EFFECT OF CHELATENG AGENTS ON POST-MOMENT CHANGES iN MEUSCL Th- {75:53 for the Degree 9? Ph. D. "TENN GATT STATE ENWEESSTY PNMP DAVTD WEENER 1957 as ITTTTTTTTTTTT TTTTI 1 Imam 3112993 01066 746 . Michigan State University This is to certify that the thesis entitled ‘ EFFECT OF CHELATING AGENTS 0N POST-MORTEM CHANGES IN MUSCLE presented by Philip David Weiner has been accepted towards fulfillment of the requirements for Ph.D. Food Science degree in MajO' Date May 26, 1967 0-169 ABSTRACT EFFECT OF CHELATING AGENTS ON POST-MORTEM CHANGES IN MUSCLE by Philip David.Weiner The primary objectives of this study were to determine the effects of various chelating agents and calcium.and.magnesium ions upon the development of rigor mortis and the subsequent quality factors of meat. This study consisted of four experiments using rabbits and.one experiment using pigs. The effect of an intravenous antemortem injection of either EDTA, CDTA, EGTA or Na Oxalate on the shortening and elasticity of the excised semitendinosus muscle from rabbits and.pigs was determined. The effect of micro-injections of Ca012 and MgClg on the shortening and elasticity of paired.semitendinosus muscles from rabbits was also studied (Experiment II and III). ATP degradation, pH changes, water holding capacity, color, cooking losses, shear values and protein fractionation were determined on the longissimus dorsi and semitendinosus muscles. The relationship between ATP levels, pH values, muscle shortening, cooking losses and shear values were also determined (Experiment II and Experi- ment V). In Experiment IV, properties of the fibrillar protein extracts were investigated.by measuring the ATPase activity and changes in turbidity and viscosity. The intravenous injection of EDTA greatly inhibited shortening of the semitendinosus muscle in Experiment I. In Experiment II, the shorten- ing of the semitendinosus muscle was significantly (P<:.OS) inhibited by an intravenous injection of either EDTA, Na Oxalate, EGTA or CDTA. Shortening of the semitendinosus muscle from pigs treated with.EDTA was Philip David weiner significantly less (P<<.OS) than for control pigs (Experiment V). In- creased concentrations of CaClg injected into the muscle resulted in a definite increase in shortening during the development of rigor. However, micro-injections of MgClg had no effect on shortening. No consistent differences in ATP levels at either 0 hr or 2h hrs postdmortem.were observed between the different treatments in either Experiment I, II or III. The ATP values for the pigs treated with EDTA were generally higher than those for controls, but the differences were not statistically significant. A definite increase in the rate of ATP hydrolysis was observed following micro-injection of Ca012 into the semi- tendinosus muscle. Treatment had no consistent effect upon the elasticity or rigidity of the uninjected semitendinosus muscle at 7 hrs postamortem (Experiment II). Muscles which were elastic after 7 hrs post-mortem had significantly higher (P<:.Ol) levels of ATP than those that were rigid and inextensible. The paired semitendinosus muscles that were injected with 0.1M Ca012 were always rigid and inextensible by 7 hrs post-mortem. The pH values of the longissimus dorsi muscle of rabbits treated with EDTA were significantly lower (P<:.OS) than those for the group treated with EGTA and the control group (Experiment II). No other signi- ficant differences in pH values were observed between treatments at eflher O or 2h hrs post—mortems Treatment had no significant effect on either the subjective color score or water holding capacity. No significant differencesin cooking losses were observed between the different treatments in Experiment I, II and V. The shear values Philip David weiner of the longissimus dorsi muscles from the rabbits treated with EDTA (Experiment I and II) were lower than the control groups, but the differ- ences were not significant. The shear values of the hams treated with EDTA.were significantly lower (P<:.OS) than.shear values obtained for control hams. However, the effect of treatment on tenderness varied between muscles. There was no appreciable change in the solubility of the sarcoplasmic protein fraction during the first 2h hrs post-mortem. There was, however, a definite increase in the amount of the fibrillar protein fraction ex- tracted at high ionic strength during the first 2h hrs post-mortems This was accounted for by an increase in the formation of actomyosin. Muscles extracted after 2b hrs post-mortem from rabbits treated with EGTA, CDTA and EDTA.had less actomyosin than the control rabbits, but the differences were not significant. A highly significant relationship (P <30l) was found between ATP and.pH values within 10 min.post-mortem for the muscles from rabbits (Experiment II) and for the muscles from.both the treated and.oontrol pigs (Experiment V). The relationship between calcium release, ATP degradation and rate of glycolysis during the development of rigor mortis was discussed, Higher ATP values at 2h hrs post-mortem in the treated pigs were significantly related (P 0.4 - 0.5) in the presence of either ATP or pyrophosphate and magnesium. He further stated that the actin that is present may be removed by a stepwise reduction in the ionic strength of the solution, thereby precipitating the actin as the acto- myosin complex. In summarizing the general properties of myosin, Szent-Gygrgyi (1951) stated that the dispersion of myosin in solution depends on the pH and the nature and concentration of the salt solution. He described myosin as being readily soluble in strong salt solutions, such as 0.6 M KCl, at pH 6.5 - 7.0. under these conditions, Szent-Gygrgyi (1951) found that myosin showed normal viscosity at a concentration of less than 0.2%. On lowering the salt concentration below 0.3 M KCl, he showed that the viscosity increased to a point of least solubility between 0.025 and 0.050 M KCl. At salt concentrations below 0.001 M KCl, he found that myosin dissolved with a great deal of swelling. Szent-Gygrgyi (1951) further indicated that salt-free solutions of myosin have an isoelectric point of 5.4, but the isoelectric point will change as the salt concen- tration is altered. Szent-Gygrgyi (1951) also pointed out that magnesium has a high affinity for myosin at a concentration of 0.11 - 0.12 M. He further indicated that bound magnesium ions will cause the adsorption of ATP, which results in the negative charge of the myosin molecule. 0n studying the components of myosin, Szent-Gygrgyi (1953) found that digestion of myosin by trypsin for a short time breaks down the molecule into two components. He referred to these components as light -11- (L) meromyosin and heavy (H) meromyosin. He showed that H-meromyosin is responsible for the ATPase activity of myosin and that it possesses the capacity of combining with actin of the intact myosin molecule. Mere recently, Huxley (1963) was able to reconstitute filaments from purified myosin, which are very similar in appearance to the thick filaments pre- sent in muscle. His results also showed that the myosin filaments are structurally polarized and possess a center of symmetry with all heads of the molecule facing away from the center. Szent-Gygrgyi (1966) stated that L-meromyosin makes up the shaft of the myosin filament, while the cross bridges are a part of the H-meromyosin. Huxley (1960) reported that there are over two hundred cross bridges extending from each thick filament. Perry and Catterill (1965) prOposed a scheme with two separate centers on each cross bridge of the myosin molecule. They stated that one of the centers on the cross bridge is involved in enzymatic activity, while the other is involved in formation of actomyosin. Levy and Ryan (1966) described a model of the myosin molecule that required both magnesium and ATP at two different sites on each cross bridge during muscle contraction. They showed that magnesium is bound very tightly at the hydrolytic site, while it is bound less firmly at the other site. Martonosi and Meyer (1964) suggested that magnesium participates in the binding of pyrophoSphate. These workers showed that the relative effectiveness of various chelating agents to inhibit the binding of pyro- phosphate to myosin increased parallel with an increase in the stability -12.. constant of the magnesium complex. They pointed out that an interesting case was that of EGTA, which was much less effective than EDTA in inhibit- ing the binding of perphosphate to myosin. They stated that EGTA forms a weak chelate with magnesium, whereas, the stability constant of its calcium chelate is equal to that of the EDTA-calcium complex. They inter- preted these results to mean that removal of magnesium from the binding site with EDTA inhibited the binding of pyroPhOSphate to myosin. From these and other results, they concluded that the binding site for pyro- phosphate on myosin is identical with a portion of the ATPase center of myosin -- presumably the site of the interaction with the phOSphate chelate of ATP. The feature of overwhelming importance in the myosin molecule is, of course, its enzyme activity as an adenosine triphosphatase. Mbmmaerts and Green (1954) showed that myosin.ATPase has a true Optimum at pH 6.4 and a minimum at about pH 7.3. The activity increased at still higher pH values, and another apparent optimum occurred at pH 9 or higher. The enzymatic prOperties are very sensitive to temperature or slight acidifi- cation (Needham, 1960). In a review, Bendall (1964) pointed out that myosin ATPase is acti- vated by calcium ions and inhibited by magnesium ions. Muhlrad at 31. (1964) suggested that magnesium inhibits ATPase activity by the formation of an unfavorable complex. Kielly gt a1. (1956) found that EDTA activates ATP splitting in the presence of monovalent cations. They further indi- cated that under these conditions, the activity is higher than in the -13¢ presence of optimal calcium ion concentrations. Szent-Gygrgyi (1960) suggested that EDTA activation is due to chelation with strongly bound ‘magnesium or calcium. He further pointed out that these ions may be involved in the binding of the nucleotide base or in its fixation in an orientation favorable to the hydrolytic process. In a review, Needham (1960) stated that there is an important differ- ence between the ATPases of actomyosin and myosin. He pointed out that actomyosin ATPase is activated by magnesium while myosin ATPase is not. He further indicated that both the ATPase of actomyosin and myosin are activated by calcium. According to Mommaerts and Green (1954), the enzymatic activity of isolated myosin ATPase was insufficient to explain the physiological utilization of ATP. They pointed out that the calcium concentration, for Optimal ATPase activity (0.04 M), never occurs in muscle. In addi- tion, they stated that the high concentration of magnesium present in muscle would result in inhibition of myosin ATPase activity. Actin Actin was first isolated by Straub (1942) from the residue left after the partial extraction of myosin from muscle. The residue was dried with acetone to denature all of the remaining proteins, except actin, and to remove the lipids. The actin was then extracted from the remaining material with distilled water. In a review of the general prOperties of actin, Bendall (1964) stated that actin extracted in this manner is present in the globular or G-form and has the appearance of -14- separate beads. He further pointed out that G-actin contains ATP, which is readily exchanged with the ATP in the medium. He then indicated that if actin is to remain in its monomeric (G-actin) form, some ATP must be present in the medium to exchange with the bound ATP. Bendall (1964) also stated that the G-form possesses the remarkable property of polymerization on the addition of neutral salts, resulting in the formation of chain-like molecules of fibrous or F-actin. He stated that there was also a simultaneous change of the bound ATP to bound ADP with liberation of a molecule of inorganic phosphate. Thus, he pointed out that G-actin is a complex of ATP with actin, while F-actin is a com- plex of ADP and actin. Purified actin (free of myosin) does not possess any enzymatic properties (Needham, 1960). In summarizing the solubility properties of actin, Szent-Gygrgyi (1960) pointed out that even though G-actin is soluble in water or low concentrations of neutral salt solutions, these solutions do not extract actin readily from muscle. He further stated that circumstantial evidence indicates that the structure of the chain-like molecule of F-actin is the same as the biologically active actin filaments. Szent-Gygrgyi (1960) thus suggested that the presence of this fibrous form of actin in muscle may explain the difficulties encountered in extraction. However, he pointed out that agents which depolymerize actin, like potassium iodide, extract actin readily. Szent-Gygrgyi (1951) stated that actin, as it commonly occurs, is a calcium actinate. Even in carefully washed preparations, actin always contains about four atoms of calcium per molecule. Thus, there is one firmly bound calcium molecule for every 18,000 g of actin. -15- Tr0pomyosin TrOpomyosin is also present in small amounts in mammalian muscle (Bendall, 1964). This protein resembles myosin in its chemical consti- tution, but differs markedly in most of its prOperties (Bailey, 1948; Tsao 25 El!) 1951). Huxley (1963) has recently demonstrated by electron microsc0py that a crystal lattice very similar to that of trapomyosin exists in very thin cross sections at the level of the Z line. In a review of muscle proteins, Bendall (1964) pointed out that tropomyosin is a highly charged protein containing 240 acidic and 150 basic groups per 100,000 g. This compares with about 160 and 140 residues respectively, for myosin and 120 and 110 for actin. Bendall (1964) also stated that trOpomyosin apparently does not possess any enzymatic pro- perties and does not combine with either actin or myosin in solution. Stroma Proteins The stroma proteins are retained in the residue after prolonged extraction of a well homogenized muscle preparation with strong salt solutions (Szent-Gygrgyi, 1960). Szent-Gygrgyi (1960) pointed out that this protein contains some material of a collagenous nature contributing to the structure of the sarcolemma and possibly to the Z membrane. -15- Interaction of Actin and Myosin In a review on the interaction of actin and myosin, Bendall (1964) stated that when solutions of F-actin and myosin are mixed at an ionic strength of about 0.6, the viscosity of the solution rises above that of the sum of the two components. He further indicated that the change in viscosity is accompanied by a strong increase in light scattering and an increase in molecular weight. The complex formed is referred to as actomyosin. Bendall (1964) pointed out that the addition of ATP or pyro- phosphate and magnesium to a solution of actomyosin at a high ionic strength results in the dissociation of actomyosin into its component parts, actin and myosin. He stated that the dissociation into actin and myosin results in an immediate fall in viscosity back to the sum of the viscosities of the two components, and also a return of the light scattering intensity to the low value characteristic of these components. The addition of F-actin to myosin also modifies the enzymatic pro- perties of myosin so that it becomes strongly activated by magnesium, but is no longer activated by EDTA (Bendall, 1964). Extensive work (watanabe and Sleator, 1957; Maruyama, 1962; Weber and Herz, 1962, 1963; watanabe gtflgl., 1964) has been carried out with various actomyosin systems in an attempt to more fully understand the mechanism of the various components involved in muscle contraction. watanabe and Sleator (1957) found that glycerinated muscle fibers shortened by about half their initial length on being treated with 4 mM ATP and magnesium. Upon addition of EDTA, ATP and magnesium, the shortened fibers relaxed rapidly to approximately their initial length. These -17.. workers pointed out that EDTA manifests its relaxing effect only in the presence of magnesium (1 mM or above) and ATP (2 mM or more). These authors further showed that the addition of calcium ions at a level less than 2 mM completely reversed the relaxing effect of 4'mM of EDTA. The shortening effect of calcium required the presence of magnesium ions. From these results, watanabe and Sleator (1957) concluded that calcium ‘must act at a different site on the protein than magnesium in producing contraction. Ebashi £5 a1. (1960) and Bendall (1964) have reported similar results with glycerinated muscle fibers. Bendall (1953) showed that the addition of 20 mM of perphosphate resulted in slow lengthening of a glycerinated, previously shortened, ‘muscle bundle. The addition of 4 mMIMgClz resulted in a five-fold in- crease in length. Ebashi 25 31. (1960) found that the lengthening of glycerinated muscle fibers by inorganic polyorthOphosphate was markedly accelerated by EDTA and its analogues. In another study using glycerol- extracted rabbit psoas muscle, Bowen and Martin (1958) found that the tension which develops in isometric contraction and the shortening which occurs in isotonic contraction increased if the concentration of MgClz was increased to 10"2 M. They showed that the ATPase activity of the fiber-bundles was not affected as the concentration of magnesium changed. Mbre recently, watanabe at 31. (1964) observed that magnesium had a triphasic effect on the ATP-induced tension of glycerinated fibers from rabbit psoas muscle. The first phase of the magnesium effect, the in- crease in tension with increasing concentrations of magnesium, was abol- ished by the metal chelators, DCTA and EDTA, but not by EGTA. They -18- pointed out that DCTA was much more effective than EDTA in abolishing the first phase. watanabe SE 31. (1964) stated that the second phase of the magnesium effect consisted of a decrease in tension with increasing concentrations of magnesium between 25 uM and approximately 1 mM. They showed, however, that when the concentration of ATP was decreased from 5 mM to about 0.5 mM, the effect of magnesium on the second phase was reduced. watanabe.g£”a1. (1964) found that the third phase, the increase in tension with increasing concentrations of magnesium above 1 mM, was also reduced by metal chelators. They pointed out, however, that EGTA gave the greatest reduction of tension and DCTA the least. From these obser- vations, these workers suggested that the first two phases are due to magnesium (probably in the form of a Mg-ATP complex) and that the third phase reflects the influence of a small amount of calcium. Filo at E1. (1965) found that the concentration of free calcium for contraction of glycerinated striated muscle fibers was 1.8 x 10"7 M. Maximum tension develOped when the concentration was slightly greater than 10"6 M. The inhibition of contraction by magnesium concentrations greater than 0.1 M was completely reversed by the addition of 10'5 M free calcium. Martonosi and Feretos (1964) attributed the relaxing effect of chelating agents and of relaxing factor extract to their ability to lower the calcium concentration of the test system below 10"6 M in the presence of 5 mM ATP and 5 mM MgClz. Using myofibrils, Perry and Grey (1956) found that EDTA strongly inhibited the magnesium-activated myofibrillar ATPase activity. They -19- showed, however, that the calcium activation of myofibrillar ATPase was much less sensitive to EDTA. More recent work by Weber and Herz (1963) showed that the addition of magnesium ions and EGTA to myofibrils re- duced the amount of bound calcium. At an ionic strength of 0.1 M; these same workers found that a highly concentrated solution of myofibrils containing a considerable amount of bound calcium was superprecipitated, even if calcium had not been added. This shows that sufficient contamin- ating calcium was present to saturate the actomyosin of the myofibrils. When 75% of the bound calcium from the same preparation of myofibrils was removed by washing with EGTA and magnesium, no superprecipitation was observed. Hasselbach (1957) and Kaldor and Gitlin (1964) have reported that magnesium ions inhibit myofibrillar ATPase activity, if the ionic strength of the solution exceeds 0.3 M KCl. In solutions of low ionic strength, however, magnesium ions activated myofibrillar ATPase. Using DCTA and EGTA to control the concentrationsof free magnesium and calcium, Barron.gtua1. (1966) studied the effect of adding actin to myosin on ATPase activity at 0.12 M KCl. At free magnesium concentrations of 2.8 x 10"9 M, they found that actomyosin.ATPase activity was very low. AS the free magnesium concentration was increased, the actomyosin ATPase activity reached a maximum at 2.8 x 10"6 M; Additional magnesium above this level decreased the enzyme activity. Perry and Cotterill (1965) suggested that modifications in the enzymatic properties of myosin by actin is of physiological significance. -20- Working with actomyosin at low ionic strengths (0.06 - 0.12 M), Weber and Herz (1962) and Yasui and Wetanabe (1964) showed that low con- centrations of magnesium (< 20 uM in the presence of 1 mM ATP) markedly increased the rate of superprecipitation. However, high concentrations of magnesium (in presence of 1 mM ATP) decreased both the speed and ex- tent of superprecipitation. Earlier results of Tonomura and Yashimura (1960) indicated that a Mg-ATP complex inhibited the superprecipitation of actomyosin by binding to a Specific site. As a consequence, ATPase activity diminished. Weber and Herz (1962) speculated that the inhibit- ory effect of magnesium on superprecipitation may be due to the bound Mg-ATP complex, since the inhibiting effect of high concentrations of magnesium is incomplete at low ATP concentrations. Mommaerts and Hanson (1956) found that the addition of ATP to 0.6 M KCl actomyosin solution resulted in the dissociation of the complex into its component proteins. They estimated that 1 mole of ATP would dissociate one mole of actomyosin. These workers also showed that pyroPhOSphate, inorganic triphosphate in the presence of magnesium and a number of other triphOSphorylated nucleotides have dissociating pr0perties. Noda and Maruyama (1958) stated that the best explanation of the change in flow birefringence prOperties on adding ATP (0.6 M KCl) to an actomyosin solution seemed to be the dissociation of the actomyosin into its component parts, actin and myosin. These workers also showed that at concentrations higher than 11M, KCl alone had dissociating effects on actomyosin. Maximal dissociation occurred at 1.5 M KCl. When KOH or KI were added to solutions of actomyosin, the effect on flow birefringence -21- was much more pronounced than with ATP (Noda and Maruyama, 1958). Marsh (1952) extracted rabbit muscle with 0.16 M KCl. Upon the addition of ATP, he noted an immediate increase in volume of the shrunken system, followed by superprecipitation. In the presence of 0.02% CaClz, the addition of ATP was followed by an immediate superprecipitation. 0n adding a water soluble protein factor from the supernatant to the ex- tracted rabbit muscle, the addition of ATP resulted in an increase in volume of the shrunken system without any subsequent superprecipitation. However, the addition of CaClz to this system resulted in superprecipita- tion. This factor has since been referred to as the relaxing factor in muscle contraction. Hasselbach (1957) found that at a low ionic strength ATP caused dissociation of actomyosin in the presence of EDTA. In more recent work, Maruyama (1962) observed the clearing response of actomyosin solutions (u = 0.06 - 0.08, pH 7.0) if various chelating agents or muscle granules were present prior to the addition of ATP. In the absence of chelating agents, superprecipitation occurred immediately. He suggested that the mechanism.of action of chelating agents appears to be the removal of trace amounts of calcium. To prove this, Maruyama (1962) showed that EDTA was not bound to the actomyosin. He also demonstrated that actomyo- sin in a calcium-free media showed a clearing response and low ATPase activity after.ATP was added. The addition of a small amount of calcium resulted in superprecipitation with high ATPase activity. Similar results and conclusions have been reported by Bargny and Jaisle (1960) and Kaldor and Gitlin (1964) using EDTA or the natural relaxing factor. -22- Maruyama and Gergely (1962) presented data showing that the clearing of actomyosin (u = 0.1 - 0.15) on the addition of ATP in the presence of magnesium is due to its dissociation into the constituent proteins, F- actin and myosin. In interpreting the significance of the double re- fraction of flow, light scattering and viscosity data in terms of dissociation, these workers assumed that there is no interaction between dissociated myosin and actin particles. Using an electron microsc0pe, Ikmmoto 25 a1. (1966) showed that onciearing a system of actomyosin (0.1 MD'with ATP, the actin and myosin filaments are clearly distinguishable and show little or no association with each other. They pointed out that the separate existence of structures corresponding to actin and myosin, confirmed earlier conclusions that were deduced in a more indirect way regarding the dissociation ofactomyosin during clearing. Maruyama and Gergely (1962) explained the changes occurring in the ATPase activity of actomyosin during clearing and superprecipitation. They suggested that in the clear phase, actomyosin exists in a dissociated form. They pointed out that the ATPase activity of the dissociated form in the presence of magnesium is very low because it corresponds to that of myosin. As the ATP concentration decreases, the association of myosin and actin begins to take place again and the ATPase becomes activated by magnesium. A number of investigators (Noda and Maruyama, 1958; Watanabe and Duke, 1960; Tonomura and Sekiza, 1961) have shown that pyrophOSphate in the presence of magnesium is as effective in clearing actomyosin as ATP. Noda and Maruyama (1958) pointed out that the failure of some workers to -23- observe the effect of perphosphate may have been due to the absence of ‘magnesium in the system. The results of Granicher-Frick (1965) indicate that the dissociating influence of the perphosphate system is conditioned by the Mg-perphOSphate complex, while the Ca-pyrophosphate complex, free pyr0phosphate ions, magnesium ions and calcium ions have no disso- ciating effect on the fibers. Muscle Contraction Hanson and Huxley (1955) prOposed a sliding filament theory of muscle contraction. This theory states that muscle contracts without an over-all permanent change in the length of the constituent molecules. The theory was based on studies with the light microsc0pe. Observations clearly indicated that shortening is brought about by a sliding movement of the actin filaments into the arrays of the myosin filaments in the A bands. The extension of the muscle reverses this movement. In a review on muscle contraction, Huxley (1960) stated that it is unlikely that ‘muscle would contract by one process over one part of its range and by a different process over another. He thus concluded that the sliding process is the fundamental mechanism over the range of shortening, and that any folding up of actin or myosin filaments is probably the result of shortening rather than the cause. From X-ray diffraction patterns of the sartorius muscle of the toad, Elliott 55 a1. (1965) concluded that there is no overall change in the 'molecular arrangement within the actin or myosin filaments during con- traction. -24- In a review of the muscle cell, Huxley (1960) pointed out that the results from early investigations with myosin, actomyosin, and glycerinated- muscle fibers suggested that ATP is intimately associated with contraction. This widely held theory has recently been confirmed by the direct demon- stration of ATP breakdown as a result of work during single contractions of isolated intact muscle (Cain and Davies, 1962). The results of Cain and Davies (1962) also make it clear that in working muscle, creatine phOSphoryltransferase can make the energy stored in GP available. Myo- kinase also Operates in the system and can make twice as much energy available from.ATP, while preventing large increases in ADP. Huxley (1960) pointed out that in living muscle at rest, the presence of ATP keeps the actin and myosin complex dissociated. He further indi- cated that the cross links of the myosin filaments are not attached to the actin filaments. Therefore, the two sets of filaments can slide past each other easily, and the muscle is readily extensible. In studying the role of calcium in muscle contraction, Niedergerke (1955) found that calcium injected into the sarcoplasm.of a muscle fiber caused local contraction at the point of injection, but did not set up a wave of activity across the fibers. Protzell gt 3;. (1964) used calcium buffers to stabilize the concentration of calcium in the solutions which they injected into the muscle fibers. Using EGTA as the buffer, these workers showed thatthreshold contraction effects were activated by a very low concentration of calcium (0.3 to 1.5 UM). The shortening re- sulting from injection of various solutions into single muscle fibers from the leg muscle of the crab was. measured by Caldwell and Walster -25- (1963). Their results showed that contraction was produced on injecting either CaClz, SrClZ, B3312 or caffeine, and that contraction was only slight or absent after the injection of distilled water, MgClz, KCl, NaCI, ATP, AMP, EDTA or potassium phosphate. Frank (1960) found that potassium-induced contraction of a muscle was reduced and eventually eliminated, if the muscle was kept in a calcium- free solution for various periods of time before testing. The Speed of inhibition was increased by adding small quantities of EDTA to the cal- cium-free solution. Edman and Grieve (1964) showed similar results with individual muscle fibers immersed in calcium-free Ringer's Solution and excited with a single current pulse. Goodwall and Szent-Gygrgyi (1953) found that the contraction and relaxation of glycerinated muscle fibers in a solution containing CP and the relaxing factor could be brought about by increasing or decreasing pH. Szent-Gygrgyi (1963) described muscle activity as the summation of the reactions taking place at the cross bridges, and indicated that each cross bridge was the site of a unit of activity. He stated that the cross bridge is the site of actomyosin formation and ATP hydrolysis during contraction. A more detailed molecular theory of muscle contraction has been pro- posed by Davies (1966). This model is based on the present knowledge of muscle structure and the properties of the various muscle proteins and their interactions with ATP, calcium, and magnesium. The theory is based on the assumption that ATP is bound to the end of a mobile polypeptide, which is part of the Hdmeromyosin cross bridge. A fixed negative charge -26- inside the cross indge repels the extra negative charge of the ionized ATP, resulting in extension of the polypeptide chain during the resting state. When the muscle is activated, depolarization of the sarcolemma and the sarc0p1asmic reticulum liberates enough calcium to form chelate links between actin and myosin. These linkages cause the neutralization of the negative charge on the extended Hemeromyosin, and thus the extended polypeptide in the cross bridge contracts, resulting in the development of tension and shortening. This process is repeated cyclically during muscle contraction. When the muscle is no longer depolarized, the cal- cium in the sarcoplasm is removed by the relaxing factor system and relaxation occurs. Results of Infante EE;El: (1964) revealed that ATP was not the direct final energy source for muscle contraction. These workers suggested that the energy from ATP is used to extend the polypeptide in the cross bridges and that the actual energy to do work during muscle contraction comes from the formation of hydrogen and hydrophobic bonds as the extended polypeptide contracts. Perry and Cotterill (1965) pointed out that the special features of the magnesium-activated ATPase of the natural actomyosin system enables the hydrolysis of ATP during the contraction-relaxation cycle to be regu- lated by the release and withdrawal of minute amounts of calcium by the sarcoplasmic reticular system. Levy and Ryan (1966) prOposed a model of muscular contraction indi- cating how the reaction of ATP at two sites on each cross bridge on myosin could cause actin and myosin to slide past each other. -27.. Rigor Mbrtis Physical changes After death, muscle changes from the highly extensible plastic condition of freshly excised muscle to the inextensible and rigid condi- tion observed in full rigor (Huxley, 1960). From a histological study of muscle in rigor, Bendall (1951) postulated that rigor mortis is essentially the same process as physiological contraction. He showed that the cross-striations of the fibers in rigor are packed at least twice as tightly as those in pre-rigor. ‘Wbrk by Marsh (1954) also tended to support the view that shortening in rigor may be regarded as a slow and irreversible physiological contraction. More recent results by a number of workers (Locker, 1959; Mauriello and Sandow, 1959; Partmann, 1963) also indicates that rigor mortis and muscular contraction deve10p through the same mechanism. Nauss and Davies (1966) pointed out that during normal contraction, the contractile components deve10p tension on stumulation. However, in rigor mortis contraction and tension mainten- ance are unusual, since the muscle contracts in the absence of external stbmulation and can bear a load in the absence of ATP. In his studies with rabbit muscle, Erdos (1943) found that full development of rigor was coincident with the complete disappearance of ATP. The results of a number of researchers (Bate-Smith and Bendall, 1947, 1949; Bendall, 1951; Lawrie, 1953) have confirmed the relationship between the onset and deve10pment of rigor mortis and the breakdown of ATP. Huxley (1960) pointed out that it is the loss of ATP that leads to the formation of fixed links between the actin and myosin filaments. The physical changes which occur during rigor have been explained by -23- Nauss and Davies (1966). They indicated that rigor is a transformation from a system.af actin and myosin filaments, which slide freely along each other, to a rigid system consisting of actomyosin joined together by cross bridges between the actin and heavy meromyosin. The cross links, which are able to break and reform during shortening, can not be broken in the absence of ATP. This rigid system maintains continuous tension in the absence of ATP breakdown. Huxley (1960) pointed out that a muscle may contract slightly as it passes into rigor, but shortening can be prevented by a relatively small force. A number of devices are available for measuring the time course of rigor mortis by following the changes in extensibility of muscle strips under controlled conditions (Bate-Smith and Bendall, 1949; Briskey gtfl§1., 1962; Partmann, 1963). Marsh (1954) and Bendall (1960) have shown that a short-time course of rigor is associated with greater contraction than a long-time course of rigor. In a study of the striation patterns of ox muscle during rigor mortis, Locker (1959) established a relationship between the striation patterns and the sarcomere length during shortening. Sink gtflal. (1965) found that muscles having a long delay phase during rigor mortis have longer sarcomeres than muscles that have a short delay phase. Consequently, these workers stated that the amount of sarcomere shortening or contrac- tion is highly dependent upon the course of rigor mortis. Herring 25 a1. (1964) showed that sarcomere length varies between muscles and can be affected by pre-rigor excision. Their results showed that sarcomere length can be markedly increased in stretched muscles and markedly decreased by thaw-rigor contracture. -29- Locker (1960) found that contraction of muscle progressively reduces tenderness. He concluded that the state of contraction is a significant factor in tenderness, where the effect of connective tissue is small. This has been confirmed by Locker and Hagyard (1963) while studying the cold shortening effect in beef muscle. Herring 25 al. (1965a, 1965b) have also shown that muscles, which shorten during rigor, have a corresponding decrease in sarcomere length, an increase in fiber diameter and a decrease in tenderness. Marsh and Leet (1966a, 1966b) reported an interesting relationship between tenderness and cold shortening in beef. These workers showed that a decrease of up to 20% of the initial excised length did not exert a significant effect on tenderness, but toughness increased rapidly with further shortening beyond this point. It reached a maximum shear reading of several times the initial value at about 60% of its original length. With still further shortening, the meat became progressively more tender, until at about 40-45% of its resting length, it was cleaved as easily as the non-contracted muscle. Ramsey and Street (1940) and Gordon gtflgl. (1964) have shown that the maximum isometric tension can be controlled by the length of the muscle. They point out that when muscle is stretched so that no overlap occurs, tension can not be develOped. When the muscle is contracted to the extent that the Z-line limits further shortening, then tension can not occur. These workers stressed that maximum tension deve10pment depends on the amount of overlap of the filaments in the sarcomere. -30- Chemical changes The main chemical changes in muscle after death include the product- ion of lactic acid by anaerobic glycolysis, the breakdown of CP and finally the breakdown of ATP (Bate-Smith and Bendall, 1956). Lawrie (1953) indicated that immediately after death, there is a rapid drop in GP, a slow decline and then a rapid decrease in ATP, which is accompanied by a parallel decline in pH. With few exceptions, data published since that time indicate that the change in extension of the muscle is not complete until the pH is at or near its ultimate value, and the ATP con- tent is reduced to a low level. Bendall and Davey (1957) reported that at 17°C, the loss of extensi- bility set in when about 3/4 of the ATP was broken down. At 37°C, however, development of inextensibility occurred when about 1/2 of the ATP was borken down. They showed that the change in extensibility occurred when the ATP level at 17°C and 37°C was 2 and 4 uM ATP per g of muscle, re- spectively. They pointed out that most of the remaining ATP is broken down as rigor is completed, but occasionally as much as 0.7 uM ATP/g may remain at room temperature for 10 to 12 additional hours. Using paper innoPhoresis, Fredholm (1963) reported 17% of the initial ATP in beef 'muscle was still present after 3-5 days of storage under refrigeration. The results of Newbold (1966), using an enzymatic method for ATP, indicated that about 0.2 uM of ATP/g of beef muscle was still present after reaching the ultimate pH. Nauss and Davies (1966) induced rigor mortis in frog sartorius muscle by incubating the isolated muscle in 2-4-dinitrof1uorobenzene (DFB) in Ringer's solution. They showed that as the muscle began to shorten, the rate of 45Ca efflux increased, and that the ATP level fell from 3.8 -31- to 0.0 uM/g. These results contradict Davies’(l963) theory of muscle contraction, which suggests that ATP is present in the actomyosin complex formed during contraction. Nauss and Davies (1966) suggested that the increased rate of calcium efflux probably resulted from the leakage of calcium from the sarc0p1asmic reticulum as a consequence of prolonged exposure to DFB. 0n the basis of their work, Bargny and Jaisle (1960) concluded that contraction is only produced when the mechanism of ATP splitting is based on the interaction between actin and myosin. Nauss and Davies (1966) postulated that the important chemical event determining the onset of the physical changes in a muscle passing into rigor seems to be the internal liberation of a sufficient amount of calcium to initiate the interaction between actin and myosin. Thus, the need for external stimulation would be bypassed. The onset of shortening in rigor mortis following the release of calcium into the sarcoplasm can be accounted for by the cyclic formation and breakage of cross links between actin and myosin, which is accompanied by the enzymatic hydrolysis of ATP due to actomyosin ATPase (Nauss and Davies, 1966). In addition, further breakdown of ATP occurs once the calcium is released into the sarc0p1asm.as a result of the ATPase activity of the sarc0p1asmic reticulum. This process is activated continuously during the deve10pment of rigor until the level of ATP is decreased. Newbold (1966) and Marsh (1966) also suggested that the release of cal- cium by the sarc0p1asmic reticulum can result in ATPase activity and sub- sequent rigor development in muscle. -32- A somewhat different interpretation of the relationship of some of the changes in rigor has been suggested by Newbold (1966). Results from his work showed that post-mortem stiffening, as measured by changes in extension, reached completion in beef sternomandibularis muscle at 1°C, while there was still an appreciable amount of ATP. He further stressed that there is a close coincidence between the completion of the change in inextensibility of muscle and the disappearance of CP. He found that this coincidence is closer than that between the completion of the physical changes and the reduction of ATP to a low level. Similar results on the longissimus dorsi muscle of the pig were obtained by Lawrie (1960). This is especially interesting in light of the results of Brown st 31. (1963) and Lee (1965), who showed that CP was necessary in order for the relaxing factor to take up calcium. The formation of lactic acid from glycogen through glycolysis plays an important part in determining muscle pH and color. The rate of post- mortem.glycolysis and its relationship to the ultimate prOperties of porcine muscle have recently been covered in a review by Briskey (1964). More recent work by Kastenschmidt gt El! (1965) showed that a sudden ante- ‘mortem.temperature change from a warm to cold environment altered the post-mortem.g1ycolytic rate and associated properties of porcine muscle. It also improved meat quality. Bodwell 25 El- (1966) found that holding pork carcasses at 37°C immediately postwmortem did not consistently pro- duce soft, watery and pale muscle. From results with pigs pre-treated with curare, Bendall (1966) postulated that the extreme variability in the rate of pH decline is due to the variable intensity of the various stimuli reaching the muscles. -33- In studies with poultry, DeFremery and Pool (1963) and DeFremery (1966) found that postdmortem glycolysis was minimized by epinephrine injections, sodium iodoacetate injections or rapid cooking. The treated meat was tender without aging. DeFremery (1966) concluded that acceleration of post-martem.glycolysis induces toughness in poultry. The pH of the longiss- imus dorsi muscle from rabbits (Paul, 1964) showed no relation to the amount of struggle post-mortem, Effect of Various Treatments on Muscle Characteristics According to Howard and Lawrie (1956), preslaughter injection of beef with sufficient magnesium sulfate markedly prolonged the delay phase of rigor mortis. Magnesium sulfate injection resulted in a decreased rate of pH fall and breakdown of ATP, but the ultimate pH was not affected. The preslaughter injection of beef with calcium shortened the delay phase of rigor mortis considerably. This treatment resulted in a lower initial pH and reduced the levels of glycogen, ATP and CP. Kamstra and Saffle (1959) attempted to evaluate the extent to which rigor contributes to toughness of meat. The normal sequence of reactions during rigor was interrupted by the infusion of sodium.hexametaphosphate into pre-rigor hams. They showed that the hams injected with sodium hexametaphosphate were more tender (P'< .01) than control hams injected with an equal volume of water. The treated hams also had higher pH values. Carpenter gt El- (1961) tried injecting rabbit legs with chelating compounds, but difficulty was encountered in obtaining an increase of 5% over the initial weight because of the massive tetany occurring upon -34- infusion. After failing in their study with rabbit muscle, the same workers conducted a study to determine the effect of an infusion of sodium hexametaphosphate solution into hot beef rounds. Tenderness, as recorded by taste panel and shear, was improved by pre-rigor infusions of 10, 15 and 20% solutions of sodium hexametaphoSphate and a 15% solution plus lactic acid (pH 5.8). Extractable nitrogen was not a reliable in- dication of tenderness in this experiment. These workers also showed that the small differences between the pH values and the glycogen levels for the treated and control samples after 48 hours indicated that infusion did not interrupt glycolysis. In a study of the ionization of some phOSphates used in food products, Batra (1965) found that sodium orthOphosphate completely dissociated in solution and that the degree of dissociation of the polyphOSphates was inversely proportional to the number of phosphate atoms in the chain or ring. The addition of calcium enhanced the dissociation of the polyphos- phate and lowered the pH in each case. Results showed that the calcium- complexing ability of sodium hexametaphosphate was the highest while that of orthOphosphate was nil. Mere recent work by weiner gt El: (1966) with hams removed from the carcass within 1 hr postwmortem and pumped with cold brine, showed lower total cooking losses and lower drip losses for treated than for control hams. iMuscles from treated hams also had significantly lower shear values than the controls. In a similar study, Mandigo and Henrickson (1966) found that hams pumped, boned, smoked and chilled within 15 hours after slaughter were not significantly different than hams processed by conven- tional procedures. -35- Extractability and Fractionation of Muscle Proteins One of the first studies reported concerning the extractability of ‘muscle proteins was by Deuticke (1932). He found that muscles, which had been fatigued by stimulation, frozen and pulverized, had less extractable protein than those freshly extracted. Bate-Smith (1934) studied the effects of a series of extracting solutions. He found that no difference could be observed between the extractability of fresh muscle and muscle in rigor. In a review of muscle chemistry, Dubuisson (1952) pointed out that the extractability of muscle proteins depends not only on the solubility characteristics of the proteins, but also upon the firmness with which the proteins are bound and upon the dissociation power of the salts used for extraction. Dubuisson (1952) has also shown that the amount of extractable myosin is reduced, if the muscle is frozen in the contracted state or while con- tracted by rigor mortis. If the muscle was allowed to relax after being contracted, it yielded the same amount of extractable protein as normal muscle. He suggested, therefore, that the changes in extractability are rapidly reversible and that the change in the structural proteins reflects some change in the contractile elements themselves. Wierbicki ggflgl. (1954) were among the first to directly approach the study of quality of meat by protein fractionation. These workers attempted to determine the amount of actomyosin in meat and relate this to tenderness changes during aging. They said their extracting solution was designed to dissolve actin and myosin and other proteins, but not -36.. actomyosin. They used a citric acid buffer of pH 5.6 with an ionic strength of 0.48. This included 0.22 M KCl. In a group of 48 beef animals, hydroxyproline values for connective tissue showed no relation to tenderness. However, a very good correlation between extractable nitrogen and tenderness was obtained. Using the same extraction procedure, Wierbicki £3 31. (1956) presented evidence indicating that tenderness ‘may be related to the degree of hydration of meat proteins. They sug- gested that post-mortem tenderization may be due to certain ion—protein or protein-protein interactions rather than classical proteolysis or dissociation of actomyosin. 0n the basis of present knowledge of the fibrillar proteins, Baliga (1962) pointed out that the citric acid buffer used by Wierbicki g£_gl. (1954, 1956) would extract only the non-protein nitrogen and the sarco- plasmic proteins and would not extract myosin and actin as suggested by the experimenters. He explained that myosin can not be extracted by the potassium chloride-citrate buffer at pH 5.6, since this is near the iso- electric point of the proteins. Baliga (1962) also pointed out that in preparation of actin, Bailey (1954) first extracted the muscle mince with salt solution at pH 6.5 to remove the sarcoplasmic fraction, and the residue was then treated with a strongly alkaline (pH 10) salt solution to dissolve out the actin. Weinberg and Rose (1960) extracted the pectoralis major muscles of young chickens with a phosphate-potassium chloride buffer (pH 7.5, ionic concentration 0.55). The SUpernatant was then diluted in steps to specific -37- ionic strengths so that the actomyosin, myosin and sarcoplasmic proteins could be separated. Using this procedure, these workers showed that the amount of nitrogen extracted increased on holding the carcass for 24 hours at 4°C. The increase was entirely accounted for by an increase in the actomyosin fraction. Saffle and Galbreath (1964) showed that as pH increased from 5.5 to 6.5, the amount of protein extracted in a 3% saline solution (0.51 M NaCl) also increased. They also showed that the amount of salt-soluble protein was 50% greater in pre-rigor beef than at 48 hours post-mortem. They further demonstrated that freezing beef reduced the salt-soluble protein in comparison with beef held for 48 hours postdmortem. Scopes (1964) found that the myofibrillar proteins from ox muscle are most soluble in 12M KCl at a pH of 6.0. The extractability was observed to drop sharply at a lower pH, owing to the insolubility of actomyosin near its isoelectric point. In another study, the proteins of the longissimus dorsi muscle of rabbits were separated by their solubility properties into sarcoplasmic proteins, non-protein nitrogen (NPN), the myofibrillar proteins soluble in 0.6 M KI-borate buffer at pH 7.5, the remainder of the myofibrillar proteins soluble in 0.1 N NaOH and the stroma proteins (Paul £5 31., 1966). The average percentage of total nitrogen for the different frac- tions was reported to be: 27% sarcoplasmic, 12% NPN, 50% myofibrillar ./ protein soluble in 0.6 MZKI buffer, 10% of myofibrillar protein soluble in 0.1 N NaOH and less than 1% stroma protein. -38.. Baliga et a1. (1962) studied the protein solubility of fresh water fish in a 5% saline solution (0.85 M NaCl) buffered with 0.02 M sodium bicarbonate. They found that the initial protein solubility of 92% decreased to 82% after five days storage. This was followed by an in- crease in solubility, reaching a maximum of 97% after 12 days storage. The solubility of the proteins then decreased to 71% at 15 days. They found that the actomyosin fraction precipitated by dilution of the salt extract to an ionic strength of 0.225 rose to a high value after 1 to 3 days, and then declined to the initial level. In another study with fish using a similar extraction media, Partmann (1963) found that the extracta- bility of structural proteins of rosefish and cod decreased at high stor- age temperatures and with advancing periods of time in freezer storage. In studying the properties of the fibrillar proteins of normal and watery pork muscle, Bendall and Wismer-Pedersen (1962) determined the amount of protein extracted by a phosphate buffer (0.55 ionic strength, pH 6.5) from fibrils washed free of soluble sarcoplasmic protein with 0.04 ‘M potassium phosphate (pH 6.5; ionic strength 0.05). Their results showed that normal fibrils were almost completely extracted, giving a highly viscous solution containing 88% of the fibrillar proteins. With "watery" fibrils, however, only 11% of the fibrillar proteins were ex- tracted and the supernatant showed a low viscosity. From additional work, they concluded that in watery meat, the main fibrillar protein, actomyosin, is in the native form.but has become covered with a layer of denatured sarc0p1asmic protein. They suggested that the layer of denatured sarco- plasmic protein covering the fibrillar protein makes it resistant to extraction at high ionic strengths. -39- The extractability of pork muscle under various conditions using the procedure of Helander (1957) has, been reported by several investi- gators (Sayre and Briskey, 1963; Briskey and Sayre, 1964; Borchert and Briskey, 1965). In this procedure, the sarc0p1asmic proteins are ex- tracted with 0.03 M potassium phosphate buffer at pH 7.4, and the total soluble proteins are extracted with 1.1 M KI in 0.1 M potassium phosphate at pH 7.4. The myofibrillar proteins were calculated by the difference between the amounts of the total soluble proteins and the sarcoplasmic proteins. These workers showed that muscle protein solubility was grossly altered by the temperature and pH existing during the onset of rigor mortis or during the first few hours after death. Hill (1962) reported values for the amount of sarcoplasmic,myofi- brillar and stroma protein as well as for NPN in porcine, bovine and ovine muscle, using a modification of Helander's (1957) extraction technique. His results showed that the amount of stroma protein was highest in bovine and lowest in porcine muscle. He also reported differ- ences in extractability of the protein fractions between individual muscles. The process of transformation of myosin and actin into actomyosin during the prolonged extraction of ground rabbit muscle with weber- Edsall solution (0.6 M KCl, 0.04 M KHC03, 0.01 M K2C03) has been recently investigated in detail using various physico-chemical techniques such as viscosity, turbidity, flow birefringence, ultracentrifugation and ATPase activity (Haga 35 31., 1965). Results of Haga £3 31, (1965) showed that -40- the formation of actomyosin began about 8-10 hours after the start of extraction and was completed in about 20 hours. In a similar study, 'Mikalyi and Rowe (1966) extracted actomyosin from rabbit muscle with the Weber-Edsall solution and followed the disappearance of ATP from the muscle mince. Their results also indicated that the Splitting of ATP is not affected in the presence of 0.2 M phosphate, but the formation of actomyosin is strongly inhibited. They proposed that ATP, as well as inorganic phosphate, preserves the myofibrillar structure, and thereby prevents the extraction of actomyosin. Hegarty.g£”§1. (1963) reported the relationship of some intracellular proteins to beef muscle tenderness. The sarc0p1asmic proteins were ex- tracted in a phosphate buffer (pH 7.6, ionic strength 0.05) and then the residue was extracted with 0.1 M NaOH to remove the total fibrillar proteins. Another sample of the same meat was extracted with weber- Edsall solution. The soluble fibrillar protein nitrogen was obtained by subtracting the sarc0p1asmic fraction from the Weber-Edsall extract. They reported that the fibrillar protein solubility was highly correlated with tenderness. In the extraction of myosin from cod flesh, Connell (1960) observed that the only method available for the quantitative extraction of myosin was that of Hasselbach and Schneider (1951). In this method the finely minced muscle is extracted repeatedly with a slightly acid salt solution containing perphOSphate ions. Connell (1960) further pointed out that perphOSphate is believed to dissociate the actin bonds existing in the muscle and to inhibit the formation of such bonds during extraction. In -41- this way myosin alone is extracted, leaving the actin adhering to the ‘muscle residue or stroma. During cold storage of cod, Connell (1960) reported that 70-80% of the myosin becomes non-extractable at a rate similar to that at which the total myofibrillar protein of the flesh be- comes non-extractable. He then discussed the implications of these findings in regards to the mechanism of cold-storage deterioration. Baliga £5 21: (1962) added pyroPhOSphate to the salt solution during the extraction of fish proteins and found that it had a dissociating effect on the actomyosin. Fukazawa £5 31. (1961) examined the effect of different inorganic phOSphates on the extractability of the myofibrillar proteins. They found that the pyrophosphate Showed the greatest improvement in the ex- tractability of fibrillar proteins, while both triphosphate and hexameta- phosphate increased the extractability of the fibrillar proteins to a lesser extent. These workers also Showed that pyrophosphate decreased the viscosity of protein solutions extracted from intact fibrils with Weber-Edsall solution. Yasiu ggugl. (1964 ) have reported similar results on the extractability of muscle protein while using these phOSphates. The results of Bendall (1954) and Helandorn (1962) showed that low concentrations of pyrophosphate in combination with NaCl increase the water binding of cooked meat. In another experiment, Bendall (1962) showed that the maximum effect of perphOSphate deve10ps rather Slowly. He also found that addition of calcium or magnesium ions at the concentra- tions found in tap-water did not significantly effect the action of pyro- phosphate. -42- Fujimaki 25 al. (1965a, 1965b) have studied the changes in actomyosin during storage of rabbit muscle. Their results indicated that the approxi- mate content of myosin and actin in actomyosin are variable. The maximum extractability of actin from actomyosin occurred 2 days after slaughter. The authors concluded that the interaction between actin and myosin in actomyosin becomes weaker as aging progresses. Recent studies by Scharph “£5 31. (1966) followed the post-rigor changes occurring in the prOperties of the actomyosin fraction of turkey muscle. Their results gave some evidence that favors the concept of dissociation of actomyosin into actin and myosin with the resolution of rigor. EXPERIMENTAL METHODS General Methods Preparation of Samples All frozen muscle Samples were powdered with pre-chilled equipment in a -20°C cold room. The frozen tissue was chipped into smaller pieces and placed in a Waring blender with an approximately equal weight of dry ice to prevent an increase in temperature. The blender was Operated at full Speed until the sample was finely powdered, usually 30 to 60 sec. The powdered sample was then passed through an 18 mesh screen and stored in glass jars at -30°C. At least 12 hours was allowed for the dry ice to sublime before using the sample for analysis. Muscle pH Approximately 1 g of powdered muscle was homogenized for 30 sec in a small waring blender containing 25 m1 of 0.005 M sodium iodoacetate. Where fresh muscle was used, approximately 2.5 g was homogenized for 1 min in a small Waring blender containing 25 ml of 0.005 M sodium iodoace- tate. All pH estimates were performed in duplicate with a Corning, Model 12, expanded scale pH meter. Adenosine Triphosphate IMuscle ATP was extracted from the frozen powdered muscle with hot (100°C) distilled water. A 0.3 to 0.6 g sample was accurately weighed and then carefully poured into a beaker of boiling water. The sample was -43- IIII‘~ I .{Ill‘ r ‘ boiled about 30 sec and then placed in a cooler at 0°C to cool. The water extract was assayed for ATP by the bioluminescence method described by Strehler and Trotter (1952) and Strehler (1953). The standard ATP'enzyme system was prepared by extracting 100 mg of vacuum dried firefly lanterns (Sigma Chemical Co.) with 10 ml of ice-cold 0.1 M sodium arsenate for 10 min, while grinding in a mortar. The ex- tract was then filtered through cheese cloth into an Erlenmeyer flask in an ice bath. The filtrate was then diluted with 15 ml of deionized water, and 48 mg.MgSO4 was dissolved in it. The enzyme preparation was stored at 0°C. Before assaying for ATP, 0.5 ml of the enzyme preparation was placed in a test tube with sufficient deionized distilled water so that after the dilution of the unknown or standard, the final volume would be 3 m1. All solutions were brought to room temperature. The reaction was started by addition of the desired volume of the unknown sample or stan- dard to the diluted enzyme solution. Fluorescence was measured using an Aminco-Bowman SpectrOphotofluorometer without a light source at 550 mu, 30 sec after starting the reaction. The standard curves were prepared each day with fresh solutions of the disodium salt of ATP (Sigma Chemical 00.). Protein Fractionation The protein fractionation procedure was adapted from that of Weinberg and Rose (1960). The principal change in adapting the procedure was the extraction of the sarcoplasmic proteins, first with 0.03 M potassium phosphate at pH 7.4. The residue from the sarc0p1asmic extraction was -45- then extracted with a phosphate buffer (pH 7.5) in 0.4 M potassium chlor- ide (total ionic strength = 0.55). All fractionation procedures were carried out at 0-3°C with cold extracting solutions (0-3°C). Details of the procedures are outlined in Figure 1. In the scheme for the quantitative determination of the muscle proteins, approximately 5 g of the powdered sample was placed in a small Waring blender containing 50 ml of a 0.3 M ph0$phate buffer (pH 7.4). This was blenderized immediately for one minute at a speed of 8000 rpm (adjusted with a Powerstat transformer setting of 40). The sample weight was then determined by difference before the material was transferred into a 250 m1 centrifuge tube. The Waring blender was then rinsed with 50 m1 of the extracting solution, which was used for the second extraction. The material in the centrifuge tube was gently mixed with a magnetic stirrer for 30 min. It was then centrifuged at 2600 rpm for 20 min. The supernatant was retained. The residue was resuspended in 50 m1 of the above rinse solution, stirred and centrifuged as described above. The volume of the combined supernatant was recorded and designated as solution A (protein solution extracted at low ionic strength). The filtrate re- sulting from the precipitation of a 15 m1 aliquot of solution A with 15 m1 of 10% TCA was used for the determination of NPN. The difference between solution A and the NPN (A-NPN) represented the sarc0p1asmic pro- tein nitrogen. The residue A, remaining from the 0.03 M phOSphate buffer extraction, was suspended in 50 m1 of a mixture of phOSphate buffer (pH 7.5) in 0.4 M KCl (total ionic strength = 0.55). The mixture was stirred gently for -46- 5 g sample powdered muscle blenderized in 50 ml of 0.03 M P04 buffer after mixing 30 min centrifuged for 20 min at 2600 nmn residue A V supernatant added 50 m1 of 0.03'M P04 buffer, mixed 30 min, centrifuged 20 min at 2600 rpm residue A supernatant added 50 ml of buffer nitrogen solution A (u = 0.55) extracted at low mixed 30 min, centri- _ ionic strength fuged 20 min at 2600 rpm residue B supernatant added 50 m1 of buffer (u = 0.55) mixed 30 min, centrifuged 20 min at 2600 rpm residue B supernatant nitrogen solu- 15 ml aliquot of (cont.) tion B extracted A treated with 15 at high ionic ml of 10% TCA for Strength NPN determination (cont.) precipitate filtrate containing (discarded) NPN Figure 1. Scheme for the quantitative determination of the muscle proteins. -47- residue B __T added 50‘ml of 0.1 M NaOH mix 1 hr, centrifuge 20 min at 2600 rpm residue C supern tant added 50 ml of 0.1 M NaOH mix 1 hr, centrifuge 20 min at 2600 rpm residue C supernatant L alkali insoluble material, connective tissue (discarded) nitrogen solution C containing remainder of fibrillar protein nitrogen nitrogen solution B 20 ml aliquot of B diluted with 29 ml deionized water (u = .225) left set overnight and centrifuge 20 min at 3000 rpm residue D { \ . Appecipitate of actomyosrn supernatant nitrogen solution D, myosin dissolve in 0.55 ionic strength buffer V solution E, actomyosin Figure 1 (cont.) Scheme for the quantitative determination of the muscle proteins. -43- 30 min and then centrifuged at 2600 rpm for 20 min. The extraction and centrifugation was repeated. The volume of the combined supernatantswere recorded and designated as solution B, the fibrillar protein extracted at high ionic strength. Twenty m1 of solution B was then diluted with 29 ml of deionized water, left overnight and then centrifuged at 3000 rpm for 20 minutes. The volume of the supernatant was recorded and designated as solution D, the myosin fraction. The residue D was resuspended in 0.55 ionic strength buffer. The volume was recorded and designated as solution E, the actomyosin fraction. Residue B from the 0.55 ionic strength buffer was resuspended in 50 ‘ml of 0.1 M NaOH, stirred gently for 1 hr, and then centrifuged at 2600 rpm for 20 mdn. Extraction and centrifugation was again repeated. The volume of the combined supernatants was recorded and designated as solu- tion C, the remaining soluble fibrillar protein. A very small amount of residue (alkali insoluble material) was discarded. All fractions were analysed for nitrogen. Nitrogen Analysis All nitrogen analyses were performed by the micro-Kjeldahl method as outlined by the American Instrument Co. (1961). For calculating protein concentrations, it was assumed that the fibrillar extract contained 16.15% nitrogen (Mikalyi and Rowe, 1966). -49- Preparation of Fibrillar Protein Extract The fibrillar protein extract of the longissimus dorsi muscle of the rabbit was prepared according to Mihalyi and Rowe (1966). The muscle was cleared of fat and connective tissue and homogenized in a waring blender with 4 volumes of weber-Edsall solution (0.6 M KCl, 0.04 M KHCOg, 0.1 M K2C03). The suspension was poured into a 250 ml centrifuge tube and left for 24 hours. At the end of this time, 2 volumes of Weber-Edsall solution ‘were added, and the sample was stirred thoroughly before centrifuging in an International Refrigerated Centrifuge at 2,600 rpm for 20 min. The myosin components of the supernatant were precipitated by diluting with 10 volumes of deionized water to remove the water soluble muscle constituents. The precipitate was collected after centrifugation for 20 min at 2,600 rpm and dissolved in 0.6 M KCl. Following 3 precipitation Steps, the myosin components dissolved in 0.6 M KCl were clarified by centrifugation for 10 min at 12,000 rpm with a Sorvall, Model 881 centri- fuge. The supernatant was then diluted to approximately 0.2% protein with 0.6 M KCl. Final protein concentrations were determined by nitrogen analysis as described above. Viscosity The viscosity of a 0.6 M KCl solution of the purified Wéber-Edsall extract was estimated by an Ostwald viscometer at pH 6.4. The viscometer used in these experiments had an outflow time for deionized water of 62 sec at 23°C and 110 sec at 3°C. Preliminary experiments were run to com- pare the effect of adding 4 mM MgC12 and to compare the results at 3° and -50- 23°C. The change in viscosity of the protein solution was measured following the addition of 1 ml of 0.1 M potassium pyrophosphate to a solution containing 1 ml of 0.01M MgClz in 23 m1 of the protein extract. The change in viscosity following the addition of ATP was measured in a similar manner. ATPase Activitj and Changes in Turbidity For measuring changes in turbidity and ATPase activity, the purified myosin components were diluted with 0.6 M K01 to a final concentration of 0.675 mg of protein per ml. Reagent solutions needed for this reaction included 0.01 M ATP, buffered at pH 6.4 with tris buffer, and 0.01 M MgClz. For determining the enzymatic activity and the changes in turbidity, Iml of 0.01 M MgClz was added to 23 m1 of the diluted protein extract. The reaction was started by adding 1 ml of the 0.01 M ATP solution to 24 ml of the MgC12 protein solution. This was mixed immediately, poured into a cuvette and changes in turbidity were measured at 350 mu (Perry and Cotterill, 1965) with a Beclonan DU spectrophotometer equipped with a Gilford, Model 220, absorbance indicator. The reference turbidity was taken from a solution containing 0.5 ml of 0.01 M MgClz, 0.5 ml of deionized water and 11.5 ml of the diluted muscle extract. ATP was determined on 1 m1 aliquots of the reaction mixture removed at apprOpriate times. The reaction mixture was inmediately pipetted into boiling water to stOp all enzymatic activity. The level of ATP was deter- mined by the method previously described. -51- water Holding Capacity Measurement of water holding capacity was made by modifying the rapid method proposed by Grau and Hamm (1956). This modified procedure utilized Whatman #1 filter paper, 9.0 cm in diameter, dried in a 110°C drying oven for 12-14 hours and then placed in a desiccator until used. When the sample was prepared for measurement, the filter paper was re- moved from the desiccator and placed on a plexiglass plate. A fresh meat sample weighing 300 mg was immediately placed on the mid portion of the filter paper. It was covered with a second plexiglass plate and pressed in a Carver Press at 5,000 pounds pressure for three min. Subsequently, the moisture area and meat area were measured with a compensating polar planimeter. Results are expressed as a ratio of total area to meat area. Cooking_Procedure All cuts were placed on broiling trays and cooked in an electric oven at 175°C to an internal temperature of 75°C. Statistical Analysis The data collected from Experiment II and Experiment V were punched onto IBM cards and analyzed in a CDC 3600. The data in Experiment II were analyzed for treatment differences by the least squares method. Duncan's multiple range test was used for comparing means. Correlation coefficients were also calculated between some of the variables in Experi- ment II. The data in Experiment V were subjected to analysis of variance. Correlation coefficients were also calculated between all the variables for the treated group and the control group. Levels of significance were used as indicated by Snedecor (1956). -52- Experimental Animals and Treatments Experiment I Eight rabbits weighing between 1.6 and 2.3 kg were used. They were randomly divided into two groups, one treated and one control with 4 animals in each group. The treatment consisted of an intravenous injec- tion of a lethal dose of the tetrasodium salt of EDTA in isotonic saline solution. The dosage varied from 65 to 132 mg of EDTA per kg of body weight. The controls received an intravenous injection of 3'ml of an isotonic saline solution per kg body weight, so that both groups were injected with approximately the same volume. All rabbits were bled within 5 min of injection. Following exsan- guination, the intact semitendinosus muscle was removed from the right leg. Shortening was recorded on a kymograph with an isotonic lever loaded with approximately 8 gm/sq cm of muscle cross section. The muscle was held at 0°C in a vertical position with two clamps over calcium-free Ringer's solution with nitrogen bubbling through it. This provided a moist atmosphere and prevented the muscle from drying out. The posterior portion of the longissimus dorsi muscle from the right side was removed immediately after bleeding and frozen in liquid nitrogen. The posterior portion of the longissimus dorsi muscle from the left side was removed after 20 hrs at 0°C and frozen in liquid nitrogen. The frozen muscle samples were then powdered and stored at -30°C for subse- quent pH and ATP determinations. The anterior portion of the loin was removed after 20 hrs, wrapped in aluminum foil, immediately frozen at -30°C and stored for subsequent cooking and tenderness studies. -53.. All loins were removed from the freezer 24 hrs before cooking and allowed to thaw at room temperature (approximately 23°C). The loins were weighed and cooked as described in general methods. After cooking, the samples were weighed and cooled overnight at room temperature. Warner-Bratzler Shear values were recorded on 3 cores 12.5 mm in diameter removed parallel to the fibers of the longissimus dorsi muscle. Experiment II A total of 22 rabbits weighing between 1.5 and 3.0 kg were used. They were randomly divided into 5 groups -- 4 treated groups and one control group. There were 4 rabbits in group I, 6 in group II, 5 in group III, 2 in group IV, and 5 in group V. The groups consisted of the following treatments: group I - EGTA; group II - CDTA; group III - EDTA; group IV - sodium oxalate; and group V - control. All rabbits were treated and handled as in Experiment I. Following exsanguination, the intact semitendinosus muscle was re- moved from both legs and shortening was recorded on separate kymographs with an isotonic lever loaded with a weight approximately equal to that of the muscle. The muscles were held under the same conditions as in iEXperiment I. After the semitendinosus muscle from the left leg was clamped into position, it received several micro—injections of a 0.1 M Ca012 solution. The total amount of Ca012 injected was equivalent to approximately 0.4 ml/kg of live weight. After 6-8 hrs, both semitendin- osus muscles were removed from the kymograph and checked for extensi- bility. The muscle from the right leg was frozen in liquid nitrogen, powdered and stored at -30°C for ATP determination. ‘u‘ I.IVI..I . ll ‘1 -54- The posterior portion of the longissimus dorsi muscle was removed from the right side immediately after bleeding and frozen in liquid nitrogen. After the remainder of the carcass was chilled at 0°C for 24 hrs, the posterior portion of the longissimus dorsi muscle from.the left side was removed and frozen in liquid nitrogen. The frozen muscle samples were powdered and stored at -30°C for subsequent pH and ATP determination and for protein fractionation studies. The anterior portion of the loin was removed after 24 hrs, wrapped in aluminum foil, immediately frozen and stored at -30°C until removed for cooking and tenderness Studies. All loins were removed from the freezer 24 hrs before cooking and allowed to thaw at room temperature. Before cooking as described in general methods, the longissimus dorsi muscle was removed from the left side of each loin, trimmed of external fat and weighed. Following cook- ing, the samples were cooled at room temperature overnight, weighed and shear values were recorded as in Experiment I. Experiment III Ten rabbits weighing between 1.5 and 3.0 kg were used. Four were randomly treated with EDTA as previously described, 1 with EGTA and l with CDTA, while the other 4 were treated as controls. All rabbits were treated and handled as in Experiment I. The semitendinosus muscles were removed and shortening was recorded as in Experiment II. The semitendinosus muscle from the left leg of the #2 and #4 control rabbits received micro-injections of 0.01 M CaClz. The left semitendinosus muscle from the other 8 rabbits in this experiment -55.. received micro-injections of 0.1 M CaClz. The semitendinosus muscle from the right leg of the #1 and #3 control rabbits received micro- injections of 0.1 M MgC12 in an amount equivalent to the 0.1 M CaClz in- jected into the paired muscle of the same rabbit. After 6-8 hrs both semitendinosus muscles were removed from the kymograph and checked for extensibility. The muscles from the #1 control rabbit were kept overnight for pictures. The 9 remaining semitendinosus ‘muscles from the right legs and 3 semitendinosus muscles from the left leg of the last 3 rabbits treated with EDTA were then frozen in liquid nitrogen, powdered and stored at -30°C for pH and ATP determinations. Experiment IV A fibrillar protein extract was prepared from the longissimus dorsi ‘muscle from the right side of the rabbit immediately after bleeding as described in the general methods. After the remainder of the carcass was chilled 24 hrs at 0°C, a fibrillar protein extract was prepared from the longissimus dorsi muscle from the left side by the same procedure. The properties of the fibrillar protein extracts were investigated by the measurement of ATPase activity, and by changes in the turbidity and viscosity as described in the general methods. In addition, changes in turbidity and ATPase activity were also investigated in the absence of added MgClz. In this study, 1 ml of 0.01 M ATP was added to 24 ml of the diluted protein extract. All other procedures were carried out as described earlier herein. -56- Experiment V Nelve crossbred Giampshire X Yorkshire) barrows and gilts were ran- domly divided into two groups, one treated and one control with 6 animals in each group. The animals ranged in weight from 92 to 110 kg at Slaughter. The treatment consisted of an intravenous injection of 250 to 310 ml of the tetrasodium salt of EDTA (0.1M) in isotonic saline solution. Following injection, the pigs were in a relaxed State. They were then shackled, hoisted and bled within 5 min. The controls were electrically stunned and bled. In all cases, a treated and control pig were slaughtered Simultaneously. The semitendinosus muscle from the right ham.was removed as rapidly as possible about 10 min postamortem. A sample from this muscle was excised and frozen in liquid nitrogen. The remainder of the muscle was hung in a vertical position at 0-3°C and shortening was recorded over a 24 hr period. After 24 hrs, another sample of the same muscle was removed and frozen in liquid nitrogen. The frozen muscle samples were then powdered and stored at -30°C until used for pH and ATP determinations. After a 24 hr chill, the hams from all carcasses were removed and trimmed. A 4 cm thick center ham slice was removed by cutting perpendi- cular to the femur, 2.5 cm from the aitch bone toward the shank. Most of the external fat was removed. The weight of the center ham slice was recorded. Following this, the ham slice from the right ham of a treated and control pig were placed on a broiling rack and cooked as outlined under general methods. After cooking, the cuts were removed from the oven and cooked weights were recorded. The ham slices from the left hams were then cooked in the same manner. -57- All ham slices were cooled overnight at 0-3°C. Warner-Bratzler shear values were then determined on 15 mm diameter cores from the biceps femoris, semimembranosus, rectus femoris and the semitendinosus muscles. At 36 hrs post-mortem, muscle samples from the biceps femoris were utilized to determine the pH and water holding capacity. ‘Muscle color of the right hamxwas subjectively evaluated at 24 hrs post-mortem. Each ham was visually rated on a 5-point scale as follows: 1 - very pale, 2 - slightly pale, 3 - grayish pink, 4 - slightly dark, 5 - very dark. RESULTS AND DISCUSSION Effect of ChelatingpAgents, Calcium and Magnesium on Various Properties of Muscle The effects of various chelating agents on the physical and chemical properties of muscles from.rabbits and pigs were investigated in five different experiments. The effecuaof added calcium and magnesium on muscle prOperties were also Studied. Results are discussed on the basis of shortening, ATP, muscle extensibility, pH, color, water holding capa- city, cooking losses, shear values, protein fractionation, ATPase activity, turbidity and viscosity. Each will be discussed separately. Shortening Fig. 2 demonstrates that the intravenous injection of EDTA greatly inhibited Shortening of the semitendinosus muscle in Experiment I. The shortening occurring in the muscle of the treated rabbits was completed at the end of 3 hrs, whereas, normal Shortening, as Shown by the controls, continued up to 7 hrs. Davies (1963) suggested that the calcium-actomyosin complex develops tension during formation, and if the sub-total of the forces develOped at any one time by the ultramicro-contractions is sufficient to overcome the extended load and the resistance of the series of elastic elements, the actin filaments will be pulled along the myosin filaments into the A band and the muscle will undergo quantal contraction. Hasselbach (1957) showed that the calcium content of the undissolved structural proteins -58- 10 -59- J.’J?:?afiafia’:aaaaaflflaa:aafi: O’BOOOOOOOOOOOOOOOOOOOOOOOOOO..%,.O%’O wwwa‘uugwuuu O O I IOOOOOOOOIOOOOOOO un50000000 0.0... O. ........ o I'o'o'o'o'o'o'o. QQOQQOOOQQQQQ.0..’O0...... O OOOOOOOOOOOOI0.000IOOCOOOCCOOOOOOO0......Oua‘ OOOQOOOOOOOQ...OOOOOOOOOCIOQOOO0.00.00.00.00. O... '01. 3‘71000000 (OIIUIUIUUIOCI v I00...0.00.0oooooooooooooooooooooooo 00000000000ooooooooooooooooooooooooo .0000.00000.000000000000000000000 O V N am u; luguauoqs IIIJIQQJIQOIQIQII 'Z§ 21 22 24. Rabbit No. Treated . Control The control treatment consisted of an intravenous antemortem injection of EDTA in isotonic 8815.116 . consisted of an intravenous antemortem injection of isotonic saline and the Effect of EDTA on the Shortening of the semitendinosus muscle. Fig. 2. -60.. from fresh muscle was diminished 50% by washing with EDTA, but that the magnesium content was not altered. Several investigators (watanabe and Sleator, 1957; Ebashi g£”§1., 1960; Bendall, 1964) have Shown that EDTA in the presence of MgClZ and ATP manifests a relaxing effect on glycerol treated fibers. Martonosi and Feretos (1964) explained the relaxing effect of chelating agents by their ability to lower the calcium concen- tration of the test system. In accord with the results cited above, the addition of EDTA in the present experiment appeared to form.a complex with free calcium and thereby inhibited shortening. Results from Experiment II Show that the intravenous injection of either EDTA, Na Oxalate, EGTA, or CDTA Significantly (P < .05) inhibited shortening of the semitendinosus muscle (Fig. 2). The decrease in the load on the isotonic lever of the kymograph in this experiment can account for the increased values for shortening in relation to those ob- tained in Experiment I. No Significant difference was observed between the different treatments, but results Show that CDTA (the poorest calcium chelator) had the least effect on decreasing shortening. In Experiment V, shortening (measured as the percentage of original length) was signi- ficantly less (P < .05) for pigs treated with EDTA (6.6%) than for con- trol pigs (10.3%). These results support the premise that chelating agents complex enough free calcium to inhibit Shortening. Additional experiments were run to determine the effect of increased levels of calcium and magnesium upon Shortening. The data in Table 1 show that micro-injections of CaClz into the paired semitendinosus muscle Shortening in mm Fig. 3. _61_ Control EGTA Na Oxalate EDTA Treatment Effect of chelating agents on the shortening of the semitendinosus muscle. The control consisted of an intravenous antemortem injection of isotonic saline and the treatments consisted of an intravenous antemortem injection of either CDTA, EGTA, Na oxalate or EDTA in isotonic saline. -62- (Experiment II) resulted in extensive shortening. Even after the injection of CaClz, however, the muscles from the treated animals Shortened less than muscles from control animals. These results would suggest that chelating agents may form a complex with calcium thereby decreasing the shortening effect of the added calcium. Table 1. Means and standard error of the means for shortening of the semitendinosus muscles of the rabbit for the various treatments in EXperiment 11.152 Right semitendinosus Left semitendinosus Treatment uninjected injected with CaClz Control 14.96a i 1.46 55.71 a 7.13 CDTA 9.00b i 1.33 32.17 i 6.53 EGTA 8.25b i 2.31 36.25 i 7.99 Na Oxalate 6.00b i 2.31 ' 27.40 i 11.33 EDTA 5.53b i 1.46 31.88 i 7.13 IShortening in mm as measured on the kymograph. 2Means with different superscripts differ significantly (P < .05). The values for shortening of the semitendinosus muscles from 4 con- trol rabbits following micro-injections of MgClz and different concentra- tionsof CaClz (Experiment III) are given in Table 2. The difference in shortening found on the addition of either 0.1M MgClz or 0.1M CaClz to the semitendinosus muscles from a rabbit are illustrated in Fig.44. The data Show that micro-injections of MgClz had no significant effect on shortening during rigor mortis. These results are in agreement with those . l‘ullvlllllll I‘llli‘lii -63- of Caldwell andKMlster'(l963)'who reported that an injection of MgClz into the muscle fibers did not produce contraction. Table 2. Effect of micro-injections of magnesium and calcium on shorten- ing of the semitendinosus muscles of the control rabbits in Experiment III.1 Right semitendinosus Left semitendinosus Rabbit Injected with Injected with Injected with number Uninjected 0.1MlMgClz 0.01M Ca012 0.1M CaClz 1 14 100 2 19 25 3 18 54 4 13 18 Average Shortening l6 16 21.5 77 IShortening in mm as measured on the kymograph. The data in Table 2 demonstrate that an increased concentration of Ca012 within the muscle resulted in a definite increase in Shortening during the deve10pment of rigor mortis. These results are in agreement with those of Niedergerke (1955) and Caldwell andwa1ster (1963), who showed that calcium injected into the sarc0p1asm of a muscle fiber caused local contraction at the point of injection. Results of the present experiments Show that the reduction of free calcium in the muscle by chelating agents will reduce the Shortening of the muscle during development of rigor mortis. On the other hand, it was also Shown that increased levels of calcium resulted in increased shortening. However, increased levels of MgC12 had no effect on shorten- ing. {##ltfll 4"" -6h— 1.50C of . 111 (221(Ila 1.5cc of Semitendinosus muscle each block 1 sq :m Fig. 4. The difference in shortening found following the addition of Mg012 and Ca012 to paired semitendinosus muscles. (Experiment III - rabbit number 1) . I‘.ll\|[ -55- ATP The data in Table 3 show that there was not any appreciable differ- ence in ATP values of the longissimus dorsi muscle from EDTA treated (4.99 uM ATP/g) and control rabbits (5.13 uM ATP/g) at 0 hr post-mortem in Experiment I. Similar results were obtained in Experiment III where the muscles from the EDTA treated rabbits contained 4.34 uM ATP/g and the control rabbits contained 4.36 uM ATP/g. In Experiment II, the ATP values at 0 hr post-mortem ranged from 3.69 uM ATP/g in the longissimus dorsi muscle from the rabbits treated with EDTA to 8.12 uM ATP/g from rabbits treated with EGTA. However, because of the large variation in the ATP levels within treatments, these values only approached signifi- cance. At 20 or 24 hrs post-mortem, no consistent difference was observed between the different groups in either Experiment I, II or III. The values obtained in the present study are in agreement with the results obtained by Bendall (1951) and Bate-Smith and Bendall (1956). In Experiment V, no significant difference was obtained between the ATP values of the semitendinosus muscle for the treated and control pigs at 10 min and 24 hrs post-mortem (Table 3). However, the ATP values for the pigs treated with EDTA.were generally higher with values at 2.42 and 0.451 uM ATP/g at 10 min and 24 hr post-mortem, respectively, as compared to 1.95 and 0.308 uM ATP/g for the controls. The ATP values at 10 min post—mortem in this eXperiment (2.42 uM ATP/g for the EDTA treated and 1.95 UM ATP/g for the controls) are in accord with the value of 1.86 uM ATP/g reported by Aberle (1967) for the longissimus dorsi muscle of the pig at 15 min post-mortem. However, much higher values have been reported aouuoauumom owe ma Nauou za.o nufiz pouoofloHN oaomsa w\mm.< moaoe WH moH.o u: n Nmnmoowpaouweom amp-H mo.H wen.o u: n mononaeaoueafim unwem moa.o oNH.o no em Hmuop om.e om.q as o anaemmwwaoq HHH mH.o Hame.o ma.o “wom.o an em mm.o a ~3.N mm.o a mm.H use on mamoaneauuae6m > ,m ,m om.oamnn.o mm.oeoH¢.o o¢.oao¢m.o mm.oaoan.a 2 om.oumNH.H no u msmooapoouaaom unwwm maodeamad oNodHHMHd wHodeoHd mHodHHNaé mHodHNiuHé an «N Hmuop eo.HHmo.m do.HHmHm.n 0H.HHONH.w mo.oa~nw.o «o.HHNnm.m as o mseammwwooa HH Noa.o ow~.o an om “when mm.q ma.m an o mseammawooq H use HHH .HH .H unoEHuodxm SH moaeu pom moaowsa.msownm> oSu pom mH< How wanna use mo House pumpooum poo mamas .m manna -67- (5.6 - 8.0 uM ATP/g) for the longissimus dorsi muscle of the pig at the time of death (Lawrie, 1960; Bendall gpugl., 1963; Bodwell g; 31., 1966; Bendall, 1966). The time of post-mortem ATP extraction from the muscle could account for some of this difference. Furthermore, most of the high values for ATP were determined as labile phosphate, thus, part of the difference in these values may be explained by the lack of corres- pondence between ATP breakdown as determined by the enzymatic method and the appearance of inorganic phosphate (Nauss and Davies, 1966). Nauss and Davies (1966) explained the above discrepancy by Showing an increase in the concentration of the other labile phosphate compounds, such as glucose-6-phosphate, fructose-6-phosphate and glucose-l-phosphate. The ATP values reported in the present experiment with pork at 24 hrs post-mortem (0.451 and 0.308 uM.ATP/g for the EDTA treated and con- trol pigs, resPectively) are consistently higher than those reported by Bodwell gpflai. (1966). These workers reported that one carcass contained '0.3 uM ATP/g at 24 hrs postemortem, although all other carcasses showed a complete absence of ATP. The higher ATP levels observed in the pre- sent study may be the result of a different extraction procedure. It should be mentioned that trouble was encountered during the injection of EDTA in the pigs #2 and #5. Consequently, these two pigs never obtained the relaxed state observed in the other treated pigs and were extremely excited at the time of Slaughter. The values listed in AppendiijEIshow that the ATP levels for these two pigs were the lowest of any of the treated animals at both 10 min and 24 hr post-mortem. A -53- comparison with other individual ATP values suggests that prOper inject- ion of EDTA resulted in higher ATP values at 10 min and 24 hrs post- mortem (Experiment V). The difference in the effect of treatment on the ATP values observed for the rabbits and pigs may be explained by the fact that the injection of chelating agents into the rabbits resulted in extreme tetany with subsequent death. This was not observed for the treated pigs. They generally showed but slight tetany for a short period of time and then became very relaxed. On using paired muscles from rabbits treated with EDTA (Experiment III), the data Show a definite increase in the rate of ATP hydrolysis following the injection of CaClz in the left semitendinosus muscle (Table 3). This effect may be explained by the presence of free calcium in the sarcoplasm resulting in the cyclic formation and breakage of the cross links between actin and myosin, which is accompanied by the enzymatic hydrolysis of ATP due to actomyosin ATPase (Nauss and Davies, 1966). In addition, Nauss and Davies (1966) pointed out that further breakdown of ATP occurs as a result of the ATPase activity of the sarco- plasmic reticulum, which is involved in the active uptake of free calcium from the sarcoplasm. 0"! I'llllllllll‘ III III" I ll]. Illl -69- Extensibility of the semitendinosus muscle and its relationship to ATP .1221 Rigor has been described as a transformation from a system of actin and myosin, which is elastic, to a rigid system consisting of actomyosin, which is joined together by cross bridges between the actin and myosin (Nauss and Davies, 1966). Observations in Experiment I show that at 7 hrs post-mortem, the muscles from the control rabbits were rigid and inextensible (Table 4). However, the muscles from the treated rabbits were elastic and extensible and could be stretched easily to about 40% of their equilibrium length and would still snap back to their original length on being released. Treatment had no consistent effect upon the elasticity or rigidity of the uninjected semitendinosus muscle as shown in Table 4 (Experiment II). Observations do Show, however, that the paired left semitendinosus muscles from the rabbits in this exPeriment, which were injected with 0.1M CaClz, were always rigid and inextensible at 7 hrs post-mortem. Similar results were obtained in Experiment III (Table 4). These results indicate that an increase of free calcium in the sarcoplasm shortens the delay phase during the development of rigor mortis. Locker (1959) and Sink g; 31. (1965) reported that a short delay phase is associated with increased shortening of the muscle during the develop- ment of rigor. In accord with these results, it is concluded that an increase in the free calcium in the sarcoplasm is responsible for the shorter delay phase observed in the deve10pment of rigor and the in- creased Shortening which occurred following the injection of CaClz. Table 4. 7 hrs post~mortem. -70- Effect of treatment on the elasticity or rigidity of muscle at Condition of Animal Treatment of 'muscle1 Experiment treatment ng, semitendinosus muscle Elastic Rigid I Control Right uninjected 2 EDTA Right uninjected 2 11 Control Right uninjected 2 3 Left injected with 0.1M CaClz 5 CDTA Right uninjected 3 3 Left injected with 0.1M CaClz 6 EGTA Right uninjected 2 2 Left injected with 0.1M CaClz 4 Na Oxalate Right uninjected l 1 Left injected with 0.1M CaClz 2 EDTA Right uninjected 3 2 Left injected with 0.1M CaClz 5 III Control Right uninjected 2 Right injected with 0.1M‘MgC12 1 1 Left injected with 0.01M CaClz 2 Left injected with 0.1M 03012 2 EDTA Right uninjected 3 1 Left injected with 0.1M CaClz 4 INumber of semitendinosus muscles from each treatment possessing an elastic and extensible condition or a rigid and inextensible condition. Huxley (1960) pointed out that the loss of ATP leads to the formation of fixed links between actin and myosin filaments as the muscle becomes rigid and inextensible. The rigid system maintains continuous tension in the absence of ATP breakdown (Nauss and Davies, 1966). In Experiment II, the level of ATP was compared between the elastic and the inextensible left semitendinosus muscles at 7 hrs post-mortem. It was found that the muscles which were elastic after 7 hrs postdmortem had significantly higher (P < .01) levels of ATP (1.544 UM/g) than those which were rigid and inextensible (0.388 uM/g). Bendall and Davey (1957) reported that with rabbit muscle held at 37°C, the onset of rigor occurred when 1/2 of the initial ATP was depleted. If held at room temperature, however, the onset of rigor occurred when 3/4 of the initial level was depleted. Nauss and Davies (1966) found that as the muscle began to shorten, the rate of 45Ca efflux increased and the ATP values fell. They suggested that the important chemical event determining the onset of the physical changes in a muscle passing into rigor seemed to be the internal liber- ation of a sufficient amount of calcium to initiate the interaction between actin and myosin. Results from the present experiments indicate that an intravenous injection of chelating agents will inhibit shorten- ing, but does not always prevent the formation of some links between actin and myosin, which will eventually result in an inextensible condi- tion of the muscle following the depletion of ATP. The injection of CaClz in these experiments increased Shortening of the muscle and lowered the level of ATP at 7 hrs postdmortem. Thus, results support the postulation (Marsh, 1966; Newbold, 1966; Nauss and -72- Davies, 1966) that the release of calcium by the sarcoplasmic reticulum is responsible for the rapid ATP degradation and the subsequent stiffen- ing of muscle during the deve10pment of rigor. pH Mean values for pH in Experiment I, II, III and V are listed in Table 5. The data from Experiment I and III Show that there was no appreciable difference in pH values of the longissimus dorsi muscle from EDTA treated and control rabbits at either 0, 20 or 24 hr postwmortem. Injection of CaClz into the muscle also had no significant effect upon pH values of the semitendinosus muscle at 7 hrs postdmortem. In Experiment II, 0 hr post-mortem pH values for the longissimus dorsi muscle of rabbits treated with EDTA (6.31) were Significantly lower (P < .05) than those recorded for the group treated with EGTA (6.67) and the control group (6.57). No Significant differences in pH values were observed between the other groups at 0 hr post-mortem. The ultimate (24 hrs) pH values of the longissimus dorsi muscle did not differ Signifi- cantly between treatments. The data in Table 5 indicate that there was no significant differ- ence in pH between the treated and untreated longissimus dorsi muscle of the pig at either 10 min or 24 hr post-mortem in Experiment V. The ulti- mate (36 hr) pH of the rectus femoris muscle did not differ Significantly between treatments. -73.. .Eounoarumoe oHa.mH «Homo 2a.0 suHs pouoonoHN .Honuo some scum economme handmonHanm no: one mudHuom unseen unonuHe memos .Amo. v my Honuo :ooo scum Hoouomme eauomonHanm one muaHHomHoesm unouomme nuH3 momQZH NH.0 H: m msmoanoouHaom umog 0H.0 NH.0 H: m mamoanoouHemm 2me 00.0 N0.m an em Hmuop 50.0 0¢.0 a: 0 mnaHmmeGOA HHH 50.0 H 00.0 50.0 H 00.0 no 00 mHHoaam mdoon 00.0 H 00.0 00.0 H 00.0 H: «N msmoGHpoouHEom 8.0 H and 85 H 3.0 5e 3 ”Emma > «0.0 H 00.0 00.0 H Nm.0 00.0 H 05.0 no.0 H 55.0 «0.0 H H0.m Ha em kuop 50.0 H 0am.0 HH.0 H 0m00.0 00.0 H on0.0 00.0 H 0m00.0 50.0 H onm.0 H: 0 moaHmmedoq HH $6 $6 an om Hes- 00.0 00.0 H; 0 msEHmmHmooA H eHnm uumamxo mz «me 0900 Houuaoo amuuoauumod edema: unoaHHomWM monogamona oEHH .> was HHH HHH «H udoaHHomxm GH moaHu pom moHomsE mnOHum> onu mo me you wanna onu mo Honuo pumpamum 0am memo: .0 vanes -74- Color score and water holding capacity The data in Table 6 Show that there was no significant difference between the subjective color score of the muscles from the pigs treated with EDTA (3.50) and from the control pigs (3.33). There was also no significant difference between the water holding capacity of the treated pigs (2.49) and the control pigs (2.60). Table 6. Means and standard deviations for color score and water holding capacity ratios in pork muscle (Experiment V). Standard Treatment Control EDTA deviation 0616: scorel 3.33 3.50 0. 67 WHC2 2.60 2.49 0.38 1Color was subjectively scored on the following 5-point scale: very pale (1); Slightly pale (2); grayish pink (3); slightly dark (4); and very dark (5). 2Water holding capacity was determined by the press method. Results are expressed as a ratio of the total area to meat area. Cooking losses and shear values Mean values for cooking losses and shear force values in rabbit muscle from Experiment I and II are listed in Table 7. These data Show that the difference in cooking losses between the rabbits treated with EDTA (22.3%).and thecontrol rabbits (25.3%) in Experiment I approached Significance at the 5% level. In Experiment II, however, the cooking losses for the rabbits treated with EDTA (30.0%) were the highest as compared to 27.6% for CDTA, 35.9% for Na Oxalate, 26.1% for the controls and 25.3% for EGTA. -75- Table 7. Means and standard error of means for cooking losses and Shear values in rabbit muscle (Experiment I and II) Experiment I Experiment II Treatment Cooking loss1 Shear values2 Cooking loss Shear values Control 25.3 i 0.92 6.38 i 0.63 26.1 i 1.16 5.64 i 0.60 EDTA 22.3 i 0.92 5.22 i 0.63 30.0 i 1.16 3.43 i 0.60 CDTA 27.6 i 1.06 4.76 i 0.55 EGTA 25.3 i 1.30 5.93 i 0.68 Na Oxalate 26.9 i 1.84 3.97 i 0.96 IExpressed as % of uncooked weight. 2Shear force values expressed in pounds -- measured by the Werner- Bratzler shear using 12.5 mm cores. No significant difference in Shear values was observed between the different treatments in Experiment I and II. However, it should be noted that shear values of the longissimus dorsi muscle from the rabbits treated with EDTA (5.22 for Experiment I and 3.43 for Experiment II) were lower than the control groups (6.38 for Experiment I and 5.64 for Experiment II) in both experiments. The muscle from rabbits treated with CDTA (4.76) and Na Oxalate (3.97) also had slightly lower shear values than the control rabbits (5.64) in Experiment II. Preliminary work using intravenous and intramuscular injections of EDTA solution into hams removed from pig carcasses at 30 min postdmortem (Experiment V) showed no effect on tenderness of the muscles as measured by the Warner-Bratzler shear. In an effort to overcome the apparent lack of permeability of the EDTA in this preliminary work, all additional experiments consisted of an intravenous injection of EDTA into the live animal. -75- The average cooking losses and shear force values for Experiment V are shown in Table 8. These data Show no Significant difference in cooking losses between treatments. Table 8. Means and standard deviation for cooking losses and Shear values in pork muscle in Experiment V. Standard Treatment Control EDTA deviation Cooking Loss1 Right ham slice 25.5 26.8 2.7 Left ham Slice 27.6 27.7 3.7 Ave. 26.52 27.28 2.45 Shear Values2 Ave. both hams 10.08 8.65** 1.22 Semimembranosus 9.54 7.93*** 1.30 Rectus femoris 9.24 7.38* 1.78 Biceps femoris 11.31 10.50 1.33 Semitendinosus 10.32 8.93* 1.34 IExpressed as % of uncooked weight. 2Shear force values expressed in pounds -- measured by the Werner- Bratzler shear using 15 mm cores. * P < .10 ** P < .05 *** P < .01 The data in Table 8 Show that the Werner-Bratzler shear values for the hams from the pigs treated with EDTA (8.65) were Significantly lower (P < .05) than the shear values obtained for the control hams (10.08). -77.. This is in agreement with the work of Locker (1960), Locker and Hagyard (1963) and Herring gpLgl, (1965a) who have shown that beef muecles which Shorten during rigor show a corresponding decrease in sarcomere length and tenderness. Marsh and Leet (1966a and 1966b) also reported that Shortening of muscle during the development of rigor mortis results in a decrease in tenderness. In recalling the effect that EDTA had on the shortening of the semitendinosus muscle in these experiments, it may be stated that the improved tenderness of the treated hams is probably the result of less shortening of the muscle during rigor mortis. Ramsey and Street (1940) and Gordon ggngl, (1964) have pointed out that the maximum tension that develOpS in muscles depends on the amount of overlap of the filaments. In accord with these results, the inhibition of Shortening resulting from the removal of some of the free calcium by EDTA in these experiments most probably decreased the formation of com- plexes between actin and myosin and seems to be associated with the improvement in tenderness that was observed. This would support the postulation of Partmann (1963) suggesting that if the interaction between actin and myosin during rigor could be completely or partly impeded, tenderness may be improved. The data in Table 8 also indicate that the effect of treatment on tenderness varied between muscles. The shear force values of the semi- membranosus muscles from the pigs treated with EDTA (7.93) were Signifi- cantly (P < .01) lower than the shear force values from the control pigs (9.54). A definite (P < .10) improvement in tenderness was also observed -78.. for the rectus femoris (7.39) and the semitendinosus (8.93) muscles from the treated animals as compared to 9.24 and 10.32 for the controls, respectively. However, no significant difference was found between treatments for the biceps femoris muscle from the treated animals (10.50) and the controls (11.31). From work with beef, Locker (1960), Herring 2.13.2.1: (1965a) and Marsh and Leet (1966a, 1966b) have concluded that the state of contraction is the major factor contributing to tenderness if the effect of connective tissue is small. The connective tissue may play a greater role in ten- derness of the rectus femoris muscle (Experiment V) than for other ‘muscles and therefore could eXplain the difference in the effect of EDTA treatment on tenderness of the different muscles. It is also possible that the difference in the effect of EDTA treatment on tenderness could be the result of differences in permeability of the EDTA in the differ- ent muscles. Protein fractionation Table 9 shows the averages for the nitrogen composition of the different fractions of the longissimus dorsi muscle of the rabbits for the five treatment groups in Experiment II at both 0 hr and 24 hr post- mortem. There was no appreciable change in the solubility of the sarco- plasmic fraction during the first 24 hrs postdmortem. There was, however, a definite increase in the amount of the fibrillar protein fraction extracted at high ionic strength (solution B) during the first 24 hrs postdmortem. According to weinberg and Rose (1960), the precipitate '1‘} .ll ' 00000000 nHouonm H0000~000 0005000 00000080» 0095 . coonuHG o00uoum so20 0000.0 0 s 00 0000000000000 a00uo0um a0000eouo0 0090 0000.0 n a 00 00000000 :00000um d0000a 0090 .suwnouum 00000 0000 00 00000Hux0 GOH000H0 000u0H0 H0000~00m 0090 .H00uo £000 scum 000H00000 0000000000000 000 0H0 mu00H00H00nm usosuHB 00002 .A00. V 00 Honuo 0000 2000 ud0u00000 0000000000000 0H0 0000Homu0asm 000000000 0003 000020 .000008 0\z 08.00000000800 0000H0020 00 0 H 00.50 00.0 H 00.0 00.0 H 00.0 00.0 H 00.0 05.0 H 00.0 50.0 H 00.00 0900 00.0 H 05.00 00.0 H 05.0 00.0 H 00.0 00.0 H 00.0 05.0 H 50.5 50.0 H 00.0 0ouuaoo 00.0 H 00.00 00.0 H 00.0 05.0 H 00.0 50.0 H 00.0 00.0 H 00.0 00.0 H 55.00 0900 m, 0.0 H 00.00 00.0 H 00.0 00.0 H 00.5 00.0 H 00.0 05.0 H 00.0 00.0 H 05.0 00000x0 02 n.00.0 H 00.00 00.0 H 00.0 00.0 H 00.0 00.0 H 00.0 05.0 H 00.0 50.0 H 00.00 090m Eouuoerumom an 00 00.0 H 05.00 50.0 H 00.0 00.0 H 00.0 00.0 H 00.0 00.0 H 055.0 00.0 H 00.00 0900 00.0 H 00.00 50.0 H 00.0 00.0 H 00.0 00.0 H 00.0 00.0 H 0000.0 00.0 H 00.0 0ouuooo 00.0 H 00.00 00.0 H 00.0 00.0 H 50.0 00.0 H 05.0 00.0 H 0000.0 00.0 H 00.00 0900 00.0 H 00.00 00.0 H 00.0 00.0 H 00.0 00.0 H 00.0 50.0 H 000.0 00.0 H 00.00 00000x0 0z 00.0 H 05.00 50.0 H 00.0 00.0 H 00.0 00.0 H 00.0 00.0 H 000.0 00.0 H 00.00 0900 aouuoelumom H0 0 50 ooHus0o0 0zmz 00 GOHun0o0 00 nOHun0om 00 000u50om n00ouuHo uaoeu0ou9 G00u000 0080000oou00 0a .00 udoaHH0mxm 00 mu0000H scum 000058.00H00 00E00000aO0 000 no mo00uo0nm uo0000000 0:0 0% 000000o0800 oomonuHa onu How a00av000 mo Houuo 000pa0um 000 m0:00> 0002 .0 00009 I I! II I #l l -80.. formed upon the dilution of this fraction to an ionic strength of 0.225 is actomyosin. Thus, the increase in the amount of the fibrillar pro- tein fraction extracted at high ionic strength can be accounted for by an increase in the formation of actomyosin (solution E). Significantly less fibrillar protein extracted at a high ionic strength was obtained from the rabbits treated with CDTA (1.77 mg N/g) than for the rabbits treated with either EDTA (2.33 mg N/g) or Na Oxalate (2.22 mg N/g) at 10 min post-mortem. The other fractions at 10 min post-mortem did not differ Significantly between treatments. The data in Table 9 indicate that there was no significant difference between treatments for any of the muscle fractions at 24 hrs post-mortem. It should be pointed out, however, that there was a big difference in the actomyosin fraction of the various treatments at 24 hrs post- mortem. The rabbits treated with EGTA had the lowest concentration of actomyosin nitrogen with a value of 0.56 mg N/g as compared to 3.49 for the CDTA treated rabbits, 3.58 for the EDTA treated rabbits and 5.05 mg N/g for the control rabbits. These results suggest that the formation of actomyosin was inhibited by injecting chelating agents into the live animal. Correlations Simple correlation coefficients between pH and ATP in rabbit muscle (Experiment II) are presented in Table 10. These data indicate that Sig- nificant relationships exist between 0 hr ATP values and pH values at slaughter (P < .01) and at 24 hr post-mortem (P < .05). A significant -31- relationship was also observed between pH at 0 hr and 24 hrs (P < .05). However, the data indicate that the relationships were not statistically significant between the ATP levels at 24 hrs post-mortem and the ATP levels at slaughter or pH at either 0 hr or 24 hrs postdmortem. Table 10. Simple correlation coefficients between pH and ATP for rabbit muscle (Experiment II). pH pH ATP 0 hr 24 hr 0 hr pH - 24 hr .50* -- -- ATP - 0 hr .78** ,45* -- ATP - 24 hr .07 .22 -.01 * P < .05 **P < .01 Table 11 contains the Simple correlation coefficients between pH and ATP for muscle from the pigs treated with EDTA and the controls in Experiment V. The pH values 10 min post~mortem for pigs treated with EDTA showed no significant relationship with pH and ATP values at 24 hrs postdmortem. The pH values at 24 hrs post-mortem also showed no Signifi- cant relationship with the ATP values at 10 min and 24 hrs post-mortem for the treated pigs. For control pigs, however, 24 hr post-mortem.pH values were significantly related (P < .05) to 10 min pH values and to ATP values at 10 min post-mortem. Also, for control pigs, a low correla- tion was observed between ATP at 24 hrs post-mortem.and pH values at 10 min and 24 hrs post-mortem. The data in Table 11 also indicated that a significant relationship existed between ATP values at 10 min and 24 hrs for both treatments. -32- Table 11. Simple correlation coefficients between pH and ATP for muscle from pigs treated with EDTA and controls (Experiment V). pH - 10 min pH - 24 hr ATP - 10 min Treatment EDTA Control EDTA Control EDTA Control pH - 24 hr .33 .76* -- -- ATP - 10 min .91** .92** -.04 .83* —- -- ATP - 24 hr .61 .47 -.02 .70 .78* .76* * P < .05 **P < .01 Correlation coefficients (Table 11) Show significant relationships between ATP values at 10 min postdmortem and pH values at 10 min post- mortem.(P < .01) for both the treated and control pigs. The high rela- tionship found between ATP and pH values within 10 min post-mortem in Experiment 11 and V verify the results reported by Kastenschmidt ggual. (1964) and Aberle (1967). In attempting to assess the significance of the results of this experiment in relation to the development of rigor mortis, the role of ATP and its hydrolytic products on glycolysis should be established. Phosphofructokinase, the glycolytic enzyme catalyzing the conversion of fructose-l-phosphate to fructose-l,6-diphosphate, has been shown to be a rate limiting step in glycolysis (Regen et a1., 1964; Ozand and Narahara, 1964). Passonneau and Lowry (1962) and Mansour (1963) reported that the activity of this enzyme is strongly inhibited by ATP at concentrations normally present in the cell. However, the results of Passonneau and -g3- Lowry (1962) and Regen gpflgl. (1964) indicated that phosphofructokinase activity and subsequent glycolysis will increase if either the ATP concentration falls or the concentration of inorganic phosphate, adenosine diphOSphate (ADP) or adenosine monOphosphate (AMP) increases. Regen ggflgl. (1964) showed that a reduction of the contractile activity of the muscle as a result of a reduced concentration of calcium was al- ways associated with a decrease in glycolysis. However, these workers found that when contractile activity is increased by electrical stimula- tion, phosphofructokinase inhibition was relieved and glycolysis increased. This was accompanied by a reduction in the ATP concentration as the amount of AMP and inorganic phosphate increased. A strong tetanic contraction on stimulation was observed by Hallund and Bendall (1965) and Bendall (1966) to result in a more rapid rate of glycolysis as indicated by an increase in the rate of pH fall. The same workers suggested that the high rates of glycolysis with the associated pH dr0p and occurrence of poor quality in pork is determined to a large extent by the long term after effect of the nervous stimuli which reached the muscle during the sticking process. The present eXperiment has shown that calcium chelating agents inhibited shortening while micro-injections of CaClz resulted in increased shortening and an increased rate of ATP hydrolysis. Results of the present experiment suggest that the amount of calcium released following the nervous stimuli on slaughtering and the subsequent hydroly- Sis of ATP by the actomyosin and sarc0p1asmic ATPase systems could be associated with the rate of ATP degradation and glycolysis. -84- Simple correlation coefficients between pH and ATP to muscle short- ening, cooking losses and tenderness for rabbit muscle are presented in Table 12. The data indicate that no Significant relationship existed between shortening of the right, uninjected semitendinosus muscle and pH and ATP values at either slaughter or at 24 hrs post-mortem. Similarly, the relationships between Shortening of the left semitendinosus muscle injected with 0.1M CaC12 and of shear values to pH and ATP values were not Significant at either 10 min or 24 hrs post-mortem. However, cooking losses were significantly related to pH and ATP values at slaughter (P < .01) and pH values at 24 hr post-mortem (P < .05). On the other hand, cooking losses were not related to 24 hr ATP values. Table 12. Simple correlation coefficients between pH, ATP, muscle short- ening, cooking losses and tenderness for rabbit muscle (Experiment II). pH - 0 hr pH - 24 hr ATP - 0 hr ATP - 24 hr Shortening right S.T.1 .24 .34 .16 .06 Shortening left S.T.2 .34 .21 .05 -.05 Cooking loss -.72** -.45* .58** .11 Shear values .39 .16 .36 -.19 * P < .05 IlRight semitendinosus muscle - uninjected. **P < .01 2Left semitendinosus muscle - injected with 0.1M CaClz. Table 13 contains simple correlation coefficients between pH, ATP, muscle Shortening, color, cooking losses and tenderness for muscle from pigs treated with EDTA and the untreated controls (Experiment V). These -85- data indicate that there was no significant relationship between color score and pH and ATP values at either 10 min or 24 hrs post-mortem. The cooking losses for the control pigs were significantly related to pH values at both 10 min and 24 hrs post-mortem. This indicates that higher cooking losses were associated with lower pH values. The data in Table 13 also Show that pH values at 10 min and 24 hrs postdmortem,were not significantly related to muscle Shortening or shear values of the right and left hams. For the control pigs, no Sig- nificant relationship was observed between ATP values at 10 min and 24 hrs post-mortem and muscle shortening or shear values of the right and left hams. However, the pigs treated with EDTA did Show a significant relationship between ATP values at 24 hrs post-mortem and muscle shorten- ing as well as with shear values of the right and left hams. These results indicate that the higher ATP values at 24 hrs post-mortem in the treated pigs was associated with less shortening of the semitendin- osus muscle and a more tender product. 00. v.m** .000005 mdmooHpao0Hamm 000 00 000000 00000000 00.0 0 m0 000000000m000wwmzm - 00.: 005.: 00.: $05.: 50. 00.: 00.: 00.: E00 0.000 % : 003”? c.0000 _ fl. *3... 0o. 00... 0m. 00... no. om: as.- new? : 000005 H0000 .. 3.- 2. N0... *8... 00.- $50.- 5m: 860 med-68 00.: 00.: 00.: 00. 00. 05. 00. 00. 00000 00000 :0. *8... 0m. 0m... 0m: 3. «0. mm... 000238-00 60932 0000000 .0900 0000000 0.9000 00.3000 0900 00.5000 0.900 000500009 00 J0 : 090 0H5 00 : 090. 00 :0 : 00, 0H8 00 : 0011 .A> 000EHH00x00 0900 0003 0000000 0000 Ho 0000 0ouucoo How 000058 How 0000000000 000 000000 0:0xooo a.30oo «0:00000000 00owsa «090 «00 0003000 000000000000 GOHu000HHoo 000800 .00 00009 -37- Properties of Weber-Edsall Extracts in Experiment IV ATPase activity and turbidity The ATPase activity and changes in turbidity following the addition of ATP to Weber-Edsall extract were measured in the presence of 0.6M KCl with and without added MgClz. In the present exPeriment, the addi- tion of ATP to the Weber-Edsall extract was followed by a high initial rate of ATPase activity, during which 50-75% of the ATP was hydrolyzed within 1 min. As shown in Fig. 5, the rapid hydrolysis of ATP was accompanied by an almost instantaneous decrease in the turbidity of the solution. Following this, the ATPase activity declined to a low rate. The turbidity remained relatively constant until the level of ATP de- creased below 15%, at which time the turbidity increased. These results are in agreement with Weber and Hasselbach (1954) who reported a high initial ATPase activity in isolated myofibrils of rabbit muscle. They indicated that the high initial ATPase activity was not affected by the accumulation of end products, decreasing concentrations of ATP, impurity in the ATP solutions or by the concentration of myofibrils. In Fig.5 it can be seen that addition of M3012 suppressed the ATPase activity of Weber-Edsall eXtract and extended the period of time during which the actomyosin was dissociated (clear phase). Kaldor and Gitlin (1963) also found that magnesium inhibited the myofibrillar ATPase activit» if 0.3M‘KC1 was incorporated into the buffer. Present results agree with those of Noda and Maruyama (1958) who showed that addition of magnesium to a cleared actomyosin solution extended the time period for the clear phase. I (I. .I 'II' [III 1: , . III III III-Il- l! ljlli III III I l -33- 40 — A"! 35 "'""""‘ Turbidity 3O 25 ’ 110 ’I ‘ ----- -- = P-.----o-r”"‘°' ,I" 'u 20 I 100. " I "' I "' I " I '3 / fi 15 ” 9o 4" IO 80 l .. J .. .v' 70 O 60 10min 30min 60min 1hr 3hr 5hr Time Fig. 5. Effect of MgClz on the ATPase activity and relative changes in turbidity of pre-rigor Weber-Edsall extract at 25°C. (6) The reaction was started by adding 1 m1 of 0.01M ATP (pH 6.4) to 24 m1 of the diluted protein extract. (C) One ml of 0.01M MgC12 was added to 24 m1 of the diluted protein extract and the reaction was started by adding 0.01M ATP. Relative Change in Tuthidity (x) Ill-Ill III! I III I I1 I‘ I! .l I : I. el -89- Maruyama and Gergely (1962) and Ikemoto (1966) suggested that the low ATPase activity of the actomyosin solution in the presence of magne- sium, as found in the present experiment, is attributable to the magne- sium inhibited ATPase of the myosin moiety. From the data in Table 14, it can be seen after five hours that the concentration of ATP in the absence of MgClz was about five times as high as the level in the presence of MgClz. This may be explained by the high affinity magnesium has for myosin if present in high concentra- tions (Szent-Gygrgyi, 1951). Szent-Gygrgyi (1951) further indicated that bound magnesium ions result in the adsorption of ATP to the myosin molecule. Results of Levy and Ryan (1966) indicated that magnesium.and ATP are bound to the hydrolytic site on myosin. Therefore, more complete hydroly- sis of ATP in the presence of.MgC12 may be the result of increased binding of ATP to the hydrolytic site. Table 14. Effect of MgC12 on the ATPase activity of the weber-Edsall extracts at 25°C. The reaction was started by adding l‘ml of 0.01M ATP (pH 6.4) to 24 m1 of the diluted protein extract. When'MgClz was added, l'ml of 0.01M MgClz was added to 23 ml of the diluted protein extract and the reaction was started by adding ATPl. Post-mortem time Added Time after adding_ATP1 of extraction MgClz l 10 15 20 2 4 5 min ‘min min min 1 hr hr hr hr 0 hr Yes 37.8 31.0 24.8 17.6 8.87 4.2 1.2 1.2 0 hr No 31.2 14.6 8.1 7.0 7.8 7.5 6.6 5.0 24 hr Yes 39.8 33.0 26.8 18.4 8.5 4.8 1.8 1.0 24 hr No 32.6 22.5 11.8 9.1 10.1 5.5 6.2 IValues EXpressed as Z of added ATP. -90- The ATPase and turbidity values for the different extraction times are shown in Tables 14 and 15. Except for the lower turbidity values at 1, 10 and 15 min for samples extracted 24 hrs post-mortem, no differ- ences were observed in ATPase activity and turbidity between the Weber- Edsall extract isolated from pre-rigor muscle and muscle in rigor. Although there was some variation among the preparations from the six rabbits, there was not any consistent difference between samples from different treatments. These results suggest that differences observed between the properties of pre-rigor and rigor muscle are not a result of a change in the actomyosin ATPase activity or the ability of actomyosin to dissociate and the actin and myosin to form complexes with each other. Robson et a1. (1966) also found little difference in enzymatic activity between myosin B isolated from pre-rigor, rigor or post-rigor muscle. Table 15. Effect of.MgC12 on the relative change in turbidity of the Weber-Edsall extracts at 25°C following the addition of ATP. The reaction was started by adding l‘ml of 0.01M ATP (pH 6.4) to 24 m1 of the diluted protein extract. When MgClz was added, l‘ml of 0.01M Mg012 was added to 23 ml of the diluted protein extract and the reaction was started by adding ATP. Time after adding ATPl Post-mortem time Added 1 10 15 30 1 2 4 5 of extraction M3012, min min min min hr hr hr hr 0 hr Yes 68.9 72.6 74.3 81.8 91.5 105.8 112 0 hr No 71.0 73.6 92.7 102.6 103.6 104.8 105.0 24 hr Yes 62.8 66.5 69.6 74.3 81.2 91.9 107.7 24 hr No 63.9 67.6 73.0 102.9 105.0 106.9 IValues expressed as relative change in turbidity. -91- However, somewhat different results have been reported by Fujimaki gt 31. (1965). At low ionic strength, they showed that extracts of post-rigor muscle stored at 4°C for 2 days had higher ATPase activity than extracts of pre-rigor muscle. Viscosity The changes in viscosity following the addition of potassium pyrophos- phate to the Weber-Edsall extract was measured in the presence of 0.6M KCl, with and without added MgClz. Preliminary results, (Fig. 6) showed that the decrease in viscosity was faster and more complete on adding MgClz. The change in viscosity was also faster and more complete at 3°C than at 23°C (Fig. 6). The data in Table 16 indicates very little differ- ence in viscosity between Weber-Edsall extracts from pre-rigor and rigor muscle following the addition of perphOSphate. Table 16. The relative changes in viscosity of the Weber-Edsall extracts from pre-rigor and rigor muscle following the addition of perphosphate. The changes in viscosity were recorded at 3°C following the addition of 1 m1 of 0.1M potassium perphosphate to 24 m1 of the Weber-Edsall extract containing approximately 0.2% protein and 0.4 mM added MgC12. Postemortem time Time of extraction 5 min 1 hr 5 hr 0 hr 47.0 47.0 47.0 24 hr 47.9 46.4 47.0 A number of investigators (Noda ananaruyama, 1958; Watanabe and Duke, 1963; Tonomura and Sekiza, 1961; Yasui SE $1., 1964; Mihalyi and ' Relative Change In Viscosity (x) -92- 100 - _ .. -‘ VI 80 ' I II A 70 B C MR 60 M'\HI\ 10ml: 3.0qu 60min 1hr 3hr {Shr 10hr 15hr Timm 50 Fig. 6. Effect of MgClz and temperature on the relative change in viscosity » of 24 hr post-mortem Weber-Edsall extract (approximately 0.2% protein). (A) Viscosity was measured at 3°C following the addition of 1 m1 of 0.1M potassiun pyrophosphate to 24 m1 protein extract. (B) One ml of 0.1M potassiun perphosphate was added to 24 m1 of the protein extract containing 1 ml of 0.01M MgClz. Viscosity was recorded at 3°C. (C) Same solution as for (B) but viscosity was recorded at 23°C. '\ 'all'llllltnlll -Illll‘l‘llllll' ‘ I . l ‘l | ‘ ‘17 Y 1‘ I II In III- lx -93- Rowe, 1966) have shown that pyrophosphate in the presence of magnesium is as effective in clearing actomyosin as ATP. Results from this experi- ment (Fig. 7) also indicate that perphosphate in the presence of magne- sium is as effective in clearing actomyosin as ATP. As the ATP was hydrolyzed, however, the viscosity of the solution returned to values greater than those obtained before the addition of ATP. 0n the other hand, samples cleared with pyrophosphate in the presence of magnesium indefinitely retained low viscosity characteristics. The dissociating influence of the perphosphate system, as observed in this eXperiment, could be conditioned by the Mg-pyrophOSphate complex (Granicher and Frick, 1965). Martonosi and Meyer (1964) concluded that the binding site for the perphosphate on myosin is identical with a portion of the ATPase center of myosin. Results of the present experi- ment suggest that perphosphate may be bound to the site on myosin that is responsible for the splitting of the actomyosin complex, but it is not involved in the formation of the complex formed between actin and myosin. -94- A 25 120 h “ '3 O 0 G > ‘100 O n B .2 . ° 80 ' O .2 I «v I 2 I = : 60 I u. 40 10min 30min 60min 1hr 2hr 3hr 4hr Time Fig. 7. The relative changes in viscosity of weber-Edsall extracts from rigor muscle. One ml of 0.1M potassiun pyrophosphate or 0.01M ATP (pH 6.4) was added to 24 m1 of Weber-Edsall extract contain- ing approximately 0.2% protein and 0.4 mM added MgClz. Changes in viscosity were recorded at 23°C for ATP (O) and at 3°C for potassiun pyrophOSphate (Q). SUMMARY The effects of various chelating agents on the physical and chemical properties of muscles from rabbits and pigs were investigated in five different experiments. The effect of added calcium and magnesium on muscle properties was also studied. The intravenous antemortem injection of various chelating agents (EDTA, CDTA, EGTA or Na Oxalate) significantly inhibited shortening of the semitendinosus muscle during the development of rigor in both rabbits and pigs. 0n due other hand, micro~injections of CaC12 into muscle resulted in a definite increase in shortening during the development of rigor. However, increased levels of MgClz had no effect on shortening. Chelating agents had no consistent effect on ATP levels of rabbit muscle. However, the ATP values for the pigs treated with EDTA were generally higher than for the controls, but the differences were not significant. There was a definite increase in the rate of ATP hydrolysis following the micro-injection of CaClz into the semitendinosus muscle. Antemortem injection of chelating agents had no consistent effect upon either the elasticity or rigidity of uninjected muscles at 7 hrs post-mortem. .Muscles, which were elastic after 7 hrs post-mortem, had significantly higher (P < .01) levels of ATP than those that were rigid and inextensible. Paired semitendinosus muscles injected with 0.1M CaClz were always rigid and inextensible at 7 hrs post-mortem. No consistent differences in pH values were observed between treat- ments. In Experiment II, however, pH values for muscles from rabbits -95- -96- treated with EDTA.were significantly lower (P < .05) than for the con- trols, or those treated with EGTA. Treatment had no significant effect on subjective color score or water holding capacity. Antemortem treatment had no significant effect upon cooking losses. Shear values of the longissimus dorsi muscle for EDTA-treated rabbits were lower than those of the control groups, but the differences were not significant. Antemortem injection of pigs with EDTA resulted in a marked improvement in tenderness as shown by significantly lower (P < .05) shear values of the ham muscles. The effect of treatment on tenderness varied between muscles. Although there was no appreciable change in the solubility of the sarc0p1asmic protein fraction during the first 24 hrs post-mortem, there was a definite increase in the amount of fflarillar protein extracted at high ionic strength. This could be accounted for by an increase in the formation of actomyosin. Muscles treated with either EGTA, CDTA or EDTA had less actomyosin at 24 hrs post-mortem than the controls, but the differences were not significant. A highly significant relationship (P < .01) was found between ATP and pH values within 10 min post-mortem for muscles from rabbits and from both treated and control pigs. The treated pigs had higher ATP values at 24 hrs post~mortem, which were significantly (P < .05) and negatively related to shortening and shear values. No consistent differences were observed in ATPase activity, turbidity and viscosity between the Weber-Edsall extract isolated from pre-rigor -97- muscle or muscle in rigor. The addition of MgClz decreased the rate 0f ATP hydrolysis and extended the clear phase of the weber-Edsall extract. Perphosphate in the presence of magnesium was as effective in clearing actomyosin as ATP. 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