, Wierbicki. POST MORTEM PROTEOL Y T l C CHANGES A F F E C T I N G MYOF I B R I I L A R P R O T E I N S 153.

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1 153. POST MORTEM PROTEOL Y T l C CHANGES A F F E C T I N G MYOF I B R I I L A R P R O T E I N S JOHN R. WH I TAKER The changes i n tenderness of beef and poultry muscle as a function of t i m e post m o r t e m are w e l l documented. I n i t i a l l y, i n t h e prerigor state, t h e muscles are very tender and, i f beef i s cooked rapidly within an hour after slaughter, t h e m e a t i s as tender as if it were aged f o r 144 hours. However, it i s quite tough i f cooked at 24 hours post mrtem (Paul e t al., 1952). Poultry i s apparently similar t o beef i n terms of post mortem tenderness changes; however, t h e t i m e involved i s much shorter. (Beef rnw require 1020 days t o reach maximum tenderness while poultry requires 1 2 t o 24 hours (de Fremery and Fool, 1960). While t h e chemical events associated with t h e increase i n toughness of meat ( r i g o r ) are, i n general, wellunderstood (Bendall, 1960, 1963), t h e events which lead t o an increase i n tenderness on aging are not w e l l understood. One might postulate t h a t t h e increase i n tenderness on aging i s due t o (1) resolution of r i g o r mortis, ( 2 ) changes i n t h e connective t i s s u e content of t h e muscle, ( 3 ) changes i n t h e hydration of t h e proteins as influenced by changes i n ph and ionic atmosphere and, ( 4 ) proteolytic enzyme action on t h e myofibrillar proteins. There i s no experimental evidence t o support t h e f i r s t two postulations. Marsh (1954) reported t h a t beef muscle i n f u l l r i g o r held under nitrogen a t 7O C. showed no increase i n e x t e n s i b i l i t y after 7 days o r after 24 hours a t 37O C. BateSmith and Bendall (1956) have reported similar results f o r rabbit muscle held at 3O C. Therefore, it appears t o be erroneous t o speak of t h e "resolution of r i g o r mortis" as t h i s implies t h e r e v e r s a l of t h e process by which r i g o r occurred. There appears t o be no evidence t h a t during t h e tenderness increase there i s a conversion of actomyosin back t o a c t i n and myosin. Wierbicki et a l. (1954) have shown t h a t there i s no change i n t h e connective t i s s u e content of m e a t on aging. This observation should be contrasted t o t h e changes found i n t h e connect i v e t i s s u e s of meat a f t e r treatment with proteolytic enzymes such as papain, f i c i n o r bromelain (Wang e t al. 1957; Miyada and Tappel, 1956; ElGharbawi and Whitaker, 1963). F i c i n can hydrolyze e l a s t i n quite readily even at low temperatures (ElGharbawi and Whitaker, 1963; YatcoManzo and Whitaker, 1962), while collagen i s not hydrolyzed by these enzymes unless it i s f i r s t denatured (Hinricks and Whitaker, 1962; ElGharbawi and Whitaker, 1963). B i r k n e r and Auerbach (1960) have reported changes i n t h e collagen but not e l a s t i n content of meat on aging but they concluded t h a t t h e changes probably do not account f o r increased tenderness on aging. This brings us t o t h e t h i r d postulation. and Hamm (1960), Hamm and Deatherage (1960 a, b) The work of Deatherage e t al., Wierbicki

2 154. (1957 a, b), Hamm (1960), Bendall and WismerPedersen (1962) as well as others leave little doubt that there are changes in the hydration of meat proteins during aging. These changes in hydration are brought about by changes in the ph and ionic atmosphere of the muscle on aging. Some of this increase in hydration could also be the result of proteolytic enzyme action as we have found that we could increase the water binding capacity of meat by 30 to 50% by treatment with the proteolytic enzyme ficin (see Hamm, 1960, p. 393). Dr. WismerPedersen will cover the subject of meat protein hydration in the next talk so we shall not dwell further on this postulation. The fourth possibility for explaining the increase in tenderness of meat on aging is through the action of proteolytic enzymes on the myofibrillar proteins. It is now generally accepted that most living cells contain small subcellular particles called lysosomes. The number of lysosomes per cell is dependent upon the f'unction of the cell. These lysosomes contain a large number of hydrolytic enzymes including the cathepsins. In order for the enzymes to be released to act upon the cellular constituents the lysosomal membrane must be broken. The strength of this membrane appears to be a function of the health of the animal and the ease with which lipid peroxidation can take place. One would expect that after death and during the aging process many of these membranes would be broken. There are two approaches that have been used to attempt to understand the role of the cathepsins in meat tenderization. The first involves the demnstration of hydrolytic changes in the proteins during the aging process and the second the isolation of the cathepsins and a study of their properties. In both approaches one must be careful that what he is dealing with is the result of action of the proteolytic enzymes of the muscle and not of the proteolytic enzymes of microbial origin. Zender et al. (1958) have demnstrated that there are electrophoretic changes in the properties of the proteins of rabbit and lamb muscle on aging under aseptic conditions. Fischer (1963) reported that there was a correlation between the increase in a watersoluble protein fraction of chicken muscle as separated on a DEAEcellulose column and tenderness as measured by a taste panel. Weinberg and Rose (1960) found that the extractable nitrogen increased during aging of chicken muscle. High dosage irradiation of unheated meat generally leads to a mushy texture and the liberation of tyrosine which is attributed to the action of proteolytic enzymes (Nickerson et al. 1950; Bty and Wachter, 1955; Kirn et al., 1956; Drake et al., 1957). Histologically, a disappearance of the crossstriations and the appearance of transverse breaks is found on aging (Birkner and Auerbach, 1960; Paul et al., 1944). Whether these histological changes are the result of cathep'tic action is not clear. It is wellknown that after rigor poultry muscle increases in tenderness much more rapidly than does beef muscle. There should be some correlation between the amount of proteolytic enzyme activity in atissue and the rate at which tenderness develops if the enzymes are involved. BandockYuri and Rose (1961), working with chickens killed and dressed under commercial conditions, found less catheptic activity in chicken than in beef muscle. However, we have found that chicken leg and breast muscle contain considerably more catheptic activity than does beef round (Ibyle and Whitaker, 1964). On the other

3 155. hand, Wierbicki and Deatherage (1956) and Locker (1960) found very little, if any, evidence for proteolysis occurring during aging. Small injuries to muscle tissue result in rapid clearance of the tissue by the increased number of phagocytic cells (Field, 1960). Whether these phagocytic cells come from the vascular system or from the muscle system is not clear. However, it is clear there is a great increase in proteolytic activity. The demonstration of hydrolytic changes in the complex protein system of muscle during aging is extremely difficult. The usual classical procedures for measuring an increase in amino or carboxyl groups as the result of peptide bond splitting are not sensitive enough. Large fragmentation of the molecules must occur before the detectable changes in amino or carboxyl group content are above experimental error. One, therefore, must rely upon the more modern techniques of electrophoresis and ionexchange chromatography. Even these alone are not sufficient. If a change in the electrophoretic and/or chromatographic properties of a protein fraction is detected during aging, it must then be demonstrated that the change is due to a splitting of peptide bonds by proteolytic enzymes rather than due to denaturation, complexation or deamidation. In other words, one must demnstrate a decrease in molecular weight and the appearance of new end terminal groups. Sephadex gel filtration should prove to be a valuable method for detecting changes in molecular weight (Whitaker 1963). While Balls in 1938 reported the presence of proteolytic enzymes in beef muscle, most of the work on the cathepsins has been carried out on enzymes from the spleen and kidney. The proteolytic enzyme content of muscle is extremely small (less than 0.01%) and is mixed up with all the proteins normally found in muscle. The purification of the cathepsins from muscle is extremely difficult. Table I summarizes some of the properties of Cathepsins A, B, C and D from beef spleen. In addition, tissues contain several peptidases including enzymes similar to pancreatic leucine aminopeptidase and carboxypeptidase A (Smith, 1948). In muscle, it is fairly wellestablished that there are two groups of proteolytic enzymes with distinct ph optima, one at ph near 5 and the other at 8 to 9 which are capable of hydrolyzing the muscle proteins (in rats, Koszalka and Miller, 1960; in beef, Landmann, 1963; and in chicken, Coyle and Whitaker, 1964). We have been particularly perplexed by the fact that there is little or no action on casein or hemoglobin of the proteolytic enzymes acting at the alkaline ph. The presence of cathepsins B and C in beef muscle has been demnstrated by Landmann (1963), cathepsin A in beef muscle by Bodwell and Pearson (1964) and cathepsins A and B in chicken muscle by Coyle and Whitaker (1964). The purification procedure used for chicken cathepsin in our laboratory is shown in Table 11. This type of work has established two important points; one, proteolytic enzymes are present in muscle and second, the ph optima of some of these is within the range of meat. However, the role, if any, which these play in meat tenderization is still not answered. Bendall (1960) has indicated that "it appears to be unnecessary to search for any other cause for the accompanying resolution than the proliferation of putref active organisms within the muscle tissue."

4 156. B a l l s, A. K Enzyme action in food products at l o w temperatures. Ice and Cold Storage 4 1 (485), 143. BandockYuri, S. and D. Rose. Technol. 15, 186. Food BateSmith, E. C., and J. R. Bendall. B r i t. Med. B u l l , Proteases of chicken b r e a s t muscle Changes i n muscle after death. Bendall, J. R Physiology and Chemistry of Muscle. Meat Tenderness Symposium, Campbell Soup Co., 33. Proceedings Bendall, J. R PostMortem Changes i n Muscle. I n "The Structure and Function of Muscle" Ed. G. H. Bourne, Academic Press, Inc., New York, Vol. 111, 227. Bendall, J. R. and J. WismerPedersen Some p r o p e r t i e s of t h e f i b r i l l a r p r o t e i n s of normal and watery pork muscle. 2. Food S e i. 27, 144. Birkner, M. L., and E. Auerbach Microscopic Structure of Animal Tissues. I n "The Science of Meat and Meat Products." W. Freeman and Co., San Francisco. p. 29. Deatherage, F. E., and R. Hamm Influence of freezing and thawing on hydration and charges of t h e muscle p r o t e i n s. Food Research 25, 623. de Fremery, D., and M. F. Pool Biochemistry of chicken muscle as r e l a t e d t o r i g o r mortis and tenderness. Food Research 25, 73. De l a Haba, G., P. S. Cammarata, and S. N. Timasheff The p a r t i a l p u r i f i c a t i o n and some physical p r o p e r t i e s of cathepsin C from beef spleen. Biol. Chem. 234, 316. z. Doty, D. M., and 3. P. Wachter Influence of gammaradiation on p r o t e o l y t i c enzyme s t i v i t y i n beef muscle. 2. Agr. Food %. 3, 61. Coyle, M. L., and J. R. Whitaker Unpublished experiments. v e r s i t y of California, Davis, California. Uni Drake, M. P., J. W. Giffee, Jr., R. Ryer, 111, and H. Harriman Proteolytic enzyme a c t i v i t y i n i r r a d i a t i o n s t e r i l i z e d meat. Science 125, 23. ElGharbawi, M., and J. R. Whitaker Factors affecting enzymatic s o l u b i l i z a t i o n of beef p r o t e i n s. J. Food Sci 28, Field, E. J Muscle regeneration and r e p a i r. I n "The Structure and Function of Muscle." Ed. G. H. Bourne, Academic Press, Inc., New York, Vol. 111, 139.

5 157. Fischer, R. L Changes i n t h e chemical and physical properties of p r o t e i n during aging of meat. Proceedings Meat Tenderness Symposium, Campbell Soup Co., 71. Fruton, J. S., and M. Bergmann On t h e p r o t e o l y t i c enzymes of animal t i s s u e. 1. Beef spleen. J. Biol. Chem , Fruton, J. S., and M. J. Mycek Arch. Biochem. Biophys. 65, 11. Studies on beef spleen cathepsin C. Greenbaum, L. M., and J. S. Fruton P u r i f i c a t i o n and p r o p e r t i e s of beef spleen cathepsin B. J. Biol. Chem , Gutmann, H. R., and J. S. Fruton On t h e p r o t e o l y t i c enzymes of animal t i s s u e s. VIII. An i n t e r c e l l u l a r enzyme r e l a t e d t o chymotrypsin. J,. Biol. Chem. 174, 851. H m, R. lo Biochemistry of meat hydration. Advances i n Food Research Hamm, R., and F. E. Deatherage. 1960a. Changes i n hydration and charges of muscle p r o t e i n s during freeze dehydration of meat. Food Research 25, 573. Hamm, R., and F. E. Deatherage. 1960b. Changes i n hydration, s o l u b i l i t y and charges of meat p r o t e i n s during heating of meat. Food Research 25, 587. Hinrichs, J. R., and J. R. Whitaker collagen. J. Food Sci. 27, 250. Enzymatic degradation of K i m, J. E., M. W. Urbain, and H. J. Czarnecki C h a r a c t e r i s t i c s of e l e c t r o n i r r a d i a t e d meats stored at ref r i g e r a t o r temperatures. Food Technol. ID, 601. Koszalka, T. R., and L. L. Miller Proteolytic a c t i v i t y of rat s k e l e t a l muscle. I. Evidence f o r t h e existence of an enzyme a c t i v e optimally at ph 8.5 t o P u r i f i c a t i o n and p r o p e r t i e s of an enzyme a c t i v e optimally at ph 8.5 t o 9.0. J,. Biol. Chem. 235, 660, 665. Landmann, W. A Enzymes and t h e i r influence on meat tenderness. Proceedings Meat Tenderness Symposium, Campbell Soup Co., 87. Locker, R. H. Proteolysis i n t h e storage of beef Agr. ll, 250. Marsh, B. B Rigor mrtis i n beef. SI. J. S c i. Food Agr. Sci. Food 5, 70. Miyada, D. S., and A. L. Tappel The hydrolysis of beef p r o t e i n s by various p r o t e o l y t i c enzymes. Food Research 21, 217. Nickerson, J. T. R., S. A. Goldblith, and B. E. Proctor A comparison of chemical changes i n mackerel t i s s u e s t r e a t e d by ionizing r a d i a t i o n. Food Technol. &, 84.

6 Paul, P., L. J. Bratzler, E. D. Farwell, and K. Knight. tenderness of beef I. R a t e of heat penetration Studies on Food Research l7, Paul, P., B. Lowe, and B. R. McClurg Changes i n h i s t o l o g i c a l s t r u c t u r e and p a l a t a b i l i t y of beef during storage. Food Research , Press, E. M., R. R. Porter, and J. Cebra The i s o l a t i o n and propert i e s of a p r o t e o l y t i c enzyme, Cathepsin D, from bovine spleen. Biochem. J. 74, 501. Smith, E. L The peptidases of s k e l e t a l, h e a r t and u t e r i n e muscle. 2. Biol. Chem. 173, 553. Snoke, J. E., and H. Neurath The p r o t e o l y t i c a c t i v i t y of s t r i a t e d r a b b i t muscle. J. Biol. Chem. 187, 127. M. E. Jones, and J. S. Fruton On t h e p r o t e o l y t i c enzyms of a n i m a l t i s s u e s. X. Beef spleen cathepsin C. J. Biol. Tsllen, H. H., Chem Wang, H., C. E. Wier, M. L. Birkner, and B. Ginger The influence of enzyme t e n d e r i z e r s on t h e s t r u c t u r e and tenderness of beef. D o c. Ninth Research Conf., g. Meat I n s t. Foundation, p. 72. Weinberg, B. and D. Rose Changes i n p r o t e i n e x t r a c t a b i l i t y during post r i g o r tenderization of chicken b r e a s t muscle. Food Technol , Whitaker, J. R Determination of molecular weights of proteins by g e l f i l t r a t i o n on Sephadex. And. Chem. 35, Wierbicki, E., V. R. Cahill, and F. E. Deatherage. 1957a. Effects of added sodium chloride, potassium chloride, calcium chloride, magnesium chloride and c i t r i c acid on mat shrinkage at 70 C. and of added sodium chloride on drip l o s s e s during freezing &a thawing. Food Technol Wierbicki, E., L. E. Kunkle, and F. E. Deatherage. 1957b. Changes i n t h e waterholding capacity and c a t i o n i c s h i f t s during heating and freezing and thawing of meat as revealed by a simple c e n t r i f u g a l method f o r measuring shrinkage. Food Technol. ll, 69. Wierbicki, E., L. E. K u n k l e, V. R. Cahill, and F. E. Deatherage Post mrtem changes i n meat and t h e i r possible r e l a t i o n t o tenderness together with some comparisons of meat from h e i f e r s, b u l l s, s t e e r s, and d i e t h y l s t i l b e s t r o l t r e a t e d b u l l s and steers. Food Technol. 10, 80. Wierbicki, E., L. E. K u n k l e, V. R. Cahill, and F. E. Deatherage The r e l a t i o n of tenderness t o p r o t e i n a l t e r a t i o n s during post mortem aging. Food Technol. E, 506. YatcoManzo, E., and J. R. Whitaker Ficincatalyzed hy&olysis of e l a s t i n. Arch. Biochem. Biophys. 97, 122.

7 159. Zender, R., C. LatasteEorolle, R. A. Collet, P. Rowinski, and R. F. Mouton Aseptic autolysis of muscle: biochemical and microscopic modifications occurring in rabbit and lamb muscle during aseptic and anaerobic storage. Food Research 23, 305. Table I Endopeptidases of Animal Tissues Name Specificity ph optimum Ref. Cathepsin A Homospecific with 5.4 (c~zgt)~ Fruton and pepsin Bergmann (1939) Cathepsin B (SH)I Homospecific with 5 (BAA)2 Greenbaum and trypsin Fruton ( 1957) Cathepsin C ( SH) Homspecif ic with 3.5 (hemglobin) Gutmann and (M=235,000) (2(. chymotrypsin 56 (GPA)~ Fruton (1948); Fruton & Mycek (1956); Tallen et &. ( 1952E De la Haba et al. (1959r Cathepsin D Same specificity as 3.1 (hemglobin) Press et a1. pepsin on oxidized 4.2 (serum (196n B chain of insulin, albumin) does not split CbzGT2 Muscle proteinase Does not hydrolyze the. 4 (hemoglobin) Snoke & Neurath specific substrates (1950) (Fe++) of pepsin, trypsin, or o( chymotrypsin Activators The following abbreviations are used: BAA, benzoyllargininamide; CbzGT, carbobenzoxylglutamylltyrosine; GPA, glycyllphenylalanindde.

8 160. Table I1 Purification Procedure f o r Chicken Skeletal Muscle Cathepsins (Doyle and Whitdser, 1964) Activity recovered (4) Froc edure 1. Supernatant l i q u i d from 80,000 x g centrifugation of homogenate a Specific activity Fraction between 45 and 70$ saturated amnmonium s u l f a t e F i r s t chromatography on CMcellulose (450 ml/hr., ph adjusted to 4.2, 5% saturated m n i u m s u l f a t e added, supernatant l i q u i d saved ph 5.2) 5. Second chromatography on CMc e l l u l o s e (100 ml/hr ph 5.2)., ~ Purification (fold) ~ a Activity measured on urea denatured hemglobin at ph 4.6 and 35.0' C. DR. SAYRE: Thank you, D r. Whitaker, f o r a very i n t e r e s t i n g presentation. I think we had better get on with t h e next speaker now. We a g d n are quite fortunate t o have with us D r. WismerPedersen. H e received h i s Ph.D. degree from McGill University and has been working f o r several years at t h e Danish Meat Research I n s t i t u t e on t h e biochemistry of post m o r t e m changes i n muscle. This p a s t year he has been a v i s i t i n g professor at Michigan S t a t e University and soon w i l l return t o t h e Danish Meat Research I n s t i t u t e. It gives m e great pleasure t o present Dr. W i s m e r Pedersen. DR. WISMERP%DERSEN: "hank YOU. ############