Denaturation Of Myosin Research Articles

Changes in physical properties of heat-induced gel on addition of gluconate associated with suppression of myosin denaturation in walleye pollack salt-ground surimi during preheating

Changes in physical properties of two-step heated gels on addition of gluconate were investigated in terms of relationships between breaking strength and gel stiffness. Regression lines between the breaking strength and the gel stiffness were extended to the x-axis (gel stiffness), and the intercept was defined as SBSO. The SBSO of the two-step heated gels increased with gluconate contents in salt-ground surimis, suggesting that the harder but less elastic gels formed on addition of gluconate were dose-dependent. Conversely, the denaturation rate constants of myosin in salt-ground surimis during preheating estimated by means of Ca-ATPase inactivation, loss of salt solubility, and decrease of denaturant solubility were considerably reduced by gluconate. Thus, the progress of myosin denaturation was strongly suppressed. Increments of SBSO (δSBSO) of the two-step heated gels on addition of gluconate were inversely correlated with the denaturation rate constants of myosin in salt-ground surimis for every index. Thus, the changes in physical parameters of two-step heated gel caused by gluconate may be associated with the sluggish progress of myosin denaturation in salt-ground surimi during preheating.

Lipid and protein deterioration during the chilled storage of minced sea salmon (Pseudopercis semifasciata)

AbstractSea salmon is a very appreciated seafood. The aim of this work was to analyze changes in lipid and protein fractions of minced muscle during chilled storage (1 ± 1 °C). Lipid oxidation was important during the first 6 days of storage according to 2‐thiobarbituric acid (TBA) values determined, decreasing mainly ω3 22:6 fatty acid content. Lipid hydrolysis was evident after 9 days of storage. Interaction compounds between oxidation products and other cellular components were analyzed by fluorescence measurements. The results obtained showed evidence of the formation of interaction products involved, mainly polar components such as proteins. Decreases in myosin and actin thermal stability and myosin denaturation were recorded by differential scanning calorimetry. Solubility of total and myofibrillar proteins decreased after 6 days of storage. The electrophoretic profile of soluble fractions showed an increase in the intensity of bands corresponding to low‐molecular‐weight polypeptides and a decrease in high‐molecular‐weight species. Available lysine content did not change during chilled storage. Copyright © 2007 Society of Chemical Industry

Hydroxyl radical oxidation destabilizes subfragment-1 but not the rod of myosin in chicken myofibrils

The thermal denaturation process of myosin in oxidized chicken myofibrils was investigated. Exposures of myofibrils to hydroxyl radical-generation systems (HRGS) resulted in an enhanced susceptibility of myosin to thermal inactivation of Ca-ATPase and a loss of salt solubility. The chymotryptic production of subfragment-1 (S-1) from myosin in oxidized myofibrils decreased more rapidly than that in un-oxidized myofibrils upon heating, which paralleled the Ca-ATPase decay. However, the heat-induced decrease in chymotryptic production of rod from myosin was not affected by the HRGS treatment. The results suggested that free radical oxidation promoted thermal destabilization of myosin in the S-1 portion instead of the rod portion. The altered myosin denaturation pattern due to hydroxyl radical oxidation was likely a cause for functionality changes in oxidatively stressed myofibrillar proteins in meat processing.

Thermal Denaturation and Aggregation Properties of Atlantic Salmon Myofibrils and Myosin from White and Red Muscles

Thermal denaturation and aggregation abilities of salmon myofibrils and myosin were studied measuring turbidity, intrinsic fluorescence, 8-anilino-1-naphthalene sulfonic acid binding, and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide cross-linking. The thermal behaviors of protein preparation from white and red muscles were compared, and the relationship with thermal gelation properties is discussed. The low gelation ability of salmon muscle proteins was related to a limited extent of protein denaturation and aggregation upon heating. These properties seemed to be carried by myosin molecules as a similar behavior was observed for both myofibrils and myosin preparations. The higher thermal stability observed for red muscle proteins with higher transition temperatures in rheological profiles was related to a shift to higher temperature in denaturation and aggregation processes. The extent of denaturation and aggregation was very similar for both muscle types as was the final rigidity of the gels formed.

Extraction of Sardine Myoglobin and Its Effect on Gelation Properties of Pacific Whiting Surimi

Myoglobin (Mb) was extracted from Pacific sardine and added to Pacific whiting surimi to measure its effects on protein gelation. The purity of Mb extract was determined by SDS-PAGE. Mb extract using ethanol showed higher purity than Mb extract using ammonium sulfate. The addition of 0.2% Mb significantly reduced breaking force. However, a synergistic effect of 1.0% Mb was observed with 1.0% beef plasma protein (BPP) to increase surimi gel strength. The highest storage modulus of gels was observed at 1.0% Mb addition, which corresponded with the nonfracture gel and also supported a synergistic interaction between 1.0% Mb and 1.0% BPP. Differential scanning calorimetry showed Mb addition might have affected myosin denaturation and aggregation.

Conformational and Rheological Changes in Catfish Myosin as Affected by Different Acids during Acid-Induced Unfolding and Refolding

Changes in the conformation of catfish (Ictalurus punctatus) myosin due to (i) anions, (ii) acid pH, and (iii) salt addition were determined using tryptophan fluorescence, hydrophobicity measurements, differential scanning calorimetry, and circular dichroism. The relationship between conformation and storage modulus (G') of acid-treated myosin was studied. Three acids, HCl, H2SO4, and H3PO4, were used for unfolding myosin at three acidic pH conditions, 1.5, 2.0, and 2.5. Unfolded myosin was refolded to pH 7.3. Denaturation and unfolding of myosin was significantly (p < 0.05) lower when salt (0.6 M NaCl) was present during acid unfolding than in the absence of salt. When salt was added before unfolding, the alpha-helix content of myosin treated at pH 1.5 was significantly lower than that treated at pH 2.5. When salt was added after refolding, the alpha-helix content of myosin was unaffected by different pH treatments. The G' of myosin increased with an increase in myosin denaturation. The G' of myosin was significantly (p < 0.05) higher when salt was added to myosin after refolding than before acid unfolding. Among the different anion treatments, the G' of acid-treated myosin decreased in the order Cl- approximately SO42- > PO43-. Among the different pH treatments, the G' of myosin treated at pH 1.5 was significantly (p < 0.05) higher than myosin treated at pH 2.5. The conditions that would result in maximum myosin denaturation and maximum G' were unfolding of myosin at pH 1.5 using Cl- (from HCl) followed by refolding at pH 7.3 and subsequent addition of 0.6 M NaCl.

Tyrosinase-Aided Protein Cross-Linking: Effects on Gel Formation of Chicken Breast Myofibrils and Texture and Water-Holding of Chicken Breast Meat Homogenate Gels

The effects of Trichoderma reesei tyrosinase-catalyzed cross-linking of isolated chicken breast myofibril proteins as a simplified model system were studied with special emphasis on the thermal stability and gel formation of myofibrillar proteins. In addition, tyrosinase-catalyzed cross-linking was utilized to modify the firmness, water-holding capacity (WHC), and microstructure of cooked chicken breast meat homogenate gels. According to SDS-PAGE, the myosin heavy chain (MHC) and troponin T were the most sensitive proteins to the action of tyrosinase, whereas actin was not affected to the same extent. Calorimetric enthalpy (DeltaH) of the major thermal transition associated with myosin denaturation was reduced and with actin denaturation increased in the presence of tyrosinase. Low-amplitude viscoelastic measurements at constant temperatures of 25 degrees C and 40 degrees C showed that tyrosinase substantially increased the storage modulus (G') of the 4% myofibrillar protein suspension in the 0.35 M NaCl concentration. The effect was the most pronounced with high-enzyme dosages and at 40 degrees C. Without tyrosinase, the G' increase was low. Tyrosinase increased the firmness of the cooked phosphate-free and low-meat chicken breast meat homogenate gels compared to the corresponding controls. Tyrosinase maintained gel firmness at the control level of the low-salt homogenate gel and weakened it when both salt and phosphate levels were low. Tyrosinase improved the WHC of the low-meat and low-salt homogenate gels and maintained it at the level of the corresponding controls of phosphate-free and low-salt/low-phosphate homogenate gels. Microstructural characterization showed that a collagen network was formed in the presence of tyrosinase. Chicken myofibrillar proteins; protein modification; cross-linking; tyrosinase; gelation; thermal stability; texture; water-holding capacity; microstructure.

Changes in protein fractions of rainbow trout ( Oncorhynchus mykiss) gravads during production and storage

Gravad fish products belong to a group of low-processed products obtained from fresh fish by sprinkling fillets with a mixture of sugar and salt and then placing them in cold storage. Little is known about changes in the tissue during the production and storage of gravads. The purpose of the present study was to investigate the type and scope of changes in rainbow trout ( Oncorhynchus mykiss) gravad proteins during processing and vacuum storage at 3 °C and −30 °C. The results showed that the solubility of protein was barely affected by the gravading process, but did change during the cold storage and freezing of the gravad. Catheptic activity decreased with gravading and further storage. A SDS-PAGE analysis revealed that the intensity of the M-protein and troponin bands was reduced, while the bands of α-tropomyosin and the myosin heavy chain increased when the trout muscle was subjected to the gravading process. Two bands with molecular weights of 255 and 135 kDa disappeared in the gravad, and new bands with molecular weights of 163 and 117 kDa appeared. An HPLC analysis revealed that gravading had a limited effect on the relative areas of peaks corresponding to low molecular weight substances of protein origin, while more significant changes were observed during gravad storage. Moreover, the gravading process caused a lowering of the actin and myosin denaturation temperature, which was proved using the DSC method. In conclusion, changes in rainbow trout muscle protein appeared to have a crucial effect on product quality.

NMR relaxometry and differential scanning calorimetry during meat cooking

By combining simultaneous nuclear magnetic resonance (NMR) T 2 relaxometry and differential scanning calorimetry (DSC) on pork samples heated to nine temperature levels between 25 and 75 °C, the present study investigates the relationship between thermal denaturation of meat proteins and heat-induced changes in water characteristics. Principal component analysis (PCA) on the distributed 1H NMR T 2 relaxation data revealed that the major changes in water characteristics during heating occur between 40 and 50 °C. This is probably initiated by denaturation of myosin heads, which however, could not be detected in the DSC thermograms obtained directly on the meat. In contrast, the DSC thermograms revealed endothermic transitions at 54, 65 and 77 °C, probably reflecting the denaturation of myosin (rods and light chain), sarcoplasmic proteins together with collagen and actin, respectively. Simultaneous modelling of DSC and NMR data by partial least squares regression (PLSR) revealed a correlation between denaturation of myosin rods and light chains at ∼53–58 °C and heat-induced changes in myofibrillar water ( T 2 relaxation time ∼10–60 ms) as well as between actin denaturation at ∼80–82 °C and expulsion of water from the meat. Accordingly, the present study demonstrates a direct relationship between thermal denaturation of specific proteins/protein structures and heat-induced changes in water mobility during heating of pork.

A kinetic study on inactivation of tilapia myosin Ca-ATPase induced by high hydrostatic pressure

Tilapia myosin (2.5 mg/ml) was treated by hydrostatic pressure (50–300 MPa) for 0–60 min to determine the inactivation kinetics of myosin Ca-ATPase. The process of the pressure-induced inactivation of myosin Ca-ATPase included two steps: the first one was an instantaneous pressure-induced inactivation, and the degrees of lost activities, called instantaneous pressure kill (IPK) values, increased with elevated pressure. The second one, the logarithm of residual activity of myosin ATPase, decreased smoothly during each pressure treatment for 10–60 min. However, D values (the time needed for 90% loss of the activity during a treatment at the same pressure) of Ca-ATPase decreased about 50% with per 50 MPa increase. In this study, 150 MPa was the pressure level that caused apparent myosin denaturation and the typical network structure formation with beyond 50% decrease of myosin Ca-ATPase activity.

Chemical composition and thermal property of cuttlefish ( Sepia pharaonis) muscle

The chemical composition and thermal property of cuttlefish ( Sepia pharaonis) muscle were studied. The head and mantle contained 11.9–14.9% protein, 0.5% fat, 1.2–1.3% ash, and 0.6–1.8% collagen. Lipids from the head and mantle contained phospholipid as the major component (78.6–87.8% of total lipid), with 10.6–19.5% diglyceride. Polyunsaturated fatty acids constituted 50.3–54.9% of fatty acids with a high content of DHA and EPA. The n-3 PUFA contents were greater than n-6 PUFA. The C16:0 and C18:0 were the most abundant saturated fatty acid in the head and mantle. The cuttlefish muscle consisted of myofibrillar proteins as the major protein (53.1–58.4%). Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that myosin heavy chain (MHC), paramyosin and actin were the major proteins and varied with the portions. Differential scanning colorimetric (DSC) study revealed three transitions corresponding to the thermal denaturation of myosin and paramyosin, connective tissues and actin, at temperatures of 49.8–50.3, 59.8–60.3 and 74.7–78.8 °C, respectively. Zinc and iron were the dominant trace minerals in both portions.

Influence of muscle type on rheological properties of porcine myofibrillar protein during heat-induced gelation

The gelation characteristics of myofibrillar proteins are indicative of meat product texture. Defining the performance of myofibrillar proteins during gelation is beneficial in maintaining quality and developing processed meat products and processes. This study investigates the impact of pH on viscoelastic properties of porcine myofibrillar proteins prepared from different muscles ( semimembranosus (SM), longissimus dorsi (LD) and psoas major (PM)) during heat-induced gelation. Dynamic rheological properties were measured while heating at 1 °C/min from 20 to 85 °C, followed by a holding phase at 85 °C for 3 min and a cooling phase from 85 to 5 °C at a rate of 5 °C/min. Storage modulus ( G′, the elastic response of the gelling material) increased as gel formation occurred, but decreased after reaching the temperature of myosin denaturation (52 °C) until approximately 60 °C when the gel strength increased again. This resulted in a peak and depression in the thermogram. Following 60 °C, the treatments maintained observed trends in gel strength, showing SM myofibrils produced the strongest gels. Myofibrillar protein from SM and PM formed stronger gels at pH 6.0 than at pH 6.5. Differences may be attributed to subtle variations in their protein profile related to muscle type or postmortem metabolism. Significant correlations were determined between G′ at 57, 72, 85 and 5 °C, indicating that changes affecting gel strength took effect prior to 57 °C. Muscle type was found to influence water-holding capacity to a greater degree than pH.

CHARACTERISTICS OF SARCOPLASMIC PROTEINS AND THEIR INTERACTION WITH MYOFIBRILLAR PROTEINS

ABSTRACT The protein solubility and molecular-weight distribution of freeze-driedsarcoplasmic proteins (SPs) from rockfish treated under low and high pH aswell as various NaCl concentrations were elucidated. The solubility of SPswas significantly suppressed at an acidic pH (2.0–4.0) and in the presence ofhigh salt concentration (0.5 M NaCl). The least amount of protein was lostwhen 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) wasalso investigated. The addition of SPs appeared to delay the thermal denatur-ation of myosin and actin. The SPs positively contributed to the gelation ofmyofibrillar proteins as judged by breaking force. INTRODUCTION A new method for preparing fish protein isolate using pH shift hassignificant advantages over conventional surimi processing. This revolutionaryapproach can reduce water usage and subsequently minimize wastewater prob-lems. The new method can also provide extremely high yields (35–40%)because it solubilizes nearly all myofibrillar and sarcoplasmic proteins (SPs)through homogenization and pH adjustment before centrifugal recovery. Incontrast, the conventional process removes SPs through washing and dewater-ing, which results in significant protein loss and excessive water usage, andcontributes to wastewater problems (Hultin and Kelleher 1999; Hultin

The use of dielectric properties and other physical analyses for assessing protein denaturation in beef biceps femoris muscle during cooking from 5 to 85 °C

Dielectric properties of beef biceps femoris muscle were recorded during heating (5–85 °C) to assess their linkage to phase changes monitored by differential scanning calorimetry (DSC) and rheology. DSC indicated endotherms between 56 and 81 °C corresponding to denaturation of actin, collagen and myosin. Matching changes in dielectric properties (dielectric constant ( ε′) and loss factor ( ε″)) were noted at radio and/or microwave frequencies though the nature of the change differed depending upon frequency. The main observation in ε′ was an increase above 65–66 °C, most likely due to fluid release on collagen denaturation. This fluid plus liquid from myosin denaturation most likely solvated ions freed during myosin denaturation which manifested as an ε″ increase. However, it must be noted that meat structural protein denaturation is compounded with physical shrinkage which can also influence dielectric properties. Rheological parameters of beef muscle heated from 5 to 85 °C also displayed marked changes relating to structural protein denaturation.

Influence of pH on rheological properties of porcine myofibrillar protein during heat induced gelation

Texture of meat products is dependent on the gelation characteristics of myofibrillar protein. Gaining an understanding of the gelation mechanism of meat gel systems is beneficial for the development of processed meat products as well as maintaining quality in meat products. The aim of this study was to investigate the impact of pH (5.6, 6.0, 6.5, and 7.0) on heat-induced gelation properties of myofibrillar proteins from porcine semimembranosus muscle. Dynamic rheological measurements were taken as the temperature increased by 1 °C/min from 20 to 85 °C, followed by a holding phase at 85 °C for 3 min to ensure complete gelation and during a subsequent cooling where the temperature dropped from 85 to 5 °C at a rate of 5 °C/min. Storage modulus ( G′) increased as gel formation occurred, but decreased after reaching the temperature of myosin denaturation (52 °C) until approximately 60 °C when the gel strength increased again. This resulted in a peak and subsequent depression in the data. This depression in the curve was more pronounced with increasing pH. Results indicate protein denaturation and gel formation are pH dependent. Furthermore, rate of gelation appears to influence water-holding capacity.

Changes in rabbit skeletal myosin and its subfragments under high hydrostatic pressure

The pressure-induced denaturation of rabbit skeletal myosin and its subfragments under hydrostatic pressure were investigated. Four nanometer of red shift of the intrinsic fluorescence spectrum was observed in myosin under a pressure of 400 MPa. The ANS fluorescence of myosin increased with elevating pressure. Changes in the intrinsic fluorescence spectra of myosin and its subfragments were quantified and expressed as the center of spectral mass. The center of spectral mass of myosin and its subfragments linearly decreased with elevating pressure, and increased with lowering pressure. The fluorescence intensity of the ANS-labeled rod did not change during pressure treatment. The present results indicate that the most pressure-sensitive portion of myosin molecule is the head. Hysteresis of the center of spectral mass of S1 appeared under pressures above 300 MPa. Changes in the center of spectral mass of S1 above 350 MPa showed stronger hysteresis. The center of spectral mass did not decrease above 350 MPa during the compression process, indicating that S1 was stable in a partially denatured state at 350 MPa under pressure. The changes in the relative intensities of ANS fluorescence of S1 were measured under pressures up to 400 MPa, and the ANS fluorescence intensity increased with elevating pressure but it did not change after pressure release. The ANS fluorescence intensity increased under constant pressure suggesting that the pressure-induced denaturation of myosin was accelerated during pressurization.

Water properties during cooking of pork studied by low-field NMR relaxation: effects of curing and the RN −-gene

Using low-field NMR transverse relaxation measurements, the physico-chemical state of water in meat was followed continuously during cooking of 20 uncured and 20 cured pork samples from RN-carriers ( n=20) and non-carriers ( n=20). The obtained relaxation data were analysed using (i) distributed exponential fitting, and (ii) principal component analysis (PCA). Distributed exponential fitting revealed transition from a system with relatively well-separated components to a less well-defined system with a wide distribution of relaxation times and merged components during cooking. In addition, distributed exponential fitting analysis implied changes in relaxation characteristics that are tentatively ascribed to denaturation of myosin and subsequent shrinkage of the myofibrillar structures. The results are further suggested to reflect formation of new compartments in the myofibrillar lattice during the shrinkage of the existing structures with a succeeding redistribution of water. Nitrite curing was found to affect the distribution of water as well as the progress in the different water populations during cooking. PCA revealed two major shifts in relaxation characteristics around 43 and 56 °C in uncured samples and around 43 and 63 °C in cured samples, and the shift around 43 °C was found to be significantly affected by RN-genotype. The strong shift in water properties around 43 °C is suggested to be a result of myosin denaturation, and thus the results imply differences in myosin denaturation in meat from RN-carriers compared with meat from non-carriers. The second shift in water properties is suggested to reflect the onset of collagen shrinkage causing longitudinal shrinkage of meat, and accordingly the shift from 56 to 63 °C in the presence of salt points towards an effect of curing on structural alterations during cooking.

Effect of storage conditions on differential scanning calorimetry profiles from thawed cod muscle

Differential scanning calorimetry (DSC) was used for measuring thermal denaturation (30–80°C) in cod fillets stored at −20°C and −30°C for up to 12 months and subsequently from 0 to 21 days in modified atmosphere at +2°C. The denaturation profiles were subjected to multivariate data analysis in order to extract the effects of the various storage conditions. The main effect of the storage conditions was the difference in the denaturation profiles between short time chill storage for all samples and long time chill storage for samples stored at −30°C. There was also a smaller effect due to the difference between short time frozen storage and long time frozen storage for samples stored at −20°C. The variation in the thermograms was primarily due to changes in size and position of the myosin, sarcoplasmatic protein and actin denaturation peaks, where variation in position could be related to changes in pH. Frozen storage at both temperatures and chill storage for samples previously stored at −30°C caused a decrease in the myosin denaturation peak. The relationship between the denaturation profiles and the water distribution (determined by low-field NMR relaxation) was found to involve mainly the two fastest relaxing populations (named III and IV), where IV is expected to contain the water most tightly bound to protein. The amount of water in pool III and IV appeared to be directly correlated to the changes in the denaturation profile during storage: the amount of M IV increased and M III decreased when myosin and sarcoplasmatic proteins denatured and the actin denaturation peak shifted to a lower temperature.

Changes in the Properties of Porcine Myosin during Postmortem Aging

Physicochemical and conformational changes, water-holding capacity (WHC) and the structure of heat-induced gel of porcine myosin were investigated to elucidate the relationship between denaturation of myosin and gelation properties during postmortem aging. The turbidity of porcine myosin upon heating increased as the period of postmortem aging increased. During postmortem aging, the increased velocity in values of aliphatic hydrophobicity of porcine myosin by heating was prominent, which suggested that porcine head was susceptible to conformational changes during storage. On the other hand, according to the changes in circular dichroism (CD) spectra of heated porcine myosin, the decrease in α-helix content was almost the same during postmortem aging, indicating no conformational changes in porcine myosin rod during aging. WHC values of porcine myosin gels showed a gradual decrease during storage. This coincided with the progressive loosening in its three dimensional ordering with increased postmortem aging period, as was revealed by SEM studies. In conclusion, conformational changes of myosin head by denaturation during postmortem aging of pork could cause a progressive loosening of heat-induced gel of porcine myosin. This result could be, in part, helpful in the subsequent use of pork in meat processing.

Changes in physico-chemical properties and gel-forming ability of lizardfish ( Saurida tumbil) during post-mortem storage in ice

Changes in physico-chemical properties and gel-forming ability of lizardfish muscle ( Saurida tumbil), stored in ice, were investigated up to 15 days. Heading and eviscerating, prior to iced storage, retarded myosin heavy chain degradation and formaldehyde formation. Additionally, denaturation of myosin and troponin was slightly impeded as monitored by the lower decrease in Ca 2+-ATPase and lower increase in Mg 2+–EGTA-ATPase, respectively. Gel-forming ability of surimi, prepared under different setting and/or heating conditions, decreased as storage time increased ( P<0.05). However, superior breaking force and deformation of surimi gel, from headed/eviscerated fish, to that from whole fish was observed throughout the storage. Whiteness of surimi gel from headed/eviscerated fish was much higher than that from whole fish, especially when the storage time increased. Therefore, storage time and pretreatment were found to be crucial factors, determining the changes in physico-chemical properties and gel-forming ability of lizardfish during iced storage.