend for loss of tryptophan residues. These results are standardized to the level of alanine residues present in the samples to compensate for any losses during processing. Alanine was chosen as this amino acid would not be expected to be oxidized to a significant extent [13].

Quantification of oxidized amino acid side-chains on proteins

The formation of the tyrosine oxidation products di-tyrosine and DOPA was quantified by HPLC after acid hydrolysis of the reaction mixtures. With both the Mb/H2O2/myosin and HRP/H2O2/myosin systems significant levels of DOPA were detected relative to reaction mixtures in the absence of H2O2 (results not shown). However, omission of myosin from the complete reaction systems resulted in similar levels of DOPA, consistent with DOPA formation occurring predominantly, or completely, on the haem protein. In contrast with di-tyrosine, significantly greater levels of this product were detected with both complete reaction systems than in the absence of either H2O2 or myosin, consistent with significant di-tyrosine formation on myosin (Figure 6). These increases were apparent when the data were expressed both as absolute values (for the myosin/HRP/H2O2 system) and when expressed relative to the parent tyrosine. Nonoxidized myosin contained insignificant levels of di-tyrosine (<0.014±0.009 mmol of di-tyrosine/mol of tyrosine, 0.122±0.009 pmol absolute concentration; n=15). The formation of di-tyrosine was rapid, with the maximum yield of these products observed at the first time point examined (30 s) after addition of H2O2. At longer time points, a small decrease in di-tyrosine was detected (Figure 6); this may be due to further oxidation of the di-tyrosine to higher aggregates (e.g. tri-tyrosine). The yield of di-tyrosine detected on myosin induced by the Mb/H2O2 system was not significantly affected by the use of thiol-blocked myosin, whereas that induced by the HRP/H2O2 system was, with significantly higher levels of di-tyrosine detected on the thiol-blocked protein compared with native myosin (Figure 6).

Formation of di-tyrosine on oxidized myosin

Figure 6
Formation of di-tyrosine on oxidized myosin

Di-tyrosine formation on myosin was quantified by HPLC after oxidation for the indicated time periods (30 s, 2 min, 5 min and 10 min) with 300 μM Mb and 300 μM H2O2 (grey bars) or 100 μM HRP and 10 mM H2O2 (white bars) with either native (non-hatched) or thiol-blocked myosin (hatched bars). Control samples of myosin yielded insignificant levels of di-tyrosine. The contributions of Mb and H2O2 alone, and HRP and H2O2 alone, to di-tyrosine formation were determined in parallel, and subtracted from the values detected in the presence of myosin. Levels of di-tyrosine are given as mmol of di-tyrosine per mol of parent tyrosine [Dityrosine formation (mmol/mmol p-Tyr)] to compensate for any loss during processing (see the text and Experimental section for details). Results are means±S.D. (n=4–6).

Figure 6
Formation of di-tyrosine on oxidized myosin

Di-tyrosine formation on myosin was quantified by HPLC after oxidation for the indicated time periods (30 s, 2 min, 5 min and 10 min) with 300 μM Mb and 300 μM H2O2 (grey bars) or 100 μM HRP and 10 mM H2O2 (white bars) with either native (non-hatched) or thiol-blocked myosin (hatched bars). Control samples of myosin yielded insignificant levels of di-tyrosine. The contributions of Mb and H2O2 alone, and HRP and H2O2 alone, to di-tyrosine formation were determined in parallel, and subtracted from the values detected in the presence of myosin. Levels of di-tyrosine are given as mmol of di-tyrosine per mol of parent tyrosine [Dityrosine formation (mmol/mmol p-Tyr)] to compensate for any loss during processing (see the text and Experimental section for details). Results are means±S.D. (n=4–6).

The formation of the oxidation product methionine sulfoxide from methionine was also assessed by HPLC after acid hydrolysis. As expected from the insignificant decrease in the level of the parent amino acid stated above, no significant increase in the level of this species was detected in the systems under study (results not shown).

Gross structural changes induced by oxidation of myosin

SDS/PAGE analysis was employed to investigate the potential formation of both fragmented and cross-linked products. Oxidation of myosin with 100 μM Mb and 100 μM H2O2 resulted in a reaction-time-dependent decrease in the band intensity of the myosin heavy chain on both non-reducing and reducing gels (Figure 7), and the formation of higher-molecular-mass material, but not lower-molecular-mass material. These observations are consistent with the formation of intermolecular cross-links, but not extensive protein fragmentation. The mass of these higher-molecular-mass bands, as judged from the molecular-mass markers, indicates that these cross-linked species are predominantly myosin–myosin species and not myosin–Mb dimers. The extent of loss of the parent myosin heavy chain was greater in the non-reducing gels than in the reducing gels, though both were observed to decrease, consistent with the formation of both reducible and non-reducible cross-links (Figure 7A). A similar pattern was observed on oxidation of myosin by HRP and H2O2 (result not shown), though the extent of modification was less extensive, as judged by densitometric analysis of the parent myosin heavy-chain band (Figure 7B).

Effect of oxidation on the structural integrity of myosin exposed to activated haem proteins as assessed by SDS/PAGE

Figure 7
Effect of oxidation on the structural integrity of myosin exposed to activated haem proteins as assessed by SDS/PAGE

(A) Myosin (13 μM) was incubated at 22 °C with 100 μM Mb and 100 μM H2O2 for 0 min (control without Mb and H2O2), 2 min, 10 min, 30 min, 60 min and 24 h before analysis by SDS/PAGE (3–8% gels) as described in the Experimental section. DTT (0.1 M) was added directly to samples when reduced conditions were required. Myosin heavy-chain (MHC) bands and cross-linked MHC (CL MHC) are indicated by arrows. (B) Quantification of the percentage of myosin heavy chains as determined by densitometric analysis after SDS/PAGE (3–8% gels) of native and oxidized myosin. Samples were incubated under identical conditions as described in (A) with either the Mb/H2O2 system (■, □), or with 100 μM HRP and 10 mM H2O2 (●, ○), with the samples resolved by SDS/PAGE in the absence of reduction (□, ○) or after treatment with DTT (■, ●). Representative results from multiple experiments with similar trends are shown.

Figure 7
Effect of oxidation on the structural integrity of myosin exposed to activated haem proteins as assessed by SDS/PAGE

(A) Myosin (13 μM) was incubated at 22 °C with 100 μM Mb and 100 μM H2O2 for 0 min (control without Mb and H2O2), 2 min, 10 min, 30 min, 60 min and 24 h before analysis by SDS/PAGE (3–8% gels) as described in the Experimental section. DTT (0.1 M) was added directly to samples when reduced conditions were required. Myosin heavy-chain (MHC) bands and cross-linked MHC (CL MHC) are indicated by arrows. (B) Quantification of the percentage of myosin heavy chains as determined by densitometric analysis after SDS/PAGE (3–8% gels) of native and oxidized myosin. Samples were incubated under identical conditions as described in (A) with either the Mb/H2O2 system (■, □), or with 100 μM HRP and 10 mM H2O2 (●, ○), with the samples resolved by SDS/PAGE in the absence of reduction (□, ○) or after treatment with DTT (■, ●). Representative results from multiple experiments with similar trends are shown.

Blocking of the thiol groups on myosin prior to oxidation caused a greater reduction in the intensity of the myosin heavy-chain band as assessed by densitometric analysis, compared with native myosin in both the Mb/H2O2 and HRP/H2O2 systems (Figure 8). These results are consistent with an increased formation of non-reducible cross-linkings in myosin with both oxidizing systems.

Effect of thiol blocking on the structural integrity of myosin exposed to activated haem proteins as assessed by SDS/PAGE

Figure 8
Effect of thiol blocking on the structural integrity of myosin exposed to activated haem proteins as assessed by SDS/PAGE

Quantification of the percentage of myosin heavy-chain bands determined by densitometric analysis after SDS/PAGE (3–8% gels) of oxidized myosin. Native (■, ●) or thiol-blocked myosin (□, ○) was oxidized with 100 μM Mb and 100 μM H2O2 (■, □), or 100 μM HRP and 10 mM H2O2 (●, ○) for 0 min (control without Mb and H2O2 or HRP and H2O2), 2 min, 10 min, 30 min, 60 min and 24 h at 22 °C. Samples were subsequently analysed by SDS/PAGE (3–8% gels) as described in the Experimental section after reduction with 0.1 M DTT.

Figure 8
Effect of thiol blocking on the structural integrity of myosin exposed to activated haem proteins as assessed by SDS/PAGE

Quantification of the percentage of myosin heavy-chain bands determined by densitometric analysis after SDS/PAGE (3–8% gels) of oxidized myosin. Native (■, ●) or thiol-blocked myosin (□, ○) was oxidized with 100 μM Mb and 100 μM H2O2 (■, □), or 100 μM HRP and 10 mM H2O2 (●, ○) for 0 min (control without Mb and H2O2 or HRP and H2O2), 2 min, 10 min, 30 min, 60 min and 24 h at 22 °C. Samples were subsequently analysed by SDS/PAGE (3–8% gels) as described in the Experimental section after reduction with 0.1 M DTT.

DISCUSSION

Previous studies have provided evidence for the modification of the heavy and light chains of myosin in both human and animal muscle and heart tissue. These alterations to protein structure have been associated with both human and animal pathologies and the loss of positive characteristics in food samples (e.g. tenderness and palatability). It has been demonstrated that myosin undergoes aggregation and, in some cases, loss of ATPase activity on exposure to a number of oxidizing (activated haem proteins, metal-ion and ascorbate systems, high-valence metal ions and peroxynitrite) or modifying agents (reactive aldehydes) (reviewed in Table 1). The mechanisms by which these changes arise, and the nature of the reactive intermediates involved, have not been examined in detail previously.

Table 1
Intermolecular cross-links formed from myosin on treatment with various oxidizing systems
Oxidizing system Type of cross-link Reference 
Fe3+/ascorbate/H2O2 Disulfide* [7
Fe3+/ascorbate Disulfide* [5
Cu2+/ascorbate Disulfide* [5
Mb/H2O2 Non-disulfide* [4
Mb/H2O2 Disulfide* [4
HRP/H2O2 Di-tyrosine*† [6]‡ 
Mb/H2O2 Di-tyrosine*† [6]‡ 
MbO2/H2O2 Di-tyrosine*† [26]‡ 
Mb/H2O2 Di-tyrosine*† [26]‡ 
Oxidizing system Type of cross-link Reference 
Fe3+/ascorbate/H2O2 Disulfide* [7
Fe3+/ascorbate Disulfide* [5
Cu2+/ascorbate Disulfide* [5
Mb/H2O2 Non-disulfide* [4
Mb/H2O2 Disulfide* [4
HRP/H2O2 Di-tyrosine*† [6]‡ 
Mb/H2O2 Di-tyrosine*† [6]‡ 
MbO2/H2O2 Di-tyrosine*† [26]‡ 
Mb/H2O2 Di-tyrosine*† [26]‡ 
*

As determined by SDS/PAGE

As determined by fluorescence spectroscopy.

Disulfide formation not investigated.

In the present study, various forms of EPR spectroscopy (direct studies at both ambient and low temperature, and spin-trapping) have been used to examine radical formation on myosin on exposure to two systems of physiological relevance: myoglobin and H2O2, and HRP and H2O2. The former system is likely to be a major source of oxidant stress in muscle tissue, owing to the high concentrations of Mb present in muscle cells; and peroxidase/H2O2 systems (for which HRP/H2O2 is a commonly used surrogate) are believed to be of importance at sites of inflammation, due to the release of myeloperoxidase and eosinophil peroxidase from activated neutrophils/monocytes/macrophages and eosinophils respectively. It is shown that both these systems give rise to multiple radicals on myosin as a result of damage-transfer from the H2O2-activated Mb or HRP. In the case of activated Mb, evidence has been provided for myosin oxidation by both tyrosine-phenoxyl and tryptophan-peroxyl radicals. With activated HRP, the nature of the species that damage myosin is less clear; this may involve reactions of the haem edge, or via undetected protein-derived radicals.

These reactions generate multiple potential radicals on myosin, with evidence obtained for tyrosine-phenoxyl and thiyl radicals; results supporting the formation of additional unidentified species have also been obtained from spin-trapping studies. These additional species are possibly alternative tyrosine- or tryptophan-derived radicals, as both tyrosine and tryptophan residues are known to be readily oxidized. No evidence has been obtained for the oxidation of methionine residues, as demonstrated either by loss of the parent amino acid, or by formation of methionine sulfoxide. Previous studies have reported the formation of long-lived radicals, probably tyrosine-phenoxyl radicals on BSA, β-lactoglobulin and casein [17,19], but no previous studies have been reported for myosin.

Previous studies on the oxidation of myosin using SDS/PAGE have indicated that the type of cross-link formed is dependent on the nature of the oxidising agent. Thus earlier studies have proposed that oxidation of myosin by hydroxyl-radical-generating systems (i.e. metal ion and ascorbate systems) results primarily in disulfide bonds, whereas haem proteins cause the formation of di-tyrosine or other non-reducible cross-links. This is surprising in the light of indiscriminate reactivity of hydroxyl radicals with protein side-chains and the considerable evidence for the formation of di-tyrosine and DOPA from tyrosine residues by this species (reviewed in [13]), and the known reactivity of activated haem proteins with thiol residues to generate thiyl radicals (e.g. [4851]).

In the present study, oxidation of myosin by both the Mb/H2O2 and HRP/H2O2 systems has been shown to result in di-tyrosine formation, oxidation of tyrosine and cysteine residues and the formation of both reducible (believed to be disulfide) and non-reducible (possibly di-tyrosine) cross-links. The occurrence of these processes indicates that the formation of thiyl and tyrosine-phenoxyl radicals on myosin are alternative competing processes, with the yield of each intermediate species dependent on the system used. Furthermore, modulation of the flux through one route appears to increase the flux through the alternative pathway. Thus the experiments with thiol-blocked myosin have provided evidence for increased formation of non-reducible cross-links and di-tyrosine with both oxidizing systems. Increased formation of non-reducible cross-linkings in myosin when thiol groups have been blocked by NEM has been observed previously [7].

It is possible that tyrosine residue oxidation proceeds via cysteine residue oxidation, based on the studies using MNP, where signals assigned to tyrosine-derived radicals were only detected at time periods after the formation of thiyl radicals. However, this may be an artifact of the time required to examine the reaction samples by direct EPR (typically >1 min as a result of the time required to tune the spectrometer) compared with that by spin-trapping, where trapping of radicals should occur immediately as a result of the presence of the spin trap in the reaction system. The reaction of tyrosine-phenoxyl radicals with cysteine residues to give thiyl radicals has been shown previously to be a reversible (equilibrium) reaction, and conclusive evidence has also been obtained for the interaction of tyrosine and tryptophan species (e.g. [52,53]). Thus tryptophan-derived radicals may also be formed on myosin. This theory is supported by the trend towards a loss of tryptophan residues in the amino acid analysis experiments and the detection of additional short-lived adduct species with MNP on myosin, which may be trapped tryptophan-derived radicals.

A proposed reaction mechanism for the oxidation of myosin by activated haem proteins is suggested in Scheme 1. This reaction mechanism includes two reaction pathways for the formation of myosin radicals: one involving direct oxidation of thiol groups to give thiyl radicals (1), a process that appears to be favoured under the reaction conditions employed in the present study, and a second that generates the (long-lived) tyrosine-derived radicals detected by direct EPR (2). This second pathway may involve the mediation of other short-lived radicals (X) that may be another population of (transient) tyrosine-derived phenoxyl radicals or possibly tryptophan-derived species. The identification of these short-lived radicals requires further study, although the hyperfine splitting constant of 15.35 G obtained for the MNP adduct would be consistent with a tyrosine-derived radical located on the surface of myosin. The formation of a population of reactive tyrosine-derived phenoxyl radicals would also be consistent with the rapid formation of di-tyrosine observed in the product studies.

Proposed damage-transfer reactions from H2O2-activated haem proteins to myosin that result in radical formation on myosin, and the consequences of these processes

Scheme 1
Proposed damage-transfer reactions from H2O2-activated haem proteins to myosin that result in radical formation on myosin, and the consequences of these processes

The myosin-X species indicated may be an alternative population of myosin tyrosine-phenoxyl radicals (myosin-TyrO) or tryptophan-derived radicals.

Scheme 1
Proposed damage-transfer reactions from H2O2-activated haem proteins to myosin that result in radical formation on myosin, and the consequences of these processes

The myosin-X species indicated may be an alternative population of myosin tyrosine-phenoxyl radicals (myosin-TyrO) or tryptophan-derived radicals.

Overall, these studies indicate that multiple reaction pathways can result in radical transfer from an activated haem protein to myosin, and that these reactions result in the generation of both thiyl and tyrosine-phenoxyl radicals on the target protein. Other radicals may also be formed (e.g. on tryptophan residues). The formation of these species results in the formation of both reducible (believed to be disulfide) and non-reducible (di-tyrosine) cross-links between myosin molecules. The significance of the reactions examined in the present study for more complex systems remains to be fully established. In particular, it might be expected that alternative reactions of the initial Mb- and HRP-derived species, particularly those involving low-molecular-mass thiols (e.g. GSH) and ascorbate, may diminish radical transfer to myosin. Thus it has been shown that both of these reductants can react with Mb-derived species, with corresponding oxidation of GSH and ascorbate [16,39,50,51]. However, there is also extensive evidence for the modification of myosin in intact tissues and myofibrils [20,2225,30], suggesting that such antioxidant systems may be inefficient or overwhelmed under certain circumstances. Thus we believe that the generation of these radical intermediates and intermolecular cross-links may account for the altered and dysfunctional myosin observed in human pathologies and degraded meat samples.

We thank Anne Mette Frederiksen (Department of Food Science, University of Copenhagen, Denmark) for establishing the procedure for myosin purification. Financial support for this work was provided by the Danish Meat Research Institute, the LMC (Levnedsmiddelcentret, Centre for Advanced Food Studies) graduate school FOOD, and the Australian Research Council (ARC) through the ARC Centres of Excellence Scheme.

Abbreviations

     
  • DTNB

    5,5′-dithiobis-(2-nitrobenzoic acid)

  •  
  • DTNP

    2,2′-dithiobis-(5-nitropyridine)

  •  
  • DTT

    dithiothreitol

  •  
  • G

    Gauss

  •  
  • HRP

    horseradish peroxidase

  •  
  • Mb

    myoglobin in the +3 (met) oxidation state

  •  
  • MNP

    2-methyl-2-nitrosopropane

  •  
  • NEM

    N-ethylmaleimide

  •  
  • TEMPO

    2,2,6,6-tetramethylpiperidine-N-oxyl

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