Changes in Some Biochemical Indices of Stability of Broiler Chicken Actomyosin at Different Levels of Sodium Bicarbonate in Presence and Absence of Sodium Chloride

Abstract

This study examined some physicochemical properties of actomyosin isolated from pre-rigor chicken breast muscle (Pectoralis major) to gain insight on its interaction with 0.2–1.0M sodium bicarbonate (NaHCO₃) either with or without 3% sodium chloride (NaCl). NaHCO₃ has been a frequent quality raiser with respect to water holding capacity of meat, which is adversely affected at low pH. This study document a remarkable inactivation of ATPases and the weakening of actin-myosin interaction with increasing molarity of NaHCO₃, irrespective of the presence or absence of NaCl. Similarly, there was a concomitant increase in turbidity of soluble fraction, increase in total protein solubility and reactive SH groups. The observations on protein solubility/insolubility are supported by SDS-PAGE profiles. Therefore, the conformational changes induced by NaHCO₃ are concentration dependent and appear different from those during thermal transitions. The findings suggest that low concentration of NaHCO₃ may be more suitable component of marinades, since it affects the main myofibrillar contractile complex less adversely.

Keywords:

• ATPase activity
• Chicken actomyosin
• Sodium bicarbonate
• Sodium chloride
• PAGE profiles

INTRODUCTION

Two of the major concerns of meat producers are consumer acceptability and good yield, which are influenced by a number of post-mortem factors including pH fall, temperature and duration of storage, tenderness or convertibility to processed products. PSE or “pale, soft, and exudative” condition faced by poultry as well as cattle meat is a pH-fall related acute problem.^[1,^] The time lag taken by chilling to reach the interior of muscle results in pH fall that denatures proteins, inflicting drip losses and texture changes.^[2,^3] To improve water holding capacity and modify textural properties of poultry meat, various phosphates and sodium chloride (NaCl) solutions have been frequently used as marinades.^[4−6] Inclusion of sodium bicarbonate in marinades of various other salts has the advantage of shifting pH to alkalinity and thus, helping overcome PSE problem and, in turn, lack of juiciness.^[7,^8] Therefore, sodium bicarbonate has become the salt of preferential use in meat industry during recent years, because at even low concentrations it restores meat pH to desirable levels.^[5,⁶,^9] According to some recent reports sodium bicarbonate has a number of other advantages also; for instance, it reduces shear force and improves water holding capacity, textural properties, and yield of the poultry meat.^[9−11] NaCl, the other salt used in the present study, has not only been reported to increase yield but enhance the effect of phosphates and carbonates which improve meat quality.^[12,^13]

Because of applied value, meat or myofibrillar products have been the objects of more intense investigations for water holding capacities, color change, juiciness, and textural properties.^[14] However, an intimate correlation can be conceived between functionality of isolated actomyosin and characteristics of muscle as food due to quantitative dominance of myosin and actin, which constitute ˜43 and 22% of the protein contents of muscle mass.^[15] It is, therefore, not surprising that most of the fundamental information on functional properties of meat originated from in vitro studies on myosin, its complex with actin (actomyosin) and regulatory and various substructural proteins. Due to decisive roles in defining post-mortem behavior of meat, these proteins have been central in understanding the events during thermal exposure and gelation, frozen storage or processing.^[16−19]

Although the basic response of actomyosin complex has been critical to understanding the functionality of muscle as food, a search of literature revealed no published information on in vitro behavior of actomyosin to widely used phosphates or bicarbonates in marination. The present investigations were, therefore, aimed at investigating some basic aspects of the effect of sodium bicarbonate and NaCl on isolated actomyosin of broiler breast muscles (Pectoralis major) and to review them with the published evidence on related aspects of myofibrillar proteins of poultry meat.

MATERIALS AND METHODS

Chemicals

Acrylamide, Bis-acrylamide, Phenyl methane sulfonylfluoride (PMSF), Adenosine 5′-Triphosphate disodium salt (ATP), Ammonium per sulphate and TEMED were procured from Sigma-Aldrich chemicals Pvt. Ltd. 1-Amino-2-naphthol-4-sulphonic acid, 5-5′-dithiobis (2-nitrobenzoic acid; DTNB), Potassium chloride, Bovine serum albumin, and Tris Buffer were purchased from SRL, India. All other chemicals and reagents used were of analytical grade.

Preparation of Actomyosin

Actomyosin was extracted according to Ahmad and Hasnain^[20] from breast muscle (Pectoralis major) of freshly sacrificed broiler chickens (n = 4), with slight modifications as per the requirement of present experiment. Instantly after excision, breast muscles (10 gm) were blended under cooling by immersing the probe of homogenizer (Make: Biospec) and the pH value of muscle (= 5.9 ± 0.1) was recorded by immersing electrode of digital pH meter (Bio-Matrix India) in the above aqueous homogenate. The pH meter was calibrated for the pH range of 4.0 to 9.1.

For each actomyosin preparation, breast muscle mince was washed three times with five volumes of sodium phosphate buffer (50 mM, pH 7.2) containing 2 mM phenyl methane sulfonylfluoride (PMSF). Addition of protease inhibitor, PMSF, was used to inactivate the proteases present in the solution. Extraction of actomyosin from washed muscle mince was carried out overnight in 0.6M KCl buffered with 20 mM Tris-maleate (pH 7.2). The viscous extract was filtered through gauze to separate out connective tissue. Actomyosin from filtrate was precipitated in ten volumes of chilled distilled water (0–2°C) and collected after centrifugation at 5000 rpm for 20 min at 4°C, using REMI, C-24 BL centrifuge. The actomyosin, dissolved in 0.6M NaCl (20 mM Tris-maleate, pH 7.2), was kept undisturbed for 30 min at 4°C. Dissolved actomyosin was washed twice with 10 volumes of chilled distilled water before finally dissolving in 0.6M NaCl (20 mM Tris-maleate, pH 7.2). After overnight dialysis with Tris-maleate buffer (20 mM, pH 7.2) containing 0.6M NaCl, the actomyosin was ready to use.

Protein Estimation

Protein content in actomyosin samples was determined by Lowry’s method^[21] using bovine serum albumin as the standard. Absorbance (optical density) was recorded at 660 nm on GENESYS 10 UV scanning spectrophotometer.

Treatment with Sodium Bicarbonate

Prior to suspension in sodium bicarbonate, actomyosin dissolved in 0.6M NaCl (Tris-maleate 20mM, pH 7.2) was precipitated with ten volumes of chilled distilled water. After centrifugation at 4000 rpm for 15 min at 4°C, actomyosin pellet was saved and suspended in chilled sodium bicarbonate (NaHCO₃) solution (pH 8.4) of different molarity (0.2M, 0.4M, 0.6M, 0.8M, and 1M) along with or without NaCl (3% v/v). Subsequently, after proper mixing, final concentration of all actomyosin solutions was adjusted to 3.5 mg/mL. Before further analysis, the mixtures were kept undisturbed for 30 min at 4°C to allow samples to reach equilibrium.

Measurement of ATPase Activity

Ca²⁺-ATPase and Mg²⁺-ATPase activities were determined following the method of Hasnain et al.^[22] with slight modifications. Briefly, 2 mL assay mixture at final concentrations contained: 25 mM Tris-HCl buffer (pH 7.5), 5 mM CaCl₂ (for Ca²⁺-ATPase assay)/or 5 mM MgCl₂ (for Mg²⁺-ATPase assay), 1 mM ATP and 0.35 mg/mL proteins. The ionic strength of KCl solution for Mg²⁺-ATPase and Ca²⁺-ATPase was 20 and 30 mM, respectively. The reaction was conducted precisely for 4 min at 20°C and terminated by adding equal volume of trichloro acetic acid (15% w/v). The mixture was centrifuged at 1500 rpm for 10 min and liberated inorganic phosphate (Pi) was determined in supernatants by the method of Fiske and Subarrow.^[23]

Estimation of Sulfhydryl (-SH) Groups

Sulfhydryl content was determined essentially by Ellman’s method^[24] using 5-5′-dithiobis (2-nitrobenzoic acid)/ DTNB with slight modifications. Briefly, 3 mL actomyosin sample was added to the mixture of phosphate buffer (2 mL) and water (5 mL) to make up the final volume 10 mL. From this mixture, 3 mL aliquot was collected and mixed with 20 μL DTNB (39.6 mg DTNB in 10 mL phosphate buffer, pH 7.2). After incubation for 20 min at 40°C, absorbance at 412 nm was recorded on spectrophotometer (GENESYS 10UV Spectrophotometer). Sample blank was prepared by replacing DTNB with water. Sulfhydryl content were calculated according to Ding et al.^[25] by using the extinction co-efficient of 13,600 L/mol.cm. C = A/E × D, where C is molarity of –SH, A is optical density at 412 nm, D is dilution factor, and E is extinction co-efficient 13,600 M⁻¹ cm⁻¹.

Determination of Solubility

For determining solubility, actomyosin samples were centrifuged at 10,000 rpm and 4°C for 20 min. Protein concentration in the supernatants was determined by the Lowry’s method^[21] as referred above (under Protein estimation) using bovine serum albumin as standard. The solubility was calculated as the ratio of protein concentration in supernatants after centrifugation and total protein concentration of actomyosin.^[26]

where, C_s is the protein concentration in the supernatant after centrifugation and C_o is total protein concentration of actomyosin.

Measurement of Turbidity

Turbidity of actomyosin samples were measured by reading absorbance at 320 nm against appropriate blank on GENESYS 10 UV scanning spectrophotometer. Absorbance was plotted against increasing sodium bicarbonate concentration.

Electrophoretic Profiling

Sodium dodecyl sulphate polyacrylamide gel electrophoreses (SDS-PAGE) of different actomyosin samples were carried out according to the protocol described by Laemmli^[27] with a few minor modifications. Resolving and stacking gels were 10% (w/v) and 4% (w/v) prepared from stock solution containing acrylamide: bis-acrylamide = 29.2: 0.8; respectively. However, resolving gels also contained 15% (v/v) glycerol, while at final concentration all the buffers contained 0.1% SDS. Actomyosin samples were prepared with SDS sample buffer containing 5% β-ME and boiled for 4 min. After loading 10 μL of each sample onto the gel slot, electrophoresis was carried out at constant voltage (20 V and 80 V) using mini vertical electrophoresis unit (Genetix, India). Following electrophoresis, gels were washed overnight in a solution containing 40% methanol and 10% acetic acid. Subsequently, the gels were stained with 0.1% (w/v) Coomassie brilliant blue R-250, in 40% (v/v) methanol and 10% (v/v) glacial acetic acid and destained in 7% acetic acid.

Documentation and Densitometric Assessment of SDS-PAGE Profiles

Stained SDS-PAGE gels were documented using SONY CYBERSHOT digital camera (Zoom-4X, 14.1 Megapixels) and by direct scanning on an all-in-one HP Deskjet (F370) computer setup. Best gel documents were used for densitometric analysis. Densitometry of the selected gel-scans was done using Scion Imaging software program (Scion Corporation; Beta release 4.0).

Statistical Analyses

All experiments were done in quadruplets and analytical measurements of each were made in triplicates. We did not find any significant variations between quantitative experiments of the same biochemical parameter (e.g., ATPase activities, relative solubility, and turbidity) while assessing effects of various concentrations of sodium bicarbonate on actomyosin complex. Student’s t-test was employed for statistical comparisons. Values were considered significant at P < 0.05.

RESULTS AND DISCUSSION

This study demonstrates the effect of increasing ionic concentrations of sodium bicarbonate (NaHCO₃) from 0.2–1.0M in the presence or absence of 3% NaCl on biochemical properties described below as actomyosin. Actomyosin was isolated from breast muscle (Pectoralis major) of broiler chicken, with pH 5.9 ± 0.1. On the other hand, pH with various concentrations of bicarbonate was recorded as 8.4.

Changes in Ca²⁺- and Mg²⁺- ATPase Activities

The effect of various concentrations of NaHCO₃ on Ca²⁺-ATPase and Mg²⁺-ATPase activities is shown in Fig. 1. It is quite obvious that, within the molarity range used here, NaHCO₃ inactivates ATPase activities. Profiles of either of the ATPase activities have certain features in common: (1) Ca²⁺- as well as Mg²⁺-ATPase activity continuously decline with the increasing concentration of NaHCO₃. (2) To increasing NaHCO₃ levels, Mg²⁺-ATPase displays more sensitivity than Ca²⁺-ATPase, as the decline is faster from 0.2–0.6M. In comparison, 0.2–0.6M NaHCO₃ minimally affects Ca²⁺-ATPase and a rapid decrease follows beyond 0.6M. (3) The presence of NaCl enhances the magnitude of decrease in both ATPase activities without affecting the trend of the curves. (4) The trend of decrease in Mg²⁺-ATPase displays higher sensitivity to NaCl as compared to Ca²⁺-ATPase activity. The sensitivity of Mg²⁺-ATPase to NaCl is more prominent at lower concentration of NaHCO₃.

FIGURE 1 Changes in relative Mg²⁺ -ATPase and Ca²⁺ -ATPase activity (%) of actomyosin of chicken breast muscle (Pectoralis major) treated with increasing concentration of NaHCO₃ (0.2M, 0.4M, 0.6M, 0.8M, and 1M) with (solid lines) or without (dotted lines) NaCl (3%). Mg²⁺-ATPase and Ca²⁺-ATPase activity of untreated actomyosin taken as control (100%) was 0.98 μmol/mg/min and 1.25 μmol/mg/min), respectively.

As for functional relevance of ATPase activities, Ca²⁺-ATPase is recognized as myosin ATPase, while Mg²⁺-ATPase represents enzymatic activity of actomyosin.^[28−30] ATPase is the function of myosin globular head while solubility is determined by rod portion.^[31,^32] Decline in Ca²⁺-ATPase activity at higher NaHCO₃ concentration may be attributed to the inter- and intra-molecular changes among interacting molecule; specifically myosin and actin. Inhibition of enzyme activity by salts as a consequence of conformational change in myosin head has been previously reported.^[33] In addition, ATPase activities are also influenced by factors such as pH, temperature, type of solutes, salt concentrations, or ions present.^[34,^35] Keeping intimate relationship of Ca²⁺-ATPase data and myosin (Fig. 1) in mind, myosin appears to be more sensitive to NaHCO₃ beyond 0.4M, since at high pH value myosin head can undergo irreversible conformational change.^[36] Removal of myosin light chain-1 (alkali LC1) at alkaline pH, which is essential for enzymatic activity^[37] from actomyosin complex support conformational changes.

Since Mg²⁺-ATPase is actomyosin ATPase activity and an index of actin-myosin interaction, more rapid decline in Mg²⁺-ATPase activity at concentrations below 0.6M NaHCO₃ is due to decreased actin-myosin interaction (dissociation) and not solely due to changes in myosin conformation, where relative solubility may also have a role. The inference is supported by the fact that except substitution of Mg²⁺ for Ca²⁺, all other experimental conditions of ATPase assay were identical. At alkaline pH of NaHCO₃, Ca²⁺- desensitization due to dissociation of regulatory proteins^[38] may also affect magnitude of inactivation of Mg²⁺-ATPase.

The state of myosin and decreased actin-myosin interaction by NaHCO₃ (as indicated by Ca²⁺, Mg²⁺-ATPases) bring in vitro findings on actomyosin close to their status in marinated muscle where NaHCO₃ is a component. More specifically, at elevated ionic strengths actin-myosin interaction is already reported to weaken in pre-rigor muscles.^[39,^40]

Variation in Solubility

Change in solubility with increasing concentration of NaHCO₃ is shown in Fig. 2a. Marked increase was observed in the total proteins solubility (˜57%) in the presence of 3% NaCl when NaHCO₃ was 0.6M, beyond which protein solubility remained almost unchanged up to 1.0M NaHCO₃. However, without NaCl, 0.8M NaHCO₃ was required to reach the maximum protein solubility (˜49%), that is 0.2M more NaHCO₃ than that needed in the presence of NaCl. Therefore, with NaHCO₃ alone, ionic strength compensation is essential, since the ionic strength to achieve highest protein solubility (˜57%) is I = 1.117, that with 3% NaCl is achieved at 0.6M NaHCO₃ (Fig. 2a). In terms of solubility, effect of NaCl on actomyosin solubility is more pronounced between 0.4M (I = 0.917) and 0.8M (I = 1.317) of NaHCO₃. Therefore, most likely reason of solubility variations appears to be the changed surface chemistry of actomyosin constituents at high salt concentrations. It is to reemphasize that myosin is the only high ionic strength soluble component of actomyosin and also the major variable constituent in SDS-PAGE profiles as in obtained SDS-PAGE profiles.

FIGURE 2 Changes in protein solubility (a); and, R-SH contents (b) of chicken breast muscle actomyosin treated with increasing concentration of NaHCO₃ (0.2M, 0.4M, 0.6M, 0.8M, and 1M) with or without 3% NaCl. a = compared to control, b = compared to lower treatment, and c = compared to (with NaCl) of respective/same molarity. *p < 0.05.

As already pointed out, solubility of muscle proteins also depend on their source, freshness, pH of muscle, type of solvent and, the ionic strengths.^[41,^42] NaCl is fairly effective in solubilizing substantial amount of major myofibrillar proteins at 2.5% concentration.^[42−44] For instance, ˜90% actomyosin proteins were solubilized from Pacific white shrimp at 0.2M NaHCO₃.^[45] Our data (Fig. 3) demonstrates that ˜57% of chicken actomyosin can be solubilized with 0.6M NaHCO₃ in the presence of NaCl. Without NaCl, even by the highest molarity of NaHCO₃ (1.0M), solubility of actomyosin was rather low (<52%).

FIGURE 3 (a) Changes in turbidity of supernatants of actomyosin obtained after treatment with various molarities of NaHCO₃ with or without NaCl. (No adjustment of protein concentration to 0.9 mg/ml was made.) (b) Change in turbidity of supernatants of actomyosin treated with NaHCO₃ with or without NaCl. (Protein concentration in all samples were adjusted to 0.9 mg/ml.)

The decreased solubility of myofibrillar proteins has been correlated with pale, soft, and exudative poultry.^[46] Solubility, on the other hand, is related to functional properties of food and is an important quality attribute of poultry meat. According to Bertram and co-workers,^[47] the use of bicarbonate assists in higher solubilization of myofibrillar proteins in marinated muscle. The amount of soluble actomyosin is important for gelation of myosin, which is the principal participant in the event of meat processing. The statement is corroborated by in vitro studies carried out with different combinations of actomyosin:myosin^[48] or with variable ratios of isolated actin:myosin.^[43] At alkaline pH, despite a probable increase in protein solubility,^[49] a number of other factors such as water absorption, decreased actin-myosin interaction and conformational status of proteins also deserve consideration, as they may influence the biochemical events related to meat quality.

Effect on Sulfhydryl Groups

The trend of reactive sulfhydryls (R-SH) of actomyosin exposed due to treatment with different concentrations of NaHCO₃, with or without NaCl, is compared in Fig. 2b. R-SH contents showed significant successive increase up to 0.6M NaHCO₃, although major increase occurred between 0.4M and 0.6M. The trend displayed by concentration dependence of R-SH profile on NaHCO₃ concentration is compatible with solubility profiles of actomyosin from 0.4M to 0.6M of NaHCO₃ (Fig. 2a). Inclusion of 3% NaCl exposed relatively higher number of R-SH; but the exposure effect of NaCl diminished beyond 0.8M NaHCO_3, as no further increase in R-SH occurred whether NaCl is included or not.

Due to greater sensitivity, most of the work on reactive thiols so far has been carried out on fish muscle or myosin with two main objectives: to compare conformational flexibility by demonstrating relative exposure of buried R-SH^[22] or their oxidation during frozen storage or thermal treatments.^[29,^50] The decline in R-SH contents due to disulfide bond formation is widely accepted index of denaturation that has an intimate correlation with the behavior of a specific muscle during storage.^[45,⁴⁷,^51] In the present study, increase in R-SH contents is probably caused by the progressive exposure of R-SH groups as a consequence of changed molecular behavior at high ionic strengths and pH.

The increase in R-SH contents with increasing salt concentration has generally been reported for fish myosin.^[52] In case of chicken breast muscle myosin, a direct correlation has been reported between oxidation of R-SH and aggregation and gelation, and viscoelastic properties of the formed gel.^[53] Since the relative stabilities of muscle proteins (specifically myosin) are major influence of behavior of a muscle, magnitude of R-SH exposure may be lower in chicken.

Variation in Turbidity

Change in turbidity with increasing concentration of NaHCO₃ is shown in Fig. 3. Turbidity of actomyosin is highest at 0.2M NaHCO₃ that gradually decreases with the increasing concentration of bicarbonate salt. Non-significant differences are observed at 0.8M and 1.0M (P > 0.05). The presence of 3% NaCl showed significant decline in turbidity at lower concentration of NaHCO₃. Actomyosin is fairly turbid or opaque protein complex and therefore, decrease in turbidity may indicate dispersion of the actomyosin complex while increase in turbidity indicates aggregation.^[54−56] Our results show that at lower concentration of NaHCO₃, actomyosin complex is comparatively less soluble (Fig. 3a) and thereby, more turbid. At higher NaHCO₃ concentration, actomyosin solubility is fairly high which seems to be the apparent cause of decline in turbidity. The data on turbidity profiles of supernatants demonstrate increase in turbidity with the molarities of NaHCO₃ in presence or absence of NaCl (Fig. 3a). However, adjusting the protein concentrations (0.9 mg/mL) reversed the trend of turbidity profile in supernatants indicating that more protein is solubilized by augmented molarities of NaHCO₃ in presence or absence of NaCl (Fig. 3b).

Electrophoretic Profiling by SDS-PAGE

SDS-PAGE analysis of actomyosin samples treated with various concentrations of NaHCO₃ and 3% NaCl did not reveal any comparative change in profiles (data not shown). All the polypeptides of known molecular weight present in the typical profiles of chicken actomyosin were intact. Therefore, similar to turbidity and solubility comparison, we attempted to fractionate actomyosin into soluble supernatant and insoluble sediment (pellet) by centrifugation. Results obtained on fractionated samples are shown in Fig. 4. Various polypeptides corresponding to approximate molecular weights documented in various previous studies were: (Myosin Heavy Chain (˜200 kD), Actin (˜44 kD), Tropomyosin (˜39 kD), Troponin-T (˜35 kD), Troponin-I (˜23 kD), Troponin-C (˜20 kD), and Myosin Light Chains I, II & III (˜25 kD, 18 kD, and 16 kD).

FIGURE 4 SDS-PAGE (10%) profiles of chicken actomyosin treated with 0.2M, 0.4M, 0.6M, 0.8M, and 1M NaHCO₃ for 30 min. Treated samples of actomyosin were fractionated into supernatant (a) and pellet (b) and the SDS samples prepared were loaded in equal volume (10 μL) in each lane. (a) Supernatant: Lanes 1–5 supernatants of actomyosin samples treated with 0.2M, 0.4M, 0.6M, 0.8M, and 1M NaHCO₃ along with 3% NaCl. MyHC = Myosin Heavy Chain; TM = Tropomyosin; TN = Troponin; MLCI = Myosin Light Chain I; TN-I = Troponin-I; TN-C = Troponin-C; MLC-II = Myosin Light Chain II and MLC-III = Myosin Light Chain-III. (b) Pellet: Lane 1 = Control; Lanes 2–6 pellet of actomyosin samples treated with 0.2M, 0.4M, 0.6M, 0.8M, and 1M NaHCO₃ along with 3% NaCl.

Figure 4a demonstrates that the increasing concentration of NaHCO₃ in the presence of NaCl causes an increase in solubility of myosin heavy chain, actin and the regulatory proteins (tropomyosin and troponin-T, troponin-I and troponin-C). Comparison of changed ratios of these proteins in the soluble (supernatant) and insoluble (residual pellet) fractions are made in Fig. 4a and 4b with the quantitative representation in Fig. 5a and 5b. Quantitation of relative intensities of various bands in SDS-PAGE profiles was made with the help of ScionImage software in terms of integrated density value (IDV). As calculated from IDV, NaHCO₃ concentration-dependent major quantitative change occurred in myosin heavy chain or MyHC (Figs. 4 and 5). According to IDV analyses gradient of changes in actin, regulatory proteins and light chains including the essential LC1 corresponded to continued increase in NaHCO₃ from 0.2M to 1.0M.

FIGURE 5 Densitometry profiles of Myosin Heavy Chain (MyHC) and Actin as stacked in 10% SDS-PAGE profiles of chicken actomyosin treated with 0.2M, 0.4M, 0.6M, 0.8M, and 1M NaHCO₃ with 3% NaCl. Integrated density value as relative IDV (%) of MyHC, actin, and the regulatory proteins along with released essential Myosin Light Chain (LC-1) in various supernatants of treated samples are compared in (a). Similarly, IDV data of pellet is compared in (b). Horizontal axis in either case is NaHCO₃ concentration. For the sake of clarity and to avoid crowding, IDV changes in other light chains are not shown in IDV diagrams.

The results thus show that not only the MyHC dimer but LCs and regulatory proteins also undergo dissolution/dissociation in course of NaHCO₃ induced changes. Extraction and subsequent solubilization of these polypeptides from chicken myofibrils by phosphates, at higher NaCl concentration, are also reported in earlier studies.^[44] However, the most important point is that the extractability and other physicochemical properties as evaluated here remained far below the fraction of actomyosin proteins which could not be solubilized (Fig. 2a). ATPase and electrophoretic data also supports this observation, since more of the proteins go with the pellet (Fig. 3a).

Although the approach applied here provides new information on the behavior of isolated actomyosin, the solvent-system used caused substantial change in physicochemical properties of actomyosin complex. High concentration of sodium bicarbonate may not be a suitable component of marinades, because of adverse effects on functional properties and integrity of actomyosin. The experimental design, however, is useful to compare differently stored muscle or under different state of rigor-mortis, where solubility and other physicochemical characteristics chosen here are matters of concern.

CONCLUSIONS

Properties of isolated myofibrillar proteins are important in adding to our understanding about their meat source. This study revealed that, under our experimental conditions, it is overall increase in salt molarity (NaHCO₃ or NaCl) which is bringing about observed inter- and intra-molecular changes in isolated actomyosin; in particular the myosin molecule. The relevance with myosin is apparent since active site is located in globular head of myosin whereas rod of the molecule determines solubility. Substructural changes, such as dissociation of myosin light chains and regulatory proteins of actomyosin complex also correspond to high molarity of NaHCO₃. Moreover, additive effect of NaCl to overall molar concentration explains the conspicuous changes in physicochemical properties of actomyosin even at the lowest concentration of NaHCO₃. Although improved solubility of myofibrillar proteins is a positive indicator for processed meat products, high sodium levels are an obvious disadvantage.

ACKNOWLEDGMENTS

The authors thank the Chairman, Department of Zoology, Aligarh Muslim University, Aligarh for providing necessary laboratory facilities. Help extended by Ms. Areeba Ahmad is also gratefully acknowledged.

FUNDING

The work was done out of the financial assistance from MRP by the UGC, New Delhi [F. No. 39-581/2010 (SR)] to AH and from UGC-Non-NET Fellowship to RS.

Additional information

Funding

The work was done out of the financial assistance from MRP by the UGC, New Delhi [F. No. 39-581/2010 (SR)] to AH and from UGC-Non-NET Fellowship to RS.