THE RESOLUTION of RIGOR MORTIS Introduction D

16.

THE RESOLUTION OF RIGOR MORTIS Introduction D. E. GOLL IOWA STATE ...... UNIVERSITY

The words "rigor" and "mortis" originate from Latin, "rigere", to be stiff, plus "or", more, and from llmortuusI', the past participle of "mori", to die. Thus, rigor mortis may be literally translated as "stiffness of death", and correct usage of the term "rigor" must be restricted to those situations involving stiffness, rigidity, or inflexibility. This stiffness or rigidity was frequently noted in corpses of exhausted subjects such as soldiers killed in battle, and the term "rigor mortis" first came into widespread use in connection with forensic medicine. I am emphasizing the close and direct relationship between rigor mortis and stiffness at the outset because, as aptly pointed out at last yeaz's Meat Industry Research Conference by Professor R. E. Davies (15), much of the confusion regarding rigor mortis, and pzrticularly the resolution of rigor mortis, has arisen because of failure to generally recognize that rigor has two closely related but quite different aspects. These two aspects are: 1) a shortening or contraction of the muscle fiber, and 2) a loss of muscle extensibility. To help clarify my discussion this morning, I wish to further divide the extensibility aspect of rigor into a macroscopic phase and a molecular phase. The macroscopic phase of extensibility can be directly observed and measured as ability of a muscle fiber or a bundle of muscle fibers to stretch under the influence of a given weight or force. The molecular phase of extensibility, on the other hand, refers to the ability of actin and myosin filaments in a single sarcomere to slide past one another. When the actin and myosin filaments in a single sarcomere interact, i.e., when actomyosin is formed, molecular inextensibility results in that sarcomere. If a whole series of sarcomeres experience loss of molecular extensibility, then macroscopic inextensibility results. The reason for making this very subtle differentiation between macroscopic and molecular extensibility will become evident later on when I present evidence that molecular inextensibility is partially reversible in post-mortem muscle whereas macroscopic measurements of inextensibility do not appear to change during post-mortem storage. In this presentation, I will distinguish between the macroscopic and molecularr phases of rigor only when necessary and hereafter, when I speak of just "extensibility", I will be referring to both the molecular and macroscopic phases. It is now clear that post-mortem muscle always experiences a loss of both macroscopic and molecular extensibility, but under certain conditions of temperature, post-mortem shortening or contraction may be so feeble as to escape detection. The thesis I wish to develop in this presentation is that, although molecular inextensibility is a necessary condition for the rigidity or stiffness observed in rigor, neither molecular nor macroscopic inextensibility is itself the primary cause of rigor, i .e., stiffening or hardness, but both are instead more closely related to adenosine triphosphate (A!I'P) depletion in post-mortem muscle. On the other hand, post-mortem shortening or contraction is a direct cause of rigor mortis since attempted shortening by two muscles on opposite sides of a 17. bone would produce a rigidity or stiffening similar to that observed in an isometric contraction. The inextensibility aspect is an essential part of rigor only because the molecular inextensibility condition keeps post- mortem muscle in the shortened state after its ATP supply has beer_ exhausted and it can no longer actively generate tension. If the sarcomeres in post-mortem muscle were not inextensible, they would be free to return to their rest length after the muscle's energy supply had been exhausted, and this would result in loss of the stiffening or rigidity caused by the shortening. Once this clear distinction between the extensibility and shortening aspects of rigor is realized, it becomes necessary to talk about resolution of rigor in terms of loss of the ability of post-mortem muscle to maintain its shortened state. I will show later in this presentation that such loss in ability to maintain a shortened or contracted condition does in fact occur in post-mortem mxxle, and it is therefore pedagogically correct to talk of a "resolution of rigor."

Chemical Changes in Post-Mortem Muscle

AS a prelude to detailing the evidence for a resolution of rigor mortis, it will be necessary to review some of the changes which occur in post-mortem muscle and to point out clearly, both in terms of measurement techniques and molecular events, the differences between the extensibility and the shortening aspects of rigor mortis. In the 1800's, the grossly observable stiffness or rigidity associated with rigor mortis was thought to originate from a coagulation of the muscle proteins caused by lactic acid, which was known in those times to appear in post-mortem muscle. Although the lactic acid theory was shown to be untenable already in 1877 by Claude Bernard * s observations that exhausted animals frequently exhibited an alkaline rigor, i.e., rigor at muscle pH values near or above neutrality, this lactic acid theory has persisted and can still be found in various mcdif ied forms in some modern textbooks. Bernard ' s observations were later confirmed and extended by Hoet and Marks (27) who, as a result of their studies, postulated that the onset of rigor mortis may be related to disappearance of one of the sugar phosphates. Then in 1943, Erdos (17) showed that the disappearance of ATP was closely related to the onse3 of stiffening in post-mortem muscle and soon afterwards the central role of ATP in rigor mortis was established, largely as a result of the work of E. C. Bate-Smith, J. R. Bendall and their coworkers at Cambridge (1, 2, 3, 4, 5, 6, 7). These early Cambridge studies elucidated many of the rela- tionships among ante-mortem stress, glycogen level, ultimate pH, ATP degradation, and the onset of rigor mortis that are generally accepted yet today. On the basis of these studies, the following chemical changes are now considered to be more or less characteristic of post-mortem muscle: 1) anaerobic breakdown of muscle glycogen to lactic acid, starting imedi- ately after death; 2) a decrease in pH due primarily to the formation of lactic acid; 3) a fall in phosphocreatine content of muscle, this fall occurring very rapidly after muscle glycogen reserves have been exhausted; 4) a decrease in ATP concentration, this decrease occurring very slowly until after the disappearance of phosphocreatine after which time it pro- ceeds rapidly usually to a level less than 20% of its initial level. 1%.

Physical Changes in Post-Mortem Muscle

The physical changes observed in post-mortem muscle will be rien- tioned only briefly, since this subject has been diseased by the preceding speaker. It was quickly recognized by the early investigators that success in rigor mortis research would depend in large part on the avail- ability of an easy and convenient method. for quantitatively and repro- ducibly determining the time of onset of rigor. A kymograph-like apparatus for doing this was developed in 1949 by Bate-Smith and Bendall (4). This apparatus alternately applied a load to a muscle strig for 8 niinutes, and then removed it for 8 minutes after which the cycle w&s repeated until rigor was complete. The extension of the muscle strip caused by application of the load was recorded, and the muscle was said to be in rigor when application of the load failed to cause any extension of the strip. This apparatus, or the extensibility principle employed by this apparatus (8, 9), has been widely adopted and until recently was almost the only method used for measuring the onset of rigor mortis. With this method, it has been shown that post-mortem muscle extensibility decreases rapidly when the ATP level falls to less than 50$ of its initial level, and that once post- mortem muscle becomes inextensible it does not fully regain its macroscopic extensibility again even though it be stored under aseptic conditions for many days. Thus, using the kymograph-type of instrwnent to measure rigor and defining rigor solely in terms of this measurement, i.e., loss of macroscopic extensibility, leads to the conclusion that there is no resolution of rigor mortis.

Recently, we have developed an instrument, which we called an "isometer", for determining the onset of rigor mortis (11,29). This instrument was designed to measure post-mortem tension development in a muscle strip held at a constant length, thereby simulating the conditions to which post-mortem muscle attached to the skeleton --in situ is exposed. The sensitivity and capacity of this method of measuring rigor has been increased through the use of isometric myographs in conjunction with an E & M Physiograph. A major improvement in versatility of the isometric measurement of rigor was made when it was realized that mmcle strips immersed in mammalian Ringer's solution exhibited the same patterns of tension development as strips in air. Immersing the strips in solution eliminates spur- ious tension patterns due to drying of the strips in air and also affords the opportunity to control and study the effects of pH, ionic environment, and the addition of fresh ATP, sulfhydryl reagents, etc. on the tension development of post-mortem muscle. This new apparatus is shown in Fig. 1, 2, and 3. It is obvious that the isometric tension measurement of rigor mortis is a direct measure first of the shortening tendency of post-mortem muscle, and then subsequently of the ability of the muscle to maintain its shortened state.

In the past several years, it has been realized that temperature has large and oftentimes unexpected effects on the onset of rigor mortis. Locker and Hagyard (30) first reported that in the case of bovine muscle, post-mortem shortening is maximal at O0C, decreases as the temperature is raised from 0-16'C, passes through a minimum at 16OC, and then increases as the temperature increases from 16-4OoC, although the shortening at 4OoC vias not as great as that at 2OC. Similar patterns of post-mortem shortening have been regorted for porcine (22) and ovine (13) muscle, whereas 19. post-mortem shortening of rabbit muscle appears to increase continuously with increasing temperature in the range O-4O0C, and very little shortening can be observed in post-mortem rabbit muscle at O°C(lO). Furthermore, as shown in Fig. 4 and 5, ATP level in bovine muscle at the time the muscle becomes inextensible is much greater at l0C than at 37OC. It is also evident from Fig. 5 that the shortening and the macroscopic extensibility aspects of rigor are easily distinguished at l0C.

Interpretation of Post-Mortem Chemical -and Physical Changes in Molecular Terms

Myofibrils are now known to consist of an interdigitating array of thick and thin filaments, arranged in a fashion thzt naturally confers a striated appearance on the muscle cell (Fig. 6). Muscle shortening or contraction is accomplished by a sliding together of this interdigit3ting array of filaments, with the force for the sliding apparently being gener- ated at the region of overlap of the thick and thin filaments. As shown in Fig. 7, the thick or myosin filaments possess a number of projections or cross-bridges extending from their surface out toward the thin or actin filaments. It is these cross-bridges which actually interact with the actin filaments and this interaction, in the presence of ATP, Mg'+, and Ca*, will produce a contraction. An impressive amount of evidence has now accumulated which suggests that ATP has a dual role in the actin-myosin interaction. On the one hand, splitting of the terminal phosphate from the ATP releases energy which can be used to drive the contractile process. On the other hand, it has been shown that high (5-10 mM or about physiological) levels of ATP in the presence of equal amounts of Mg" (also about physiological levels), and very low (less than lom6M) levels of Gaff will prevent interaction of the thick and thin filaments, thereby causing relaxation. Recognizing these two roles of ATP and in terms of the thick and thin filament structure of muscle, it is reasonable to assume that the molecular inextensibility aspect of rigor results when the ATP level in post-mortem muscle falls to a level too low to effectively prevent the interaction between the actin and myosin filaments. When this happens, almost all of the cross bridges on the myosin filament will be bound and "locked" to some site on the actin filaments, and the interdigitating filaments will no longer be free to slide past one another. The macro- molecular manifestation of this "locking" of the interdigitating filament array in a whole series of sarcomeres is macroscopic inextensibility of the fiber. It is obvious, therefore, that there is an easy and direct connection between A!TP level and the extensibility aspect of rigor, and furthermore that since post-mortem muscle will always eventually lose mosf, of its ATP, post-mortem muscle will also always undergo a loss of both macroscopic and molecular extensibility. Moreover, since post-mortem muscle does not itself possess the means for regenerating ATP after its glycogen reserves have been exhausted, it would appear that the extensibility aspect of rigor is irreversible unless exogenous agents are added, and that in terms of the extensibility criterion, there is no resolution of rigor mortis. However, as will be shown later in this presentation, there appesrs to be some weakening of the actin-myosin interaction in post-mortem muscle and this weakening can apparently lead to some "slippage" between the actin and myosin filaments, even in the absence of ATP. This implies that there is a partial reversal of molecular inextensibility even though it is clear that the present measurement techniques cannot detect any reversibility of the macroscopic phase of inextensibility in post-mortem muscle. It is also 20. clear, when considering the thick and thin filament structure of muscle, that "locking" of the interdigitating filaments in post-mortem muscle can occur when a muscle is at rest length or at any stage of contraction from slightly to severely shortened. It is difficult to envision how z simple "locking" of filaments at either rest length, or at lengths greater than rest length, would by itself cause much increase in rigidity or hardness of the cadaver. Indeed, Cassens (12) has stated that in some cases muscle may not exhibit any detectable change in stiffness or hardness during post-mortem storage. Such muscle has probably not undergone any appreciable post-mortem shortening and is therefore in only very weak "rigor", at least in the sense of stiffness or rigidity. These observations suggest that the extensibility aspect of rigor is not the primary cause of the stiffness or rigidity which by definition constitutes rigor mort is.

The shortening or contractile aspect of rigor is very poorly understood, but it ostensibly depends on the occurrence of a contractile- producing stimulus at some time before ATP content of the post-mortem muscle fiber is exhausted. The nature of this contractile-producing stimulus can only be speculated upon at the present time, but it is quite possible that in many cases it involves the release of Ca? from the sarcoplasmic reticulum. The important thing to note, however, is that this stimulus may or may not occur depending on post-mortem temperature and possibly on other, as yet, unknown effects. If this contractile-producing stimulus occurs soon post-mortem while ATP levels in the muscle are still relatively high, considerable shortening and appreciable post-mortem tension will result. If, on the other hand, the contractile-producing stimulus occws later post-mortem, after the muscle A_Tp level is considerably lower than its original value, very little shortening and only weak tension development will result. There will be some very slight shortening even in the absence of the contractile-producing stimulus, because when ATP concentration in post-mortem muscle falls to a level too low to prevent interaction between the actin and myosin filaments, the very small amount of ATP remaining at this time may be used by the interacted filaments to provide energy for a very limited amount of sliding. The fact that both the shortening and the extensibility aspects of rigor are related to muscle ATP levels and therefore often appear to proceed almost in parallel in post-mortem muscle has quite possibly contributed to the failure of many investigators to distinguish between these two separate phenomena. However, in terms of our present concepts of muscle structure and biochemistry, it is obvious that post-mortem shortening mGst occur before total loss of molecular extensibility since the latter event designates that the interacting filaments have now become "locked" at some fixed position and cannot slide past one another. There will be some loss of molecular extensibility accompanying the shortening process itself because by definition, shortening is caused by an interaction between thick and thin filaments. However, Huxley and Brown (28) have shown that in contracting muscle only about 203 of the cross-bridges on the myosin filament are actually bound to the actin filament at any given instant. There- fore, post-mortem muscle in its shortening phase should be about five fold more extensibile than post-mortem muscle after completion of the extensibility phase. The preceding considerations demonstrate that at the molecular level, the shortening and extensibility aspects of rigor may be clearly distinguished from one another, both in time and in mechanism. 21.

Rigor Mortis and Tenderness Before presenti% evidence for the existence of a resolution of rigor, I wish to point out how a clear distinction between the extensibility and shortening aspects of rigor may also lead to clarification of the relationshlp between tenderness and rigor mortis. For many years now, meat scientists have believed that muscle immediately after death is quite tender, whereas muscle in rigor is extremelytough. After 2-3 days of post-nortem storage, this toughness appears to pass and the muscle gradually becomes tender again. This increase in tenderness has been described by the meat scientist as a "resolution of rigor." When the Cambridge investigators reported that there was not any large increase in macroscopic extensibility after 2-3 days post-mortem and thus no "resolution of rigor", at least in the macroscopic extensibility sense, the cause of the post- mortem tenderization seen so clearly by the meat scientist became shrouded in controversy and mystery. If, however, rigor mortis is defined in terms of shortening and the ability to maintain the shortened state, there is a clear relationship between post-mortem tenderness changes and the state of rigor in bovine muscle stored at Z°C. It has already been mentioned that Locker and Hagyard (30) have shown that bovine muscle stored at Z0C under- goes a substantial amount of post-mortem shortening, and as I am sure Dr. Herring will point out in his presentation, there is a general relationship between degree of shortening and tenderness for bovine muscle stored at Z0C. It now becomes evident, therefore, that the decreased tenderness observed in bovine muscle after 6-24 hrs post-nortem at 2O is in fact due primarily to the circumstance that this muscle is shortened substantially and is not due to the loss of extensibility in this muscle. ,/ This fact was first clearly demonstrated in 1964 when we observed that bovine muscle that was excised from the skeleton immediately after death and was therefore free to shorten exhibited the classical pattern of post- mortem tenderness changes, becoming extremely tough after 6-24 hrs post- mortem at 2O, and then gradually increasing in tenderness up to 13 days post-mortem (23). On the other hand, bovine muscle which was not excised from the skeleton and was therefore not free to shorten, did not undergo any post-mortem toughening but instead gradually increased in tendersness with increasing time of post-mortem storage at Z°C (Fig.8). This differ- ence in post-mortem tenderness pattern occurred even though both excised and intact muscles became inextensible at approximately the same time post- mortem. These results clearly show that post-mortem tenderness changes are not determined primarily by extensibility changes in post-mortem muscle but we in fact directly related to the shortening aspect of rigor.

Resolution of Rigor

Now that the distinction betwen the extensibility and shortening aspects of rigor has been made clear and the reasons for selection of the shortening aspect as the primary cause of rigor mortis have been discussed, I wish to present two lines of evidence that a resolution of rigor mortis does in fact occur and that this resolution involves, at least in part, a weakening, although not a complete dissociation, of the interaction between the actin and myosin filaments. The first of these two lines of evidence involves our isometric tension measurements on post-mortem muscle. As shown in Fig. 9, muscle held isometrically will begin to develop tension immediately after death, this tension development increasing to a maximum 22. after some varying period of post-mortem storage. Both the exact time at which maximum isometric tension development is attained and the amount of tension developed per unit cross-sectional area of the m-dscle depends on temperature, species, and other conditions such as physiological state of the animal at the time of slaughter, etc. The important feature in Fig. 9, however, is that regardless of species or temperature of post-mortem storage, the ability to maintain isometric tension slowly declines after the point of maximum tension development, and if the experiments are done carefully and over a sufficient period of time (usually 48-72 hours), isometric tension development will return to zero and stay there. These results are somewhat different from our earlier findings that a decrease in isometric tension development occurred only when the muscle was held at 2' (11,29) . We have now ascertained, however, that these early results were due to the lower sensitivity of our original isometer as well as to some unavoidable drying of our strips in air at the higher temperatures. We feel that the decline in the ability of post-mortem muscle to maintain isometric tension development is analogous to loss of the ability to maintain a contracted or a shortened state and therefore corresponds directly to a resolution of rigor. Hereafter in this presentation, when I talk of resolution of rigor, I will be referring to this period of declining isometric tension development .

The second line of evidence suggesting that a resolution of rigor occurs in post-mortem muscle is the observation that sarcomeres which have undergone extensive post-mortem shortening will, after three to four days post-mortem, actually lengthen a.gain. This lengthening was first observed independently and almost simultaneously by Gothard --et al. (25) working with bovine muscle at Louisiana, by Stromer working with bovine muscle in our laboratory (33, 34, 35), and by Yasui and coworkers (38) working with chicken muscle in Japan. Fig. 10, 11, and 12 show electron micrographs of samples taken from a single bovine animal at-death and after 24 and 312 hours of post-mortem storage at 2'. It is obvious that the sarcomeres of this muscle have first shortened considerably and have then spontaneously, and in the absence of ATP, lengthened or "relaxed." The micrographs also clearly show that both shortening and "relaxation" have occurred by means of a sliding of interdigitating filaments past one another. The observation that post-mortem r'relaxationt' involves a sliding of filaments strongly suggests that in post-mortem muscle there is some weakening or slight dissociation of the interaction between the thick and thin filaments, even in the absence of added ATP. This weakening or slight dissociation corresponds to a partial reversal of the molecular inextensibility phase of rigor. The dissociation or reversal cannot be complete since we have observed, in confirmation of the early Cambridge results, that post-nortern muscle fibers never fully regain their original macroscopic extensibility. It should be noted, however, that the macroscopic extensibility measurement may not be a very useful test for detection of partial dissociation of the actin-myosin interaction in post-mortem muscle. Although it seems reasonable to assume that such partial dissociation may best be detected as a small increase in macroscopic extensibility under the influence of heavier weights or larger forces than those normally used, application of such weights or forces to post-mortem muscle strips causes rupture or breaking of the strips, even though the same weight or force was supported without any ill effects by at-death muscle strips of the same size. This increased fragility of post-mortem muscle strips has thus far prevented us from 23. directly testing by macroscopic extensibility measurements the theory of a weakened actin-nyosin interaction in post-mortem muscle.

What Causes Resolution of Rigor

The two lines of evidence just presented plainly show that a resolution of rigor does indeed occur in post-mortem muscle and the ques- tion now becomes "what are the changes in the molecular architecture of the myofibril that cause this resolution?". Some recent experiments, both in our laboratory and in Dr. Yasui's and Fujimaki's laboratories in Japan have suggested to us that resolution of rigor is caused primarily by two kinds of alterations in post-mortem muscle. I should emphasize at this point that we certainly do not exclude the possibility that there are other changes also contributing to the resolution process nor do our data conclu- sively link the two changes I shall mention to resolution. However, the data are very suggestive and it may be instructive at this point to review these data and the reasoning that led us to our hypothesis.

The two alterations in post-mortem myofibrils that we feel are primary contributors to the resolution of rigor mortis are: 1) loss of Z-line structure and weakening and eventual rupture of the bonds between the I and Z filaments; and 2) weakening of the actin-myosin interaction, this weakening possibly being caused by a very specific and limited proteolysis of myosin, actin, and/or one of the regulatory proteins.

The first of these two changes, loss of Z-line structure, was initially observed independently and almost simultaneously by Stromer (34,35) working in our laboratory with bovine muscle and by Yasui and coworkers (21) working with chicken muscle in Japan. Cook and Wright (14) had earlier shown that Z-lines were severely disrupted in prerigor frozen ovine muscle, but these findings were difficult to interpret in terms of post-mortem Z-line degradation because of possible effects of freezing and thaw contracture on Z-line structure. Recently, Henderson (26) in our laboratory has shown that the Z-line in post-mortem porcine muscle also loses its characteristic zigzag appearance and appears in the form of large amorphorus clumps (Fig. 13, 14, and 15). Post-mortem chicken muscle, on the other hand, appears to lose its Z-line entirely (Fig. 16 and 17) although some of this loss may be because the studies with chicken muscle were done on washed myofibrils whose degraded Z-lines had been removed by the washing process. It is interesting to note that loss of Z-line structure in bovine muscle requires several days if the muscle is stored at temperatures of 2 or 16OC, but will occur within 8-24 hrs post-mortem if the muscle is stored at 25 or 37OC. That these times correspond closely to the times of increased tenderness in bovine muscle at these storage temperatures suggests that Z-line degradation may be related to the tenderness increase observed in post-mortem muscle. In his extensive examination of Z-line ultrastructural changes in post-mortem muscle, Henderson (26) has discovered some remarkable species differences in susceptibility of the Z-line to degradation during post-mortem storage. Z-lines from both porcine and rabbit muscle appear to be much more labile to post-mortem muscle conditions than Z-lines from bovine muscle. It is not uncommon to find that Z-lines in even unwashed porcine or rabbit myofibrils are completely absent after 24 hours of post-mortem storage at 25'. In many cases in such muscle, crystalline Z-line material may be seen lying in the sarcoplasm 24. next to the fibrils (Fig. 18). These observations show that the bonds between the I and the Z filaments may be completely ruptured in post-mortem porcine or rabbit muscle, even in the absence of any washing or homogeniza- tion.

Further evidence for a post-mortem degradation of Z-lines comes from phase microscopic observations of myofibrils prepared from post-mortem muscle. Both Yasui * s group (38) and Stromer (33) and Henderson (26) have noticed that myofibrils prepared from muscle after 2-3 days of post-mortem storage appear highly fragmented, with many of them being only one, two, or three sarcomeres in length, whereas myofibrils prepared from zt -death muscle contain eight to ten or more sarcomeres (Fig. 19 and 20). This fragmentation of post-mortem myofibrils appears to occur principally at the level of the Z-line, suggesting that post-mortem myofibrils have been weakened at this point so that they break or rupture during the homogeniza- tion procedure used in myofibril preparation.

The structural evidence just described clearly shows that considerable degradation of the Z-lines together with weakening and rupture of the I-Z junction occurs in post-mortem muscle. However, the exact cause of the Z-line degradation remains an enigma. Since the Z-line has been shown to be very susceptible to removal by proteolytic enzymes such as trypsin or chymotrypsin, proteolysis by cathepsins may be a likely explanation for post-mortem Z-line degradation. Fukazawa and Yasui (Zl), however, have reported that addition of cathepsins to chicken myofibrils does not cause any noticeable structural alterations in the Z-line, and de Lumen (16) in our laboratory has confirmed this finding using purified lysosomes on myofibrils. It may be that proper conditions for catheptic degradation of Z-lines have not yet been found in our --in vitro studies, or it is possible that Z-line degradation originates primarily from prolonged exposure to nonphysiological temperatures and pH values. Stromer --et al. (36, 37) hzve shown that it is possible to completely extract Z-lines from glycerinated muscle by the use of low ionic strength solutions alone. Regardless of its origin, it is evident that degradation of the Z-line in post-mortem muscle would cause increased fragility of the muscle fiber, both during homogeni- zation and with respect to the ability of the fiber to support weight without breaking. In addition, Z-line degradation may also contribute to increased tenderness and to loss of the ability to maintain isometric tension In short, Z-line degradation may be responsible for many of the characteristic changes observed in post-mortem muscle.

The second alteration which may be a primary contributor to the resolution of rigor mortis in post-mortem muscle is a proteolytic weakening of the actin-myosin interaction. I wish to enphasize that we are not pro- posing that the interaction between the actin and myosin filaments is completely dissociated in post-mortem muscle, although this proposal has been made by Partmann (32) and was earlier discussed by Weinberg and Rose (39) and Wierbicki --et al. (40). The fact that post-mortem muscle never fully regains its original macroscopic extensibility appears to argue con- clusively against a complete dissociation of the actin-myosin interaction during the resolution of rigor. Rather, what we are suggesting is that in post-mortem muscle, the actin-myosin interaction is weakened so that residual traces of ATP, adenosine diphosphate, or other agents present in -situ may cause some "slippage" or partial dissociation at the points of- 25. interaction of the myosin cross-bridges with the actin filaments. A weakening of the actin-myosin interaction in post-mortem muscle was first proposed by Fujimaki, Arakawa, and coworkers (18, 19, 20) working with myosin B in Tokyo. These investigators showed that, in the presence of 1 mM Mg" and 600 mM KCI, myosin B prepared from muscle after 7 days post- mortem was dissociated to actin and myosin by as little as 0.1 mM A!TP, whereas 0.6 mM ATP was required to cause this same dissociztion in myosin B prepared from at-death muscle. The cause for this weakened actin-myosin interaction in post-mortem muscle, however, could not be ascertained from Fu jimaki ' s and Arakawa ' s experiments .

Meanwhile, in our laboratory at Iowa, we were adopting a slightly different approach to the study of post-mortem muscle. Whereas nost studies of rigor mortis involve sampling of the muscle at-death and then again after some period of post-mortem storage, we chose simply to prepare myofibrils from at-death muscle and then to expose these myofibrils to conditions which would be likely encountered in post-mortem muscle. This approach allows us to study the effects of different post-mortem conditions, singly and in controlled combinations. We initidly assumed that some proteolysis may be occurring in post-mortem muscle, so a number of our experiments involved incubation of myofibrils with a proteolytic enzyme followed by careful examination of a number of the characteristic properties of myofibrils to see whether proteolysis produced any consistent changes in these properties. These experiments have shown that very brief treatment with trypsin has a number of quite unique and interesting effects on myofibrils and that these effects all point to the conclusion that very brief trypsin treatment causes a weakening, but not a complete dissociation of the actin-myosin interaction. I will summarize only a few of these effects today and will point out how these alterations caused by trypsin closely parallel the changes observed in post-mortem myofibrils.

One effect caused by tryptic treatment of at-death myofibrils is a characteristic increase in the Ca*- and Mg++- modified ATPase activities (Fig. 21). This increase occurs very quickly, within 30 seconds after the addition of trypsin. Longer incubation with trypsin causes a decrease in the Mg++- modified activity whereas the Ca"- modified activity remains approximately constant. These results may be compared to the ATPase activities of myofibrils prepared from muscle after varying times of post- mortem storage (Table 1). The Ca+'- and Mg"- modified ATPase activities of myofibrils prepared from muscle after 24 hours of post-mortem storage at either 2O or 16OC are 2040% higher than the corresponding activities from at-death muscle (24) . Moreover, further paralleling the situation observed in trypsin-treated myofibrils, longer post-mortem storage times (312 hours in Table 1) cause the Mg++- modified ATPase activity to decrease whereas the Ca?- modified activity remains nearly the same.

A second effect of trypsin is its ability to lengthen or "relax" highly contracted myofibrils in the absence of ATP. In this experiment, myofibrils exhibiting the banding pattern typical of that for relaxed mscle and having an average sarcomere length of 2.1~were prepared from at-death muscle (Fig. 22, a). These myofibrils were then contracted to an average sarcomere length of 1.01-1 'by the addition of ATP to a final concentration of 0.1 mM in the presence of 0.05 mM Mg", 0.01 mM Ca?, and 120 mM KCI. This treatment resulted in myofibrils with the typical supercontracted appearance in the phase microscope (Fig. 22, b). The supercontracted 26. myofibrils were sedimented at low speed, weshed with 120 mM KCI, and rcsus- pended in 120 mM KCI and 0.1 mM ca++. The washing and resuspension did not affect the structure or the sarcomere lengths of the supercontracted myofibrils. The resuspended myofibrils were then treated with trypsin for 4 minutes at 25OC. This treatment caused a lengthening or a ''relaxation" of the supercontracted myofibrils back nearly to a relaxed banding pattern and to sarcomere lengths of 1.7~(Fig. 22, c and d) . This lengthening or "relaxation" caused by trypsin bears much resemblance to the lengthening or "relaxation" seen in post-mortem myofibrils (cf. Fig. 10, 11, and 12) in that both occu in the absence of measurable amounts of ATP.

The close similarity between the effects of trypsin on myofibrils and the effects of post-mortem storage on myofibrils suggests that the increased ATPase activity and the lengthening or "relaxation" observed in post-mortem muscle may be due to a very limited and specific proteolysis of myosin, actin, and/or one of the regulatory proteins, this proteolysis probably originating from catheptic enzymes. In fact, in the last several months, de Lumen (16) in ow laboratory has succeeded in demonstrating that it is possible to cause a 30-60$ increase in the Ca'+- and Mg"- modified ATPase activities of at-death nyofibrils simply by incubating them with a purified preparation of lysosomes. Since other evidence not detailed here clearly indicates that the effect of brief tryptic proteolysis is to weaken the actin-myosin interaction, it is tempting to speculate that this is also the result of a limited catheptic proteolysis in post-mortem muscle. This would provide an easy and direct explanation for Fujimaki and Arakawa's (18, 19, 20) finding that myosin B from post-mortem muscle is more easily dissociated by ATP than myosin B from at-death muscle. Furthermore, both the lengthening or "relaxation" observed in post-mortem myofibrils and loss of the ability of post-mortem muscle to maintain isometric tension could be easily explained in terms of a weakened actin-myosin interaction, this weakening resulting in a "slippage" at the points of interaction of the myosin cross-bridges with the actin filaments.

Conclusions

In conclusion, if rigor mortis is defined in terms of stiffness, rigidity, or hardness, and resolution of rigor is then loss of this rigidity or stiffness, there are a number of lines of evidence which clearly indicate that a resolution of rigor mortis occurs in post-mortem muscle. This resolution is most easily seen as loss of the ability of post-mortem muscle to maintain isometric tension, but is also indicated by the lengthening or "relaxation" of contracted sarcomeres in post-mortem muscle and by the increased tenderness observed in muscle after 2-3 days post-mortem. Loss of the ability to maintain isometric tension and lengthening of contracted sarcomeres probably originates primarily from a weakening or a partial dissociation of the actin-myosin interaction, and perhaps also in part from a degradation of the sarcomere at the level of the Z-line. The increased tenderness of post-mortem muscle, on the other hand, may be primarily due to the degradation at the level of the Z-line. Although it is now possible to demonstrate that a resolution of rigor mortis occus and to show that this resolution can be associated with certain alterations in the molecular architecture of muscle, the underlying cause of these alterations remains uncertain. Further studies of the effects of lysosomes and pH changes on post-mortem myofibrillar proteins 27. nay clarify the nature of these alterations and eventually provide us with a clear understanding of what causes the resolution of rigor mortis.

I wish to express my sincere appreciation to a number of my colleagues and students who have supplied much of the data cited In this review and who have also contributed to many of the ideas presented here. I aa particularly indebted to Dr. M. H. Stromer, Dr. D. W. Henderson, Dr. N. Arakawa, Dr. F. C. Parrish, Richard M. Robson, Wayne A. Busch, and Ben de Lumen for long and valuable discussions and to Joanne Temple for devoted and expert laboratory assistance. Research results which are reported in this review and which originated in the author's laboratory were supported in part by Public Health Service Grant No. GM-12488 and by Iowa Agricultural Experiment Stztion Project No. 1549. LITERATURE CITED

1. Bate-Smith, E. C. Changes in elasticity of mammalian muscle undergoing rigor mortis. J. Physiol. -96, 176 (1939). 2. Bate-Smith, E. C. The physiology and chemistry of rigor mortis, with special reference to the aging of beef. Adv. Food Res. -1, 1 (1948). 3. Bate-Smith, E. C., and J. R. Bendall. Rigor mortis and adenosine triphosphate. J. Physiol. -106, 177 (1947).

4. Bate-Smith, E. C., and J. R. Bendall. Factors determining the time course of rigor mortis. J. Physiol. -110, 47 (1949). 5. Bate-Smith, E. C., and J. R. BendaJ.1. Changes in muscle after death. Brit. Med. Bull. L12, 230 (1956).

6. Bendall, J. R. The shortening of rabbit muscles during rigor mortis: its relation to the breakdown of adenosine triphosphate and creatine phosphate and to muscular contraction. J. Physiol. -114, 71 (1951).

7. Bendall, J. R. Post-mortem changes in milscle. In "Structure and Function of Muscle", Vol. 111 (G. H. Bourne, ed7 Academic Press, Inc., New York, N. Y. p. 277 (1960).

8. Briskey, E. J. Etiological status and associated studies of pale, soft, exudative porcine musculature. Adv. Food Res. -13, 89 (1964).

9. Briskey, E. J., R. N. Sayre, and R. G. Cassens. Development and application of an apparatus for continuous measurement of muscle extensibility and elasticity before and during rigor mortis. J. Food Sci. -,27 560 (1962).

10. Busch, W. A. Effect of temperature on adenosine triphosphate degradation, shear resistance, and tension development in post-mortem rabbit and bovine striated muscle. M. S. Thesis, Iowa State University Library, Ames, Iowa (1966). 28.

11. Busch, W. A., F. C. Parish, and D. E. Goll. Molecular properties of post-mortem muscle. 4. Effect of temperature on adenosine triphosphate degradation, tension parameters, and shear resistance of bovine muscle. J. Food Sci. 28, 680 (1967).

12. Cassens, R. G. General aspects of post-mortem changes. In "The Physiology and Biochemistry of Muscle as a Food" (E. J. BrEkey, R. G. Cassens, and J. C. Trautman, ea.) University of Wisconsin Press, Madison, Wisconsin, p. 151 (1966).

13. Cook, C. F., and R. F. Langsworth. The effect of pre-slaughter en- vironmental temperature and post-mortem treatment upon some charac- teristics of ovine muscle. 1. Shortening and pH. J. Food Sei. -731 497 (1966).

14. Cook, C. F., and R. G. Wright. Alterations in contracture band patterns of unfrozen and prerigor frozen ovine muscle due to varia- tions in post-mortem incubation temperature. J. Food Sei. -31, 801 (1966) .

15. Davies, R. E. Recent theories on the mechanism of muscle contraction and rigor mortis. Proc. Third Annual Meat Industry Res. Conference, American Meat Institute Foundation, Chicago, Illinois. p. 39 (1966).

16. de Lumen, B. Personal communication. (1968).

17. Erdos, T. Rigor, contracture and ATP. Stud. Inst. Med. Chem. Univ. Szeged -3, 51 (1943).

18. Fujimaki, M., N. Arakawa, A. Okitani, 0. Takagi. The changes of "myosin B" ( 'tactomyosin") during storage of rabbit muscle. 11. The dissociation of "myosin B" into myosin A and actin, and its interaction with ATP. J. Food Sci. -30, 937 (1965).

19. Fujimaki, M., N. Arakawa, A. Okitani, and 0. Takagi. The dissociation of the "myosin B" from the stored rabbit muscle into myosin A and actin and its interaction with ATP. Agr. Biol. Chem. -29, 700 (1965). 20. Fujimaki, M., A. Okitani, and N. Arakawa. The changes of "myosin B" during storage of rabbit muscle. Part I. Physico-chemical studies on ''myosin B." Agr. Biol. Chem. -29, 581 (1965).

21. Fukazawa, T. and T. Yasui. The change in zigzag configuration of the Z-line of myofibrils. Biochim. Biophys. Acta -140, 534 (1967).

22. Galloway, D. E., and D. E. Goll. Effect of temperature on molecular properties of post-mortem porcine muscle. J. Lima1 Sci. -26, 1302 (1967) e

23. Goll, D. E., D. W. Henderson, and E. A. Kline. Post-mortem changes in physical and chemical properties of bovine muscle. J. Food Sei. -'29 590 (1964). 29.

24. Goll, D. E., and R. M. Robson. Molecular properties of post-mortem nuscle . 1. Myof ibri1I.a.r nucleosidetriphosphatase activity of bovine muscle. J. Food Sci. -32, 323 (1967).

25. Gothard, R. E., A. M. Mulllns, R. F. Boulware, and S. L. Hansard. Histological studies of post-mortem changes in sarcomere length as rebted to bovine muscle tenderness. J. Food Sei. L31, 825 (1966).

26. Henderson, D. W. Effect of temperature on post-mortem structural changes in rabbit, bovine, and porcine skeletal muscle. Ph. D. Thesis, Iova State University Library, Ames, Iowa (1968) .

27. Hoet, J. P., and H. P. Marks. Observations on the onset of rigor mortis. Proc. Royal SOC.B. -100, 72 (1926).

2%. Huxley, H. E., and W. Brown. The low-angle X-ray dizgram of vertebra,te striated muscle and its behavior during contraction and rigor. J. Mol. Biol. -30, 383 (1967).

29. Jungk, R. A., H. E. Snyder, D. E. Goll, and K. G. McConnell. Isometric tension changes and shortening in muscle strips during post-mortem aging. J. Food Sei. -32, 158 (1967). 30. Locker, R. H., and C. J. Hagyard. A cold shortening effect in beef muscles. J. Sei. Food Agr. -14, 787 (1963).

51. Newhold, R. P. Changes associated with rigor mortis. In "The Physiology and Biochemistry of Muscle as a Food" (E. J.Briskey, R. G. Cassens, and J. C. Trautman, ed.) University of Wisconsin Press, Madison, Wisconsin, p. 213 (1966).

52. Partmann, W. Post-mortem changes in chilled and frozen muscle. J. Food Sei. -'28, 15 (1963).

33. Stromer, M. H., and D. E. Goll. Molecular properties of post-mortem muscle. 2. Phase microscopy of myofibrils from mine muscle. J. Food Sei. -32, 329 (1967). 34. S-tromer, M. H., and D. E. Goll. Molecular properties of post-mortem muscle. 3. Electron microscopy of myofibrils. J. Food Sei. -'32 386 (1967).

35. Stromer, M. H., D. E. Goll, and L. E. Roth. Morphology of rigor- shortened bovine muscle and the effect of trypsin on pre- and post- rigor myofibrils. J. Cell Biol. -34, 431 (1967).

36. Stromer, M. H., D. J. Hartshorne, and R. V. Rice. Removal and reconstitution of Z-line material in a striated muscle. J. Cell Biol. -35, C23 (1967).

37. Stromer, M. H., and R. V. Rice. Further studies on 2-line reconstitution in a striated muscle. Federation Proc. -27, 300 (1968). 30.

38. Takahashi, K., T. Fukazawa, and T. Yasui. Formation of myofibrillar fragments and reversible contraction of sarcomeres in chicken pectoral muscle. J. Food Sci. -32, 409 (1967). 39. Weinberg, B., and D. Rose. Changes in protein extractability during post-rigor tenderization of chicken breast muscle. Food Technol. 14 -7 376 (1960).

40. Wierbicki, E., L. E. Kunkle, V. R. Cahill, and F. E. Deatherage. The relation of tenderness to protein alterations during post-mortem aging. Food Technol. -8, 506 (1954). 31

0, p. rl 0 0 0 0 0 0

+I +I -1-1 co d cc) Lo* cu ri 0 0 0 0 0

* + rl rl 0 0 0 0 0

+I +I +I b F a) d 2 ri 0 0 0 0 0

co cs) rl 0 0 0 0 0 0

+I +I -!-I N cu cs) 2 cu rl 0 0 0 0 0

Lo F rl 0 0 0 0 0 0

+I +I +I m rl N cs) a) N rl 0 0 0 0 0

N 0 0 $ 0 0 0 + +I +I (D rl 01 0 Lo cu rl 0 0 0 0 0

+ + +cd 2 u k w % % Ln cu 32.

Figure 1 Ovcrrtll view of E & M Phy;iogr:*?h setup for ric-t;irin.g icornetric tension dnvoloprwrit in post-nortc 'I rwxlt .jc controlied tempera- tues.

Figure 2

Isometric rnyographs and setup used to nieaswe isometric tension devcloprncnt of rnuscle strips immersed in physiological saline. 33.

Figure 3

Close-up view of six channel E & M Physiograph.

P" 0 0- ACI D- LAB I LE RiOSPHORUS 7.0 --*O, 20

- CI 10 6.5 c-9- z- 60 E I e 6.C u)z 80 e X w

- 5.: 100 HOURS

Figure 4

Chemical and physical changes in beef sternomandibularis muscle held at 37OC. Zero time was 105 minutes post modem. (Taken from Newbold (31). Reproduced by permission of the University of Wisconsin Press .) 34.

SHORTENING 100 - 0

7. a 20

Y x' U 40 6.5 s W - k z (2: 0 60 E I- X 6.C w 80 I >r 0 Y 5.5 100 2, 0 4 8 12 16 20 24 HOURS Figure 5

Chemical and physical charges in beef sternomandibulxris muscle held at l0C. Average of four muscles. Average total shortening was about 35% of the initial length. (Taken from Newbold (31). Reproduced by permission of the University of Wisconsin Press.)

I II I I II I I II I I I 11- II I ON: SAIRFOMFRE-I I III I I I I II I I II I I II I I II I I If I e/BfiND7-A E/A,NDI,I+/ BAND+! I 1 1 I I II I I II I I I II I I II I

,. -I ~ MILIhE I l~~~~~~~~s - - - - II II I1 - II II I1 Z PSEUDO Z LINE H ZONE LINE Figure 6

Electron micrograph of frog skeletal muscle with an accompanying. schematic of the interdigit6ting thick and thin filament structure that is responsible for the striated appearance of skeletal muscle. (Reproduced by the kind permission of Dr. H. E. Huxley.) 35.

BRIDGE-FREE REGION

/ MYOSIN BRIDGES FILAMENT FI LAM EN T

Figure 7

Schematic of the thick and thin filament structure of muscle showing how nyosin cross-bridges can make contact with actin filaments. (Reproduced by the kind permission of Dr. H. E. Hwrley .)

EXCISED FROM SKELETON IMMEDIATELY POST - MORT EM G+ ATTACHED TO SKELETON UNTIL SAMPLED

.\ -0

I,lllllllllllllll~~~~"~~1 0 24 48 72 96 168 192 216 240 264 250 312 I2O +?$E (HRS)

Figure 8

Post-mortem changes in Warner Brztzler shear-force values of bovine semitendinosus muscles either excised from the skeleton immediately after death or left attached to the skeleton. Post- mortem storage temperatwe = ZOC. t Rabbit L. Muscle

U 4 40 0 Y

TIME POST MORTEM (hr.1

Figure 9

Post-mortem changes in isometric tension development of rabbit -L. dorsi muscle. Isometric tension measured on muscle strips immersed in 50mM KCI, 60 mM K phosphate, pH 6.8, 5mM MgCI2, by using the instrumentation shown in Fig. 1, 2, and 3.

Figure 10 Electron micrograph of bovine semitendinosus muscle sampled at death. (Reproduced by the kind permission of Dr. M. H. Stromer.) 37.

Figure 11

Electron micrograph of bovine semitendinosus muscle excised from the skeleton immediately after death, stored at Z0C, and sampled after 24 hours post-mortem. This m-dscle is severely shortened (supercontracted) to the point that I-bands are no longer evident and the thick fi1m:ents are crumpled up against the 2-lines to produce heavy dark areas at the level of the Z-line. (Reproduced by the kind permission of Dr. M. H. Stromer.)

Figure 12 Electron micrograph from the sane muscle shown in Fig. 11 but after 312 hours of post-!iortern storage at Z°C. The supercontracted muscle seen in Fig. 11 has now lenzthened to the moderately contracted state seen here. (Reprodu-ced by the kind permission of Dr. M. H. Strorier.) 38.

Figure 13-X28,500 Electron micrograph of porcine L. dorsi muscle sampled at death. The Z-lines axe intact and the characteristic zigzag structure of the Z-line can be seen in several places. X28,500. (Reproduced by the kind permission of Dr. D. W. Henderson.) 39.

Figure 14-X22,500

Electron micrograph of porcine L. dorsi muscle excised from the skeleton immediately after death, stored at ZOC, and sampled after 24 hours post-mortem. Several breaks are starting to appear in the Z-lines although for the most part, Z-line structure is relatively intact and the zigzag structure can still be seen in several places. X22,50C. (Reproduced by the kind permission of Dr. D. W. Henderson.) 40.

Figure 15-X34,COO

Electron micrograph of the same porcine &. dorsi shown in Fig. 14 but after 120 hours of post-mortem storage at 2OC. Z-lines are now severely disrupted even though the mitochondrion in the micrograph appears relatively intact. X34, COO. (Reproduced by the kind persmission of Dr. D. 'VJ, Henderson.) 41.

Figure 16

Electron micrograph of myofibrillar frzgment prepared from chicken muscle which had been left attached to the skeleton at 0% for 24 hours. The zigzag structure of the Z-lines is com?letely gone in this myofibril. (From Fukezawa and Yasui (21). Reproduced by permission of Elsevier Publishing Co., Amsterdam. )

Figure 17

Electron micrograph of sincgle sarcomeres in myofibrillar prepmat ions of chicken muscle. The Z-line is gene in these sarcomeres. (Fro.- Fukazawa and Yasvi (21). Reproduced by permission of Elsevier Publishing Co., Amsterdam. 42.

Figure 18-X40,00O-Inset -X68,000

Electron micrograph of rabbit L. dorsi muscle excised from the skeleton immediately after deazh, stored at 25OC, and sampled after 24 hours post-mortem. The Z-lines are almost totally gone in this muscle and netlike structures resembling cross-sections of the Z-line can be seen in the sarcoplasm surrounding the myofibrils. X40,OOO.. Insert. a higher magnification of the netlike structures in the sarcoplasm. X68,OOO. (Reproduced by the kind permission of Dr. D. W. Henderson). 43.

Figure 19-X2,000

Phase micrograph of myofibrils prepared from porcine --L. dorsi immediately after death. Most of the myofibrils in this preparation are ten or more sarcomeres in length. X2,OOO. (Reproduced by the kind permission of Dr. D. W. Henderson). 44.

Phase nicrograph of myofibrils prepared from porcine --L. dorsi mascle after 168 hours of post-mortem storage at Z°C. Notice that the Z-lines are absent and that this preparztion contains many myofibrils that are only three, four, or five sarcomeres in length. Some myofibrils only two sarcomeres in length are also visible. X’2,OOO. (Reproduced by the kind pernission of Dr. D. W. Henderson). 45.

ATPase

O'I0 t 2mM EDTA - 0 Y 0 I5mMMg,2mM EGTA I I I 1 I 1 0.001 5 IO 15 20 25 30 CONTROL TIME OF TRYPSIN TREATMENT (MIN )

Figure 21

Effect of trypsin treatment on ATPase activity of myofibril sus- pensions. Myofibrils were incubated with trypsin (1 part of trypsin to 100 parts of myofibrillar protein, w/w) for times shown on the abscissa. Control samples were not subjected to any addition of either trypsin or soybean trypsin inhibitor, zero-time samples had trypsin which was first mixed with soybean trypsin inhibitor added to them. Conditions for trypsin treatment: 2.8 mg myofibrillar protein/ml, 110 mM KCI, 110 mM Tris-HCI, pH 7.6, 25OC. Reaction was stopped with a four-fold addition of soybean trypsin inhibitor, the suspension diluted, and aliquots removed for imme- diate ATPase assay. Conditions for ATPase assay: 108mM KCI, 46 mM Tris-HCI, pH 7.6, 0.8 mg myofibrillar protein/ml, activator as shown, 5 mM ATP, 25OC. (Taken from Goll, Robson, and Henderson, submitted). 46.

Figure 22-X2,000

Phase micrographs of myofibrils before and after supercontraction with ATP and after "relaxation" with trypsin. a) Control myofibrils before any treatment, b) Myofibrils after supercontraction with ATP; c) Myofibrils from "b" but after treatment with trypsin for 4 minutes, d) A "clump" of myofibrils from "c" showing that almost all myofibrils in the preparation are "relaxed" by 4 minutes of trypsin treatment. Experimental conditions: supercontraction was done by addition of M ATP to 5.0 mg myofibrillar protein ml in 120 mM KCI, 25mM Tris-HCI,. pH 7.6, 5 x Mg++, and lo-' M Ca* . Trypsin "relaxation" was done in 120 mM KCI, 100 mM Tris-HCI, pH 7.6, 0.1 mM Ca++ with 5 mg myofibrillas protein/ml by treatmnt with 1 part of trypsin to 200 parts of myofibrillar protein w/w for 4 minutes at 25OC. (Taken from GoU, Robson, and Henderson, submitted).