Thermal denaturation of proteins in Post rigor muscle tissue as studied by differential scanning calorimetry

Post rigor bovine M. semimembranosus was analysed by differential scanning calorimetry (d.s.c.). After extractive removal of sarcoplasmic proteins, subsequent pH adjustment and manual connective tissue removal, d.s.c. yielded reproducible thermograms which permitted investigation of the individual major myofibrillar proteins in various pH and salt environments without prior isolation. The positions of two major peaks, interpreted as myosin transitions, proved to be strongly pH dependent. At pH 5.4, the peak maxima occurred at 58 and 65°C, respectively, at a heating rate of 10°C min-¹. Above pH 6.5 their order of denaturation was reversed. In the pH range 5.4–6.5 the peak ascribed to actin had its maximum near 80°C in intact muscle. Above this pH range it was displaced to lower temperature. The thermal stability of actin was studied after treatment of the muscle tissue with different salt solutions. At equal ionic strengths (μ = 0.15) at pH 5.5, calcium chloride and sodium chloride caused 6.5°C and 4°C displacement to lower temperature, respectively. The thermograms of bovine semimembranosus muscle were compared to those of two red and two white muscle types (bovine cardiac and rabbit soleus muscles, chicken breast and rabbit semimembranosus muscles, respectively) at two pH levels. Greater myosin differences were found between red and white muscles than between muscles from different animal species. All muscles gave similar actin transitions, with exception of the heart muscle where the actin peak appeared at 3 °C lower temperature. The necessity of a strict pH control in order to obtain reproducible muscle thermograms is demonstrated.     

Rheological properties of myosin-gelatin mixtures

ABSTRACT:  This study was designed to investigate changes of dynamic rheological properties of myosin and gelatin mixtures during heating and cooling. The rheological and calorimetric attributes (G', G", tan δ, and heat flow) of myosin and gelatin mixtures were measured during heating and cooling. The results indicated that gelatin could increase the G' and G" values and decrease the denaturation temperature of myosin. The G' and G" values of myosin and gelatin mixtures decreased with pH over 5.5 to 7.5 and ionic strength over 0.3 to 0.6. Therefore, it is advisable to add gelatin into chicken meat comminuted products in that it could improve the rheological attributes of myosin.     

Filamentous structures of bovine myosin in diluted suspensions and gels

Turbid solutions and fine-stranded gels of myosin from bovine semi-membranous muscle were investigated by transmission and scanning electron microscopy. Evidence is given that the turbidity was caused by filament formation upon dialysis to pH 5.5 and 0.25 M KCl and to pH 4.0 and 0.6 m KCl at 4°C. The filaments formed at pH5.5 and 0.25 m KCl had a backbone with a diameter of c. 25 nm with the myosin heads located close to the filament backbone. The total width of these filaments was c.45nm. The filaments were prepared for electron microscopy by adsorption on various substrates, negative staining, or freeze drying and rotary shadowing. Variations in the preparation technique did not affect the appearance of the filaments. The filaments formed at pH 4.0 and 0.6 m KCl had a more irregular appearance, and the total filament width varied between 20 and 45 nm. Fringes of globular material surrounding the filament backbone were seen but also clusters of myosin molecules protruding further out from the backbone and from the filament ends. Comparison of heat-treated filaments in dilute solutions with strands of the gel network confirmed that the gel strands originated from filaments formed upon dialysis prior to gelation. Typical features of the network structure were junction zones formed by parallel alignments of filaments in pairs and by end-to-side interactions forming so-called Y-junctions. At pH5.5 and 0.25 m KCl these interactions resulted in a rather loose and open network structure. At pH 4.0 and 0.6 M KCl the filaments often interacted approximately at right angles, which resulted in a denser network than that observed atpH5.5 and 0.25 m KCl. The efficient network formation at pH 4.0 gave rise to spontaneous gel formation upon dialysis without any heat treatment. Additional heating did not change the character of the network, and no differences could be observed between unheated and heat-treated gels at low magnifications. At higher magnifications it could be seen that heating resulted in loss of details of the filaments at both pH values and ionic strengths. The shape of the myosin heads was lost, and the heads fused together on the filament backbone.     

Ionic Strength Effects on Heat-induced Gelation of Myofibrils and Myosin from Fast- and Slow-twitch Rabbit Muscles

The effects of ionic strength on myofibrils and myosin from rabbit fast-twitch Psoas major (PM) and slow-twitch Semimembranosus proprius (SMp) muscles before and after heating were studied by electron microscopy and thermal scanning rheometry. The direct suspension of proteins in low ionic strength (0.2M KCl; pH 6.0) led to very weak gels, whereas a gradual lowering of the ionic strength (by dialysis against 0.2M KCl; pH 6.0) of 0.6M KCl protein solutions induced strand-type networks at low temperature and strong heat-induced gels. As shown by transmission and scanning electron micrographs, in low ionic strength, SMp myosins formed shorter filaments before heating and thinner and shorter structures in heat-induced gels, as well as a lower gel porosity than PM myosins.     

CHARACTERISTICS OF SARCOPLASMIC PROTEINS AND THEIR INTERACTION WITH MYOFIBRILLAR PROTEINS

The protein solubility and molecular‐weight distribution of freeze‐dried sarcoplasmic proteins (SPs) from rockfish treated under low and high pH as well as various NaCl concentrations were elucidated. The solubility of SPs was significantly suppressed at an acidic pH (2.0–4.0) and in the presence of high salt concentration (0.5 M NaCl). The least amount of protein was lost when SPs were treated at pH 2.0 or 3.0 followed by precipitation at pH 5.5. The interaction of SPs with Alaska pollock surimi (myofibrillar proteins) was also investigated. The addition of SPs appeared to delay the thermal denaturation of myosin and actin. The SPs positively contributed to the gelation of myofibrillar proteins as judged by breaking force.     

Differential scanning calorimetric study of muscle and its proteins: Myosin and its subfragments

The thermal denaturation of rabbit skeletal muscle myosin and its subfragments was investigated by differential scanning calorimetry. The thermal denaturation of myosin was shown to occur via three (at least) partly independent cooperative endothermic processes. The temperatures at which these processes occurred (312, 317 and 323 K at pH 6.0 and I=1.0) were shown to vary with pH (5.5–8.0) and I (0.05–1.0). The apparent enthalpy of denaturation of myosin was also shown to be dependent on pH and I. By comparing thermograms of myosin with those of the isolated myosin subfragments, the three major processes associated with the thermal denaturation of rabbit myosin could be tentatively assigned to different regions of the myosin molecule, namely, the helical tail, the ‘hinge-region’ and the globular heads. The ‘hinge-region’ thermal denaturation was shown to be reversible at pH 6.0 and I=1.0. Investigations of the effects of ortho-, pyro-, and tripolyphosphate on the thermal denaturation of myosin showed that added pyrophosphate destabilised the myosin molecule by about 9 K compared to the effects of ortho and tripolyphosphate, even though the latter was probably hydrolysed to ortho and diphosphate.     

Salt‐ and pyrophosphate‐induced structural changes in myofibrils from chicken red and white muscles

Myofibrils isolated from post‐rigor chicken Pectoralis major (PM, white) and Gastrocnemius (Gas, red) muscles were irrigated with various concentrations of NaCl (0.1–1.0 M ) with or without 10 mM sodium pyrophosphate at pH 5.5 and 6.0. Structural changes were examined using phase contrast microscopy. PM myofibril samples tended to show more definitive H‐zones but obscure Z‐lines compared to Gas myofibrils. Significant myofibril swelling, accompanied by a pronounced protein extraction, occurred in 0.5 M NaCl solution. The extent of swelling as well as protein extraction increased with the NaCl concentration up to about 0.8 M . Addition of pyrophosphate facilitated myofibril swelling and reduced the minimal NaCl concentration for swelling to 0.4 M . Without pyrophosphate, protein extraction for both PM and Gas myofibrils occurred along the A‐band, sometimes starting from the centre, but when pyrophosphate was added, the extraction began from the ends of the A‐band. At pH 5.5, protein extraction was similar for PM and Gas, but at pH 6.0, PM myofibrils were more extractable and their architecture changed more extensively than Gas myofibrils, especially when pyrophosphate was present. The results may explain the different water‐imbibing abilities of white and red meat when processed with salt and phosphate. © 2000 Society of Chemical Industry     

Heat-Induced Aggregation and Denaturation of Egg White Proteins in Acid Media

Heat-induced aggregation and denaturation of egg white proteins adjusted to pH 5.5, 4.5, 3.5 and 2.5 were investigated by vertical flat-sheet polyacrylamide gel electrophoretic and differential scanning calorimetric methods. The fractional and step-wise aggregation of egg white proteins was caused by heating. As the acidity was increased from pH 5.5 to 2.5, ovotransferrin, ovomacroglobulin, globulin G3A, globulins A1 and A2, and ovalbumin became much more unstable to heat. However, ovomucoid and ovoinhibitor did not aggregate in the acidified egg white under heat treatments of 3 min at 90°C or 20 min at 74°C. The heat-induced aggregation of flavoprotein was slightly greater at pH 4.5 and 3.5 than at pH 5.5 and 2.5.     

Comparative study on acid-induced gelation of myosin from Atlantic cod (Gardus morhua) and burbot (Lota lota)

Physicochemical and rheological properties of myosin from Atlantic cod and burbot during acid-induced gelation at room temperature (22–23 °C) by d-gluconic acid-δ-lactone (GDL) were monitored. Turbidity and particle size of both myosins increased and salt soluble content decreased when pH decreased, suggesting the formation of protein aggregates caused by acidification. The formation of disulphide bonds in myosin gelation was induced by acid. Ca²⁺-ATPase activity of myosin decreased (p < 0.05), while surface hydrophobicity increased during acidification (p < 0.05). Furthermore, the decreases in maximum transition temperature (T_max) and the denaturation enthalpies (ΔH) were found in both myosins. During acidification, the increases in storage modulus (G′) and loss modulus (G″) of myosin were observed (p < 0.05), revealing the formation of elastic gel matrix. Thus, gelation of myosin from Atlantic cod and burbot could take place under acidic pH via denaturation and aggregation. However, myosin from Atlantic cod was generally more favourable to gelation than was burbot myosin.     

Thermal stability of fish myofibrils: a differential scanning calorimetric study

The variation in myofibrillar protein thermostability was compared for various fish species, using differential scanning calorimetry. The tropical fish, catfish ( Clarius gariepinus ), carp ( Cyprinus carpio ), Nile perch ( Lates niloticus ), red snapper ( Lutianus sebae ), red mullet ( Parpeneus barberinus ), sea bream ( Gymnocranius rivalatus ), and cold-water reared trout ( Salmo gairdneri ) and cod ( Gadus morhua ) were analysed. Onset temperature of myofibrillar protein denaturation occurred at up to 11°C higher for tropical species (43.5°C, catfish), than cod (32.6°C) at pH 7 and low ionic strength (I). As pH (6.0-8.0) and I (0.05-1.00) were increased, thermal denaturation temperatures of myosins from tropical, but not cold-water, species decreased. Enthalpies of myofibrillar denaturation decreased for all species with increasing pH and I. Only one thermal transition was detected for myosin at pH 6 and low I, increasing to three as pH and I were increased. Changes in thermal characteristics of myosin subunits over iced and frozen storage suggest more rapid deterioration in cold-water than in tropical fish. The differences in myofibrillar stability of fish from different habitat temperatures have implications for the processing and storage of tropical fish.     

Thermal Stability and Gel-Forming Ability of Shark Muscle as Related to Ionic Strength

Thermal stability and gel-forming ability of silvertip, hammer-head and thresher shark as affected by ionic strength were investigated. Low temperature endothermic peaks (LTEP, between 30 and 44°C) were observed in differential scanning calorimetric (DSC) thermograms of the shark muscles and surimi samples. Increases in ionic strength reduced the thermal stability of surimi as shown by depression of transition temperature, denaturation enthalpies and LTEP. Although gel strength of heat-treated surimi increased with increasing NaCl concentration, the three species had different sensitivities to ionic strength. The highest NaCl-induced gel-forming ability of surimi was achieved at 0.5M salt concentration for silvertip and thresher; at 1.0M for hammer-head shark.     

Protein Denaturation in Pork Longissimus Muscle of Different Quality Groups

Differential scanning calorimetry (DSC) was used to investigate the protein denaturation characteristics of pork muscles from four quality groups namely RFN (red, firm, and non-exudative), RSE (red, soft, and exudative), PFN (pale, firm, and non-exudative), and PSE (pale, soft, and exudative). The thermograms indicated three endothermic peaks between 45°C to 90°C, corresponding to denaturation of myosin (peak I), sarcoplasmic proteins (peak II), and actin (peak III). The myosin peak was much reduced in PSE samples, while the actin peak remained almost identical in all groups. RFN and RSE samples were found to have very similar protein denaturation characteristics and were not significantly different in their thermodynamic protein denaturation parameters. PFN samples showed similar myofibrilar protein denaturation but significantly different sarcoplasmic protein denaturation characteristics compared to normal (RFN) samples according to their DSC thermograms. Based on these findings, it was suggested that the pale color in PFN pork is linked to sarcoplasmic protein denaturation.     

Myosin denaturation in pale, soft, and exudative (PSE) porcine muscle tissue as studied by differential scanning calorimetry

Myofibrillar tissue from pale, soft, and exudative (PSE) pork was compared to tissue from normal pork by differential scanning calorimetry at pH 5.4. Thermograms of myofibrillar tissue from normal pork were characterised by three major peaks with temperature maxima at 58 and 66°C, associated with myosin denaturation, and at 78°C, associated with actin denaturation. In thermograms of PSE pork, the peak at 58°C was markedly reduced, and appeared as a shoulder. When the thermograms were divided into segments corresponding to the three major peaks, the area of the low temperature myosin segment was shown to be reduced by about 50% in PSE pork, as compared to normal pork. This indicates approximately 50% denaturation of the least thermostable parts of the myosin molecule. The more thermostable parts of the myosin molecule were largely unaffected, as was actin.     

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.     

Suppression of thermal denaturation of myosin and salt-induced denaturation of actin by sodium citrate in carp (Cyprinus carpio)

The effect of sodium citrate (Na-citrate) on myosin and actin denaturation in myofibrils was investigated. Na-citrate significantly suppressed the thermal inactivation of Ca²⁺-ATPase of carp myosin in a concentration-dependent manner. The effect was greater than that of sorbitol. A similar effect was observed with myofibrils in which myosin is stabilized by F-actin binding. Na-citrate dissolved myofibrils at lower concentration than NaCl. Nevertheless, Na-citrate at 1 M failed to denature F-actin in myofibrils, while 1 M NaCl denatured F-actin almost completely. Na-citrate suppressed the NaCl-induced F-actin denaturation. Sorbitol did not show such protective effect on F-actin denaturation. Moreover, Na-citrate suppressed the freeze denaturation of myofibrils at lower concentration than sorbitol. Thus, Na-citrate was proved to be superior to sorbitol. It was suggested that Na-citrate alone could substitute sorbitol as cryoprotectant in surimi and NaCl as dissolving reagent of myofibril in thermal gel production.     

Biochemical and conformational changes of myosin purified from Pacific sardine at various pHs

ABSTRACT:  Biochemical and conformational changes of purified sardine myosin were investigated at various pHs. The purity of myosin, as determined by SDS-PAGE, was approximately 94.6%. One major band at 205 kDa, corresponding to myosin heavy chain, and 3 light chains at 31, 24, and 23 kDa were observed on the SDS-PAGE gel. The greatest myosin protein solubility was observed at pH 7 and remained constant up to pH 11. Sardine myosin showed no solubility at pHs 2.5 to 5.0. Three endothermic peaks were obtained for samples prepared at pHs 7 and 10, while no peaks were shown for pH 2 samples, indicating chemical denaturation of myosin occurred before thermal treatment. The greatest Ca²⁺-ATPase activity was observed at pH 7, while no activity was observed between pHs 2 and 5 and at pH 11. Total sulfhydryl content was not measured at pHs 2.5 to 4 while the greatest measure was obtained for samples at pH 5.5. Surface hydrophobicity was not detected from pHs 2.5 to 5.0; thereafter the content remained consistent through pH 11. Storage modulus, indicating the elastic element of myosin gels, was minimally affected at pH 2, indicating myosin was chemically denatured before the temperature sweep treatment. However, at pH 10, the thermal exposure of myosin, as evidenced by dynamic thermograms with deeper valleys at 40 to 60 °C, was noted, indicating myosin was not damaged by adjustment to pH 10 and therefore was still able to undergo thermal gelation.     

Liquid Loss as Effected by Post mortem Ultrastructural Changes in Fish Muscle: Cod (Gadus morhuaL) and Salmon (Salmo salar)

This study was performed in order to assess the effect of early post mortem structural changes in the muscle upon the liquid-holding capacity of wild cod, net-pen-fed cod (fed cod) and farmed salmon. The liquid-holding capacity was measured by a low speed centrifugation test. Transmission electron microscopy was used to discover ultrastructural changes both in the connective tissue and in the myofibrils. Differential scanning calorimetric thermograms of the muscle proteins were recorded to elucidate whether fundamental differences did exist between the proteins of the raw material tested. Multivariate statistics were used to explicate the main tendencies of variations in the thermograms. The salmon muscle possessed much better liquid-holding properties than the cod muscle, and wild cod better than fed cod regardless of the storage time. Both fed cod and farmed salmon, underwent the most severe structural alterations, probably caused by the low muscle pH values. The higher liquid-holding capacity of the salmon muscle was related to species specific structural features and better stability of the muscle proteins. The myofibrils of the salmon muscle were denser and intra- and extracellular spaces were filled by fat and a granulated material. The differences in thermograms of muscle from wild and fed cod were largely explained by the variations in pH. The severe liquid loss of fed cod is due to a low pH induced denaturation and shrinkage of the myofibrils. Post mortem degradation of the endomysial layer and the sarcolemma may have further facilitated the release of liquid.     

Species dependence of fish myosin stability to heat and frozen storage

The thermal stabilities of the proteins of a range of fish muscles of different habitat temperatures were determined by differential scanning calorimetry before and after frozen storage at 20°C.
In whole muscle a clear relationship was seen between habitat temperature and the thermal denaturation of myosin, which persisted when isolated myosins were analysed under conditions close to physiological pH and ionic strength. the ionic environment of the myosin molecules in the whole tissue will, however, not be exactly the same as in the myosin solution.
No significant correlations were seen between habitat temperature, ultimate pH and other analytical parameters.
After frozen storage, the myosin transitions in red snapper, a warm water species, was markedly changed in whole muscle but not in isolated myosin, suggesting the post mortem development of an interaction with other muscle proteins. In contrast, in cod, a cold water species, changes in myosin transitions were very similar, both in whole muscle and isolated myosin.
The implications of species differences in the 'melting' of myosin domains and changes in textural quality during frozen storage are discussed briefly.     

Microbial transglutaminase-induced structural and rheological changes of cationic and anionic myofibrillar proteins

This study investigated the effects of microbial transglutaminase (TG) on structural changes in porcine myofibrillar protein (MP) under varying pH (2.0–6.0) and two ionic strength conditions (0.1 M versus 0.6 M NaCl). Lowering the pH below the isoelectric point (pI) of myosin induced protein unfolding as revealed by surface hydrophobicity and differential scanning calorimetry. Although the MP solubility at the low ionic strength (0.1 M NaCl) was maximal at pH 3.0, both SDS-PAGE profiles and dynamic rheology indicated TG could not cross-link MP under this condition. Based on the carboxyl group content, the TG-catalyzed deamidation was dominant at a pH lower than the pI of myosin (pH 5.0) while cross-linking occurred at higher pH. Moreover, deamidation had no effect on rheological properties of MP. The results indicate that the TG reaction was governed by the pH of substrate protein, and the reaction intensity was related to the solubility of protein.     

Slow lowering of pH induces gel formation of myosin

It has been found that solutions of myosin (10 mg/ml) form gels at 5°C if the pH is decreased slowly, by dialysis, to a value in the region of 2.5 to 5.5. Gel strength displays strong dependence on final pH, having a maximum at about pH 4.5. Salt (KCl) concentration was found to affect gel strength positively and linearly. Differential scanning calorimetry revealed that the myosin of pH-induced gels absorbed no thermal energy when heated, implicating acid-induced denaturation as the basis of gel formation. By comparison with heat-induced gelation of myosin and from the fact that low pH is conducive to filament formation, it is suggested that filaments may also be involved in the gelation process.