Monitoring Myosin Degradation During Conditioning in Chicken Meat Using an Immunological Method

ABSTRACT: Changes of chicken breast myosin during storage at 2°C and 37°C were monitored immunochemically. Anti-myosin subfragment-1 (S-1) monoclonal antibody, which recognized epitopes within the 27 kDa fragment of S-1, and the anti-myosin rod polyclonal antiserum, were prepared. Myosin degradation products were not detected in muscle extracts stored for 3 weeks at 2°C. In contrast, storage at 37°C brought about the degradation of myosin heavy chain to immunologically detectable small fragments. While, myosin rod produced during the conditioning period was not decomposed into any small filaments. Namely, storage of muscle at 37°C resulted in minor amounts of myosin heavy chain degradation, with initial conversion to rod and S-1 fragments, and subsequent breakdown occurred in the S-1 region only. Immunoblot assay also suggested that the pattern of changes in myosin heavy chain in muscle incubated at 37°C was similar to that produced by in vitro digestion with cathepsin D.     

Thermal Transitions of Myosins/Subfragments from Black Marlin (Makaira mazara) Ordinary and Dark Muscles

Thermal transitions were studied by means of differential scanning calorimetry (DSC) and a spectrophotometric method. Three endothermic peaks (40, 43, 50°C: ordinary muscle; 46, 54, 62°C: dark muscle) were observed in DSC thermograms of both myosins. Thermograms of S-l fragments showed one peak (41°C: ordinary muscle, 43°C: dark muscle). But ordinary and dark muscle rod fragments gave two peaks (41, 62°C) and one peak (58°C), respectively. The spectrophotometric results also showed two thermal transitions for both myosins and one transition for their S-1 fragments. However, the rod from ordinary muscle myosin had two transitions, whereas that from dark muscle myosin had one transition.     

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.     

ROLE OF MUSCLE PROTEINASES IN MAINTENANCE OF MUSCLE INTEGRITY AND MASS

Current evidence indicates that, of the thirteen known lysosomal peptide hydrolases, only seven, cathepsins A, B, C, D, H, L, and lysosomal carboxypeptidase B are located inside skeletal muscle cells. Only one of the reported neutral and alkaline proteases is located inside skeletal muscle cells', this neutral protease is the Ca²⁺-dependent proteinase, CAF. With the possible exception of cathepsin N, which can degrade collagen, it seems probable that any protease that contributes to postmortem tenderization needs to be located inside muscle cells. Because very little degradation of myosin or actin occurs in postmortem muscle, most of the small amount of proteolytic degradation of the myofibrillar proteins that occurs during postmortem storage must be due to CAF, which is unique in being unable to degrade myosin and actin. It is not certain that postmortem proteolysis by CAF causes increased tenderness; some recently discovered actin-fragmenting proteins could be involved.     

HEAT-INDUCED GELATION OF MYOSIN IN THE PRESENCE OF ACTIN

ABSTRACT The rabbit muscle contractile proteins, myosin, actin and reconstituted actomyosin were mixed in 0.1–1.0 M KCl, 20 mM buffers, pH 5.0–8.0, and were tested quantitatively for thermally induced gelation properties by measuring the rigidity (shear modulus) of the system at 20–70°. Scanning electronmicroscopy (SEM) was also used to study the structure of the gels formed by gelation of myosin in the presence of F-actin. Under the standard condition, i.e. at 0.6 M KCl, pH 6.0 and 65°, decrease of the myosin/actin mole ratio to about 1.5–2.0 in the reconstituted acto-myosin system resulted in substantial augmentation of the rigidity of the gel formed. Further decreases in the myosin ratio relative to F-actin reduced the rigidity value of the gel to close to the level of myosin alone. Gel-formability of the reconstituted actomyosin was maximal at pH 5.5–6.0 and between 0.5 and 0.8 M KCl and decreased considerably at other pH values and KCl concentrations. The SEM studies revealed progressive changes in three dimensional ordering as actin concentration in the actomyosin varied. These were in concordance with the results of gel strength.     

RELATIONSHIP OF pH AND TEMPERATURE TO DISRUPTION OF SPECIFIC MUSCLE PROTEINS AND ACTIVITY OF LYSOSOMAL PROTEASES

From a review of the literature, and from specific data presented in this paper, it was concluded that both postmortem temperature and pH have effects on meat tenderness and on disruption of specific myofibrillar proteins. Increased postmortem temperature porduces more tender muscles and increases the disruption of troponin-T, myosin, Z-lines, connectin and gap filaments. Elevated postmortem temperature also increases the activity of enzymes which cause the disruption of myofibrillar proteins. Higher ultimate postmortem pH (above 6.0) produces more tender muscle, but also produces dark-cutting meat Except for one experiment, lower pH in the first few hours postmortem (in muscle with normal ultimate pH; i.e., 5.8 or below) improves meat tenderness. High pH increases the activity of CAF and low pH increases the activity of lsosomal cathepsins. Both high and low pH increase the degradation of troponin-T, Z-lines, gap filaments and connectin, but the degradation of these proteins (except for Z-lines) is greater at a low pH. Low pH increases the degradation of myosin; conversely, high pH retards it degradation.     

Structural studies of rigor bovine myofibrils using fluorescence microscopy. I. Procedures for purification and modification of bovine muscle proteins for use in fluorescence microscopy

The myofibril is a fully competent fragment of the contractile apparatus of muscle, and its structure can be investigated with fluorescent-protein probes. Several purification and chemical modification techniques were developed for the synthesis of fluorescent myosin subfragment-1 (S1), actin and alphaactinin to be used as fluorescent probes of myofibril structure. Myosin was isolated from both rectus abdominis (RA) and cutaneus trunci (CT) and subjected to chymotryptic digestion to give S1 and myosin rod. Analysis of the time course of digestion by SDS-PAGE showed that S1 from RA myosin contained at least three heavy chain fragments while myosin from CT had essentially one. The S1 obtained from CT myosin was enzymatically characterized and used for fluorescent conjugation. Methods were adapted to conjugate thiol-specific fluorophores to actin, alpha-actinin and S1. These fluorescent probes may be useful for structural studies on the isolated myofibril and on muscle in relation to its properties as a food.     

MOLECULAR PROPERTIES OF POST-MORTEM MUSCLE: EFFECT OF SULFHYDRYL REAGENTS AND POST-MORTEM STORAGE ON CHANGES IN MYOSIN B

Studies to determine the relationship of SH groups to certain changes of the myofibrillar proteins of post-mortem muscle were carried out with myosin B from at-death and post-mortem stored rabbit skeletal muscle (2° C and 25° C for 3 days) and with SH reagent modified myosin B from at-death and post-mortem stored muscle. Quantitative SH analysis, ATPase activity, turbidity rate and analytical ultracentrifugation were employed to determine the changes in myosin B associated with changes in SH groups. Post-mortem storage of muscle at 2°C for 3 days had no effect on SH content of myosin B; a decrease in SH groups, however, was observed in myosin B from muscle stored at 25°C for 3 days and for iodoacetamide (IAA) and N-ethylmaleimide (NEM) modified myosin B. ATPase activity was inhibited by reacting myosin B with enough NEM or IAA to block all SH groups. Dialysis of myosin B from at-death and post-mortem muscle against MCE to restore SH groups resulted in partial reversal of Mg++ and EGTA-activated ATPase of myosin B from post-mortem muscle and a less rapid rate of turbidity development. These results suggest that the state and nature of SH groups are partly involved in the actin-myosin interaction of post-mortem muscle; other constituents, however, in addition to SH groups are evidently modified and, in some instances, irreversibly modified, under certain post-mortem storage conditions.     

The surface active properties of myosin and its proteolytic fragments

The surface active properties of myosin and its proteolytic fragments, light meromyosin (LMM), heavy meromyosin (HMM), subfragment-1 (S-1) and myosin rod, at initial bulk phase concentrations in the range of 10(-4)% to 10(-2)% w/v were determined by the drop volume method. Overall, S-1 was the most effective surface tension depressor, whereas the tail portions of myosin, i.e. LMM and myosin rod were less surface active than the parent myosin molecule. The surface pressures attained after 40 min, at an initial bulk phase concentration of 10(-2)% (w/v), were 22·00, 21·77, 21·02, 16·77 and 16·77 mNm(-1) for S-1, HMM, myosin, LMM and myosin rod, respectively. Furthermore, S-1 effected the most rapid change in surface pressure during the initial 5 min period.     

Changes in firmness and thermal behavior of ice-stored muscle of jumbo squid (<Emphasis Type="Italic">Dosidicus gigas</Emphasis>)

Changes in firmness, muscle total protease activity, and the thermal behavior of jumbo squid (Dosidicus gigas) were measured throughout 15 days of ice-storage. A significant decrease (p<0.05) in the shear force of raw mantle muscle was observed after 7-days ice-storage. The highest total protease activity detected was 1.24 U/g mantle. The thermograms obtained at day zero showed four transition states. The first three transition states were endothermic and correspond to myosin (50 °C), sarcoplasmic proteins (69 °C), and actin (79 °C). The fourth transition state was exothermic at 107 °C, and was probably associated with protein aggregation. The thermal behavior of the muscle showed a decreasing trend in temperature and enthalpy of transition for myosin, sarcoplasmic proteins, and actin with storage time. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis analysis showed a change in the myofibrillar protein pattern, which with the shear force, and differential scanning calorimetry data, suggests a partial denaturation of that protein fraction during ice-storage.     

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.     

SOME PROPERTIES OF MYOFIBRILLAR PROTEINS OBTAINED FROM LOW IONIC STRENGTH EXTRACTS OF WASHED MYOFIBRILS FROM PRE- AND POST-RIGOR CHICKEN PECTORAL MUSCLE

SUMMARY— Changes in extractability of the proteins associated with the fragmentation phenomenon of myofibrils in chicken pectoral muscle were studied. The results indicate that the protein fractions extracted by neutralized water from muscle residue. from which water-soluble proteins have been washed out, increase in post-rigor muscle. The extracts from pre- and post-rigor muscle were fractionated with ammonium sulfate into two fractions: the fraction precipitated by 1.7 M ammonium sulfate (Fr.1) and the supernatant (Fr. 2). Depressing effect on the onset of ATP-induced superprecipitation of trypsin-treated myosin 6 which was initially present in Fr. 2 from pre-rigor muscle decreased to a great extent in that from post-rigor muscle, whereas promotive effect on gelation of F-actin and superprecipitation of the myosin 6 which was little in Fr. 1 from pre-rigor muscle appeared in that from post-rigor muscle. It is proposed that an increasing amount of protein which indicates α-actinin activity is released along with the destruction and final dissolution of the Z-line structure during postmortem storage of chicken pectoral muscle.     

The change of thermal gelation properties of horse mackerel mince led by protein denaturation occurring in frozen storage and consequential air floatation wash

This study investigated the protein deformation of horse mackerel muscle after frozen storage and consequential air-floatation wash (AFW) and its effect on thermal gelation property change. As frozen storage period of whole fish increased, the rigidity and gel strength of surimi products decrease, and longer washing time was needed for surimi products to gel. The reactive sulfhydryl group decreased with frozen storage time but increased with AFW time. Moreover, the addition of reducing agent (NaHSO₃), which was able to reduce disulfide bond, could effectively improve the gelling ability of surimi made of the muscle with long term frozen storage but not significantly of the one made of unfrozen AFW treated mince. The chemical bonding behavior and the thermal analysis results indicated that the myosin rod was unfolded after AFW to interact with other protein, unlike the effect of frozen storage which resulted in denaturation and oxidative disulfide bond formation.     

Effect of ageing on the properties of myofibrils of rabbit muscle

The effect of ageing of rabbit muscle at 4° and 15–18° on the extractability and adenosine triphosphatase activity of the myofibrils has been examined. The amount of protein extracted by M-KCL-4 mM sodium glycerophosphate (pH 6.2) and by 0.1 M sodium tetrapyrophosphate-4 mM MgCl₂-10 mM-KH₂PO₄ (pH 7.2) increased as the muscle aged. By using the amount of Ca²⁺ adenosine triphosphatase extracted, i.e. the enzyme associated with myosin, as a measure of the amount of myosin in the actomyosin extracted, it was possible to show that the buffered potassium chloride did not extract all the actomyosin from the myofibrils of pre-rigor muscle. As the muscle aged, more actomyosin was extracted, together with some tropomyosin. Pyrophosphate, however, extracted all the myosin from the pre-rigor muscle, and the increase in the protein extracted from aged muscle was due to actin and tropomyosin in addition to myosin. It is suggested that these changes are caused by a disruption of the Z-band structure during ageing, perhaps due to the hydrolysis of tropomyosin by proteolytic enzymes. The specific Ca²⁺ adenosine triphosphatase activity of the myofibrils was unaltered by ageing but the specific Mg²⁺-activated adenosine triphosphatase, i.e. the enzyme associated with actomyosin, was reduced by about one-third. This latter result may be caused by a change in the mode of linkage of actin to myosin.     

Myosins from red and white bovine muscles: Differences measured by turbidimetric,calorimetric and rheological techniques

The aim of this study was to elucidate the functional performance of the most abundant protein component in meat, ie myosin, which is recognised as important for binding in meat products. As several genetic variants of skeletal myosin exist, myosins from two bovine muscles of different fibre type composition, M masseter and M cutaneus trunci were compared with respect to filament forming properties and denaturation characteristics. The principal methods used were turbidimetric measurements, which were used to monitor filament formation, calorimetry and rheology. The myosin systems were examined at two different salt levels (0.2 and 0.6 M NaCl) and at pH 5.5–7.0. The method of preparing myosin suspensions/solutions was also examined. Differences in the filament-forming process for the two myosins were detected. Measurements of turbidity revealed that at conditions of low pH and low ionic strength white myosin had a higher ultimate turbidity compared with red myosin. Early in the transition from low to high turbidity, red myosin had a higher turbidity than white myosin corresponding to reduced solubility. The turbidity increased with time of storing the myosin suspensions/solutions. This change was attributed to formation of filaments and further association of filaments. White myosin had a smaller apparent enthalpy of denaturation than red myosin. The calorimetric measurements recorded in 0.2 M NaCl suggested that the head and the rod of white myosin were less stable than the corresponding parts of red myosin. However, exceptions to this rule were found at pH 6.0. In 0.6 M NaCl the identification of the transitions for red myosin was more difficult. The method of preparing myosin suspensions affected calorimetric and rheological measurements. In 0.6 M NaCl and pH 6.0 calorimetric thermograms of both myosins were affected by the preparation method. At pH 5.5 this change was interpreted as caused by denaturation promoted by the dilution/rapid titration technique compared with dialysing the systems to pH 5.5 from pH 7. Differences in the filaments formed might, however, also contribute to the variations seen in the calorimetric ther-mograms. The gelling properties of white myosin were most sensitive to the preparation method used. Systems prepared by dialysis gave stronger heat-induced gels than those prepared by ‘dilution’. White myosin always produced stronger gels than red myosin independent of the preparation technique. The rheological properties (at 80°C) of red myosin were less affected by the preparation method than were those of white myosin. At lower temperatures, however, there was more variation in the shapes of the rheological thermograms (? versus temperature) for red myosin than in the corresponding thermograms of white myosin.     

Characterization of Myosin Subfragment-1 of Summer and Winter Silver Carp (Hypophthalmichthys molitrix) Muscle

Myosin subfragment-1 (S1) was prepared from myofibrils of summer and winter silver carp by chymotryptic digestion in the presence of ethylenediaminetetraacetic acid (EDTA). Two S1 heavy chain isoforms with different molecular sizes of 91 kDa and 95 kDa were detected in the fast skeletal muscle from summer and winter silver carp, respectively. ATPase inactivation assay indicated that winter S1 was about 20-fold unstable comparing to summer S1. Matrix-assisted laser desorption/ionization time-of-flight/mass spectrometry (MALDI-TOF MS) further confirmed that summer and winter myosin S1 heavy chain isoforms were homologous to myosin high-temperature type and myosin low-temperature type S1 heavy chain, respectively. Moreover, both types of myosin S1 heavy chain isoforms were identified at the intermediate stage. The results indicated that myosin was expressed in a season-specific manner; different types of myosin isomer expressed in different seasons, showing different thermostabilities. Practical Application: Silver carp, Hypophthalmichthys molitrix, is one of the most abundant freshwater fish species in China. The structure thermal stability of myosin rod from silver carp was affected by season change. The gel-forming abilities of surimi prepared in different seasons were different. This study investigated the seasonal differences in structure thermal stability of myosin S1 which is vital for gel formation of myosin. The results of this study will aid understanding of the relationship between the structure and function of myosin, and effective production of surimi from freshwater fish species in different seasons.     

Presence of an unidentified myosin isoform in certain bovine foetal muscles

Genetic variation in the establishment of bovine muscle fibre types was studied by comparing muscle differentiation at 210 days of foetal life in normal cattle and in ‘culard’ animals, which have muscular hypertrophy. The different fibre types were determined by histochemical and immunohistochemical analyses with monoclonal antibodies specific to different myosin heavy chain isoforms. The isoforms were separated by electrophoresis and quantified by the ELISA method. Four muscles with different contractile and metabolic characteristics were studied: Semitendinosus, Longissimus thoracis, Masseter (slow) and Cutaneus trunci (fast). Muscle fibres recognized by none of the antibodies used were observed in ‘culard’ foetuses in all the muscles studied and also in the Cutaneus trunci of normal animals. Electrophoretic analysis showed no particular myosin isoform in these muscles. It is possible therefore that the fibres contained a mysosin isoform until now unidentified in cattle, with a molecular weight the same as that of known isoforms. This newly observed isoform seems to be specific to muscles rich in IIB fibres such as Cutaneus trunci and to the muscles of adult ‘culard’ cattle.     

Myosin is solubilized in a neutral and low ionic strength solution containing l-histidine

Myosin, one of the major myofibrillar proteins, is insoluble at low and physiological ionic strength and soluble at high ionic strength. In this study, the behavior and morphology of myosin solubilized in a low ionic strength solution containing l-histidine (l-His) was investigated. More than 80% of myosin was solubilized in a low ionic strength solution with dialysis against a solution containing 1 mM KCl and 5 mM l-His. Transmission electron microscopy with rotary shadowing demonstrated that the rod of myosin in a low ionic strength solution containing l-His is longer than that of myosin in a high ionic strength solution. The elongation of the myosin rod in a low ionic strength solution containing l-His would inhibit the formation of a filament, resulting in the solubilization of myosin.     

Seasonal Expression of 2 Types of Myosin with Different Thermostability in Silver Carp Muscle (Hypophthalmichthys molitrix)

Myofibrils were prepared from dorsal muscle of silver carp monthly from Jul 2003 to Jun 2004. Myofibrils of silver carp in the summer season were much more stable than those in the winter season as measured by (Ca‐ATPase) thermal inactivation rate (k_D) at 0.1 M KCl. The k_D of myofibrils measured at 2 M KCl was referred as 1 for myosin without protection by F‐actin. It was proved that myosin itself in summer season fish (summer‐type) was very stable compared with that in winter season fish (winter‐type). Winter‐type myosin easily denatured upon storage in ice. Analysis of the thermal inactivation profiles of myosin monthly prepared demonstrated that myosins in December, January, February, and March were winter‐type, and ones in June, July, August, and September were summer‐type. It was also demonstrated that both winter‐type and summer‐type myosin at different ratios coexisted in the transit season (April, May, October, November), showing the breaking point in the inactivation profile. It was concluded that silver carp expresses 2 types of myosin with different k_D seasonally and that the exchange of the expression completed in 2 mo.