:44... 1.. . . 4.4.4:; 2.4... 4.1.4.. ”thu..4._w iwwuw.«k..rmm. 4.7.»: gram 1.2.4.... 1341.4«33373247. 4.49.0 .42 1%“; 4’ 1.444414 3:15.... . 1.; s41 . 53.24.5114 1..:=x.«nnt..4 4 .. .4 . L r .5 :51: ,4 1 . a . . . . 4.4....m...4_4 an... . . . . . . 1- .1 44 J i’vcob. : . . . . 4... i. 1.4 : 2.3,. , . . 1 1: v '- (W1. 2 . .3: .69.}... 5m: 1 a. . .339» v 3V 341:“. 4.4.. . 4....” .4... 44:44.43. . . . — r n. A .i. z. a 5.... . 1.9.2,: 4.144....nw4 ... 43w 3 any... mymwwfib wsfiifiyu. 4.4.. 12%.:19: .... infini‘m'u: ? L1}? E15 .5 "‘1 Michigan 3mm: This is to certify that the thesis entitled Proteolysis of Porcine Muscle By Clostridium Perfringens presented by Lynn Tilley Hapchuk has been accepted towards fulfillment of the requirements for Ph. D. degree in Food Science and Human Nutrition ,4. 74g Cid/W Major professor Date W4 0-7639 .115 ABSTRACT PROTEOLYSIS OF PORCINE MUSCLE BY CLOSTRIDIUM PERFRINGENS BY Lynn Tilley Hapchuk The objective of this study was to determine the mechanisms by which muscle proteins break down during growth of g. perfringens. An enzyme from a culture filtrate of this organism was isolated and its action upon muscle was compared with that of the intact organism. g. perfringens (ATCC 12915 and 13124) were grown in amino acid and peptone media. Since only g. perfringens ATCC 13124 produced measurable quantities of proteolytic enzyme(s) in these media, it was used in the majority of these studies. Crude enzyme was partially purified from a culture filtrate of g. perfringens ATCC 13124 by Zn Cl 2 precipitation, followed by disodium phosphate extraction and ammonium sulfate precipitation. The crude enzyme solution was further purified by gel filtration on Bio— Gel P-lOO, followed by ion exchange chromatography on DEAE-cellulose and gel filtration using Bio-Gel P-200. Enzyme recovery was 11% with 158-f01d purification and a final specific activity of 79 units/mg protein, with a unit being arbitrarily defined as the amount of Lynn Tilley Hapchuk enzyme required to raise the absorbance of azocoll supernatant by 0.1 unit in 15 min. When subjected to disc gel electrophoresis, the purified enzyme solution contained five measurable protein peaks, indicating a lack of homogeneity. Aseptic muscle samples were inoculated either with Q. perfringens or with enzyme solution and incubated at 30 or 37°C. After incubation, the samples were analyzed for sarcoplasmic, myofibrillar and non-protein nitrogen. The sarcoplasmic extract was subjected to disc gel electrophoresis, SDS gel electrophoresis and iso- electric focusing. The myofibrillar extract was subjected to disc gel electrophoresis in urea, SDS gel electro- phoresis and isoelectric focusing in urea. After incubation with g. perfringens, the quantity of non-protein nitrogen increased, which indicated proteolytic breakdown into small nitrogen containing compounds. Total sarcoplasmic nitrogen decreased after incubation with g. perfringens, which also suggested proteolytic breakdown. Disc gel electrophoresis of the sarcoplasmic fraction indicated that growth of Q. perfringens was responsible for the production of three new protein peaks and for reduction in the concentration of several other peaks. Reduction in some protein peaks was also apparent upon electrophoresis in SDS gels. Lynn Tilley Hapchuk There was a consistent reduction in the concen- tration of troponin in muscle samples incubated with g. perfringens. This was shown by both disc gel and SDS gel electrophoresis. A reduction in the amount of actin was noted upon disc gel electrophoresis in urea, but was not apparent with SDS gel electrophoresis. The concen- tration of tropomyosin declined upon incubation of muscle with Q. perfringens as measured by SDS gel electrophoresis and by a disc gel electrophoresis procedure utilizing urea. Electron micrographs also indicated that Q. perfringens destroyed the thin filaments, which are composed mainly of actin, tropomyosin and troponin. The appearance of new peaks in the myofibrillar fraction, presumably break- down products,was noted with both SDS gel electrophoresis and disc gel electrophoresis in urea. This investigation clearly indicates that g. perfringens is capable of degrading both sarcoplasmic and myofibrillar proteins from muscle. However, the isolated enzyme exerted its major action on only the sarcoplasmic proteins. Since the isolated enzyme frequently did not breakdown the same proteins as the viable organism, it is presumed that g. perfringens also produces other enzymes causing proteolysis. PROTEOLYSIS OF PORCINE MUSCLE BY CLOSTRIDIUM PERFRINGENS BY Lynn Tilley Hapchuk A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science and Human Nutrition 1974 ACKNOWLEDGEMENTS The author wishes to express her appreciation to Dr. A. M. Pearson for his guidance and assistance in the preparation of this thesis. The author is also indebted to Dr. J. R. Price for his assistance in obtaining aseptic muscle samples and in providing the stock cultures of g. perfringens. She also wishes to thank Mr. Maxwell T. Abbott for his excellent electron microscopic work. Appreciation is also expressed to the other members of her guidance committee, Dr. L. R. Dugan, Dr. G. A. Leveille, Dr. 0. Mickelsen, Dr. B. S. Schweigert and Dr. J. E. Wilson. Gratitude is especially expressed to her husband, John, and daughter, Melissa, whose unwaivering support encouraged the completion of this work. ii TABLE OF CONTENTS INTRODUCTION . . . . . . . . . . . . . REVIEW OF LITERATURE . . . . . . . . . Characterization of C. perfringens Occurrence of C. perfringens . . . Infections Caused by C. perfringens Food poisoning . . . . . . . . . Gas gangrene . . . . . . . . . . Toxins Produced by g. perfringens . Alpha toxin . . . . . . . . . . Beta toxin . . . . . . . . . . . Epsilon toxin . . . . . . . . . Theta toxin . . . . . . . . . . Kappa toxin . . . . . . . . . . Lambda toxin . . . . . . . . . . Mu toxin . . . . . . . . . . . . Other toxins . . . . . . . . . . Changes in Mucle Caused by g. perfringens MATERIALS AND METHODS Culturing and Growth of C. perfringens Stock culture . . . . . . . . . Culturing for toxin production . Peptone medium . . . . . . . Amino acid medium . . . . . . Growth studies . . . . . . . . . Enzyme Preparation and Purification Preparation of culture filtrate Zinc chloride precipitation . . Disodium phosphate extraction . Ammonium sulfate precipitation . Bio-Gel P-lOO column . . . . . . DEAE- cellulose column . . . . . Bio-Gel P- 200 column . . . . . Carboxy methyl cellulose column Enzyme production in amino acid medium Azocoll Method for Measuring Enzyme Activity Measurement of Protein . . . . . . Lowry method . . . . . . . . . . Biuret method . . . . . . . . . Kjeldahl method . . . . . . . iii Muscle Spoilage Studies . . . . . Preparation of C. perfringens inoculum . Preparation of enzyme solution for inoculation . . . . . . . . Muscle sample preparation . . Preparation for Kjeldahl analysis. . . . Preparation for electrophoresis Electrophoretic Studies . . . . . Disc gel electrophoresis . . . Sample preparation . . . . Gel preparation . . . . . . Sample application . . . Electrophoretic conditions Staining and destaining . . Quantitation of protein bands . . . . Disc gel electrophoresis in urea . . . . Sample preparation . . . . Gel preparation and sample application Electrophoretic conditions Staining, destaining and quantitation of protein bands . . . . Sodium dodecyl sulfate gel electrophoresis Sample preparation . . . . Gel preparation . . . . . . Electrophoretic conditions Staining and destaining . . Quantitation and characterization of protein bands . . . . . . Isoelectric focusing . . . . . Sample preparation . . . . Gel preparation . . . . . . Electrofocusing conditions Determination of the pH gradient . . Staining and destaining . . Characterization of protein bands . . Electron Microscopy . . . . . . . Fixation and embedding . . . . Sectioning and staining . . . Observation and photography of muscle Sections 0 I O O O O O O O 0 RESULTS AND DISCUSSION . . . . . . . . Determination of Culturing Conditions . . . Selection of the organism . . O O C O 0 Time requirement for enzyme production . Selection of the medium . . . Determination of Assay Conditions Enzyme Purification . . . . . . . Bio-Gel P—lOO fractionation . DEAE-cellulose fractionation . Bio-Gel P-200 fractionation . CM—cellulose fractionation . . Disc electrophoresis of enzyme iv fractions Page 35 35 35 35 37 39 41 41 41 42 43 43 43 44 45 45 45 45 46 46 46 47 47 48 48 48 49 49 49 50 50 50 50 51 51 52 53 53 53 53 54 57 57 60 60 64 64 67 Page Changes in Nitrogen and Protein of Incubated Muscle . . . . . . . . . . . . . . 75 Measurement of nitrogen by Kjehldahl analysis . . . . . . . . . . . . . . . . . 75 Changes in the sarcoplasmic extract . . . 77 Changes in myofibrillar extract . . . . . 77 Changes in non-protein nitrogen . . . . 79 Measurement of protein by Lowry and biuret methods . . . . . . . . . . . . . . . . . . 79 Electrophoretic Analysis of Sarcoplasmic Proteins . . . . . . . . . . . . . . . . . . . 80 Disc gel electrophoresis . . . . . . . . . . 80 SDS gel electrophoresis . . . . . . . . . . 88 Isoelectric focusing . . . . . . . . . . . . 97 Electrophoretic Analysis of Myofibrillar Proteins . . . . . . . . . . . . . . . . . . . 97 Disc gel electrophoresis with urea . . . . . 98 SDS gel electrophoresis . . . . . . . . . . 109 Isoelectric focusing in urea . . . . . . . . 120 General Observations on Electrophoresis . . . . 121 Electron Microscopy . . . . . . . . . . . . . . 121 Changes in the ultrastructure of incubated muscle . . . . . . . . . . . . . . . . . . 122 Appearance of C. perfringens . . . . . . . . 132 Importance of Spoilage Studies . . . . . . . . 132 SUMMARY . . . . . . . . . . . . . . . . . . . . . . 135 BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . . 137 APPENDIX . . . . . . . . . . . . . . . . . . . . . . 144 Table LIST OF TABLES Page Purification Scheme for a Protease Produced by Clostridium perfringens ATCC 13124 O O O O O O O O O O O O O O O O O 59 Relative Mobility and Peak Areas of Sarco- plasmic Proteins after Electrophoresis in 7.5% Acrylamide, Stained with Amido Black . . . . . . . . . . . . . . . . . . . 81 Relative Mobility and Peak Areas of Sacro- plasmic Proteins after Electrophoresis in 7.0% Acrylamide, Stained with Coomassie Blue . . . . . . . . . . . . . . . . . . . . 85 Molecular Weight and Peak Areas of Sarco- plasmic Proteins after SDS Gel Electro- phoresis with 0.27% Cross Linking . . . . . 89 Molecular Weight and Peak Areas of Sarco- plasmic Proteins after SDS Gel Electrophoresis with 0.135% Cross Linking . 94 Relative Mobilities and Peak Areas of Myofibrillar Proteins after Electrophoresis and Staining with Amido Black . . . . . . . 100 Relative Mobilities and Peak Areas of Myofibrillar Proteins after Electrophoresis and Staining with Coomassie Blue . . . . . . 104 Average Molecular Weights and Peak Areas of Myofibrillar Proteins Separated by SDS Gel Electrophoresis . . . . . . . . . . . . 115 vi LIST OF FIGURES Figure Page 1 Enzyme purification scheme . . . . . . . . 26 2 Preparation of muscle fractions for Kjeldahl analysis . . . . . . . . . . . . 38 3 Preparation of muscle fractions for electrophoretic studies . . . . . . . . . 40 4 Growth and enzyme production by C. perfringens ATCC 13124 and 12915 in amino acid medium . . . . . . . . . . . . 55 5 Growth and enzyme production by C. perfringens ATCC 13124 in peptone medium . 56 6 Effect of substrate concentration upon the azocoll reaction at different incubation times . . . . . . . . . . . . . . . . . . 58 7 Bio-Gel P-lOO fractionation . . . . . . . 61 8 Stepwise DEAE—cellulose fractionation . . 62 9 DEAE—cellulose gradient fractionation . . 63 10 Bio-Gel P-200 fractionation . . . . . . . 65 11 CM-cellulose gradient fractionation . . . 66 12 Disc gel electrophoresis pattern of culture filtrate . . . . . . . . . . . . . 68 13 Disc gel electrophoresis pattern of zinc chloride precipitate . . . . . . . . 70 14 Disc gel electrophoresis pattern of ammonium sulfate precipitate . . . . . . . 71 15 Disc gel electrophoresis pattern of Bio-Gel P—100 fraction . . . . . . . . . . 72 16 Disc gel electrophoresis pattern of DEAE—cellulose fraction . . . . . . . . . 73 vii Figure Page 17 Disc gel electrophoresis pattern of Bio-Gel P-200 fraction . . . . . . . . . . . 74 18 Nitrogen content of fraction from the incubated control and C. perfringens inoculated muscle as measured by Kjeldahl analysis . . . . . . . . . . . . . . . . . . 76 19 Molecular weights of sarcoplasmic proteins determined by comparison with standard proteins using 0.27% cross-linked SDS gels . . . . . . . . . . . . . . . . . . . . 91 20 Molecular weights of sarcoplasmic proteins determined by comparison with standard proteins using 0.135% cross-linked SDS gels . . . . . . . . . . . . . . . . . . . . 95 21 Molecular weights of myofibrillar proteins determined by comparison with standard proteins using 0.135% cross-linked SDS gels . . . . . . . . . . . . . . . . . . . . 110 22 Densitometric tracing of the unincubated myofibrillar fraction electrophoresed upon a 0.135% cross-linked SDS gel with tentative peak identification . . . . . . . . . . . . 111 23 Electron micrograph of white or inter- mediate fiber from control muscle incubated for 1 day at 37°C . . . . . . . . . . . . . 123 24 Electron micrograph showing red fibers from control muscle after incubation for 1 day at 37°C . . . . . . . . . . . . . . . . . . 124 25 Electron micrograph of white or inter- mediate fibers after incubation for 1 day with C. perfringens at 37°C . . . . . . . . 126 26 Electron micrograph showing red fibers from muscle incubated for 1 day at 37°C with g. perfringens . . . . . . . . . . . . 127 27 Electron micrograph of red fiber from muscle incubated for 4 days at 37°C . . . . 128 28 Electron micrograph showing red muscle fibers incubated at 37°C for 4 days with g. perfringens . . . . . . . . . . . . . . . 130 29 Electron micrograph showing a longitudinal section of C. perfringens grown in muscle for 1 day at 37°C . . . . . . . . . . . . . 133 viii LIST OF APPENDIX TABLES Table Page 1 Composition of Peptone Medium . . . . . . . 144 2 Composition of Amino Acid Medium . . . . . . 145 3 Formulation of 7.5% Polyacrylamide Gel Electrophoresis System . . . . . . . . . . . 146 4 Formulation of 6.25% Polyacrylamide Gel Electrophoresis System with Urea . . . . . . 147 ix LIST OF APPENDIX FIGURES Figure Page 1 Conductivity of NaCl solutions . . . . . . . 148 2 Standard curve for determining nitrogen using Nessler's reagent . . . . . . . . . . 149 3 Standard curve for determining protein using Lowry's method . . . . . . . . . . . . 150 4 Standard curve for determining protein using the biuret method . . . . . . . . . . 151 INTRODUCT ION Spoilage of meat has been an economic and public health problem 'since muscle was first used as a food. The role of microorganisms in muscle spoilage is well recognized, but the mechanism of spoilage is, as yet, not clearly defined. Some workers (Jay and Kontou, 19 67; Lerke gt; a}: , 1967) originally concluded that microorganisms degraded only small compounds, such as amino acids, peptides and nucleotides, but did not break down muscle proteins. More recent work has shown that several species of bacteria cause proteolysis of muscle proteins (Ockerman _e_t_ a1” 1969; Hasegawa 313 31., 1970). Clostridium perfringens has been reported to be a common contaminant of muscle foods. In addition to its known role as a causative agent of enteritis, g. per;- fringens organisms are known to cause serious disruption 0f muscle, such as occurs in gas gangrene infections. Several workers have described the gross and microscopic appearance of muscle infected by C. perfringens (Robb- Smith, 1945; MacFarlane and MacLennan, 1945; Strunk gt 31. , l96-7) . Hasegawa e_t_ 31. (1970) demonstrated that proteolysis Of the sarcoplasmic and urea soluble fractions of muscle occurred when it was inoculated and incubated with Q. Pgrfringens. C. perfringens is known to produce multiple extra- cellular enzymes (toxins), and as a result causes complex changes in infected muscle. Various workers have attempted isolation and purification of the toxins from g. perfringens in order to elucidate their role in gas gangrene infections. While much has been learned regarding the in yiyg effect of growth of C. perfringens, little is known of their bio- «chemical action upon individual muscle proteins. It may be possible to find new methods of preventing Ineat spoilage by studying the nature and sequence of events .leading to microbial spoilage. New techniques to block the aaction of microbial enzymes, which are presumably responsi- Iale for the deleterious effects of microorganisms, can only hue discovered after the specific mechanism of action is determined . This investigation was undertaken to determine the efflfects of g, perfringens growth upon the proteins of ENDlrcine muscle. Furthermore, a proteolytic enzyme produced by C. perfringens was isolated and its influence upon muscle PIKDteins was studied. REVIEW OF LITERATURE Characterization of (_I. Perfringens Clostridium perfringens, which was previously known as Clostridium welchii and Bacillus aerogenes capsulatus, is a short, plump, non-motile, anaerobic, gram-positive rodwith rounded ends and well marked capsules (MacLennan, 1962). Yamamoto et a1. “(1961) reported that the C. perfringens organism is proteolytic (liquifying gelatin), catalase negative and reduces nitrate. They further reported that C. perfringens produces acid in sucrose, glucose, maltose and lactose media, but does not produce indole. They observed that it caused a "stormy" fermentation in iron milk. MacLennan (1962) in a review stated that _C_:_. Pfi-‘fringens is essentially a saprophyte, producing twelve toxins, which are not important to its metabolism or economy. He reported that the toxins have been named “'13 r y, 5 , c, n, e, 1, K, A, u and v. Not all strains of 9.- I&rfringens produce all toxins, but most strains produce at least one to several (Hauschild, 1971) . MacLennan (1962) reported that there are five recog- nized types of _C_. perfringens, designated A, B, C, D and E. He Stated the classification is based on the nature of the toxins produced. Hauschild (1971) reported that a sixth type, F, was actually a form of type C. Occurrence of g. Perfringens (_3. perfringens is generally considered to be an ubiquitous microorganism, and is found in many environments, such as food, soil, and in human and animal feces. Upon sampling foods in Madison, Wisconsin, Strong g a_l_. (1963) reported that C_. perfringens is present in frozen prepared foods, home prepared foods, spices, market meats, ground and spiced meats, and organ meats. Canada and Strong (1964) found (_2. p_erfringens in 26 per cent of the samples of bovine liver purchased in a commercial market, while only 12 per cent of the livers from newly slaughtered cattle were contaminated. They concluded that the con- tamination did not originate from the bile. Hall and Angelotti (1965) in a survey of the Cincinnati area found C. perfringens to be present in 58 per cent of the raw unprocessed meat specimens. The meat they sampled included veal, beef, chicken, lamb, and pork. Nearly 20 per cent of the processed meat and meat products contained C. perfringens. Those products, which require little or no cooking by the consumer, were found to contain fewer C_:. perfringens cells. The more a meat product is handled, the greater the possibility of contamination with C_. perfringens. Baltzer and Wilson (1965) found C_I. perfringens contamination of pork occurred during the butchering process, after scalding, after scraping, and after inspection. They also found contamination in the scald tank water and on skin sections. They concluded that contamination from machinery was at least partially responsible for the increase in mi crobial numbers . Lepovetsky _e_E §_1_. (1953) isolated C_. perfringens from bovine lymph nodes and bone marrow. They speculated that lymph nodes may be the source of "deep spoilage" in meat. Yamamoto §_t_ E. (1961) found that 16.4 per cent 0f the feces from mature chickens and 15.5 per cent of the feces from turkeys were contaminated with C_. perfringens. They also isolated C_I. perfringens from the feces of 9.6 Per cent of frying chickens. They reported that C_. @rfringens were found in the livers of four fryers and Orlee fowl. Solberg and Elkind (1970) reported 20 to 50 per Cent of C_. perfringens survived heating for 30 to 48 minutes in inoculated frankfurters. They found growth Occurred during storage at temperatures of 12°C or higher. Gough and Alford (1965) determined that Q. per- W survived in cured smoked hams when the final salt Concentration was 3% NaCl, 500 ppm NaNO3 and 200 PP!“ NaN02 They further observed that C_. perfringens remained viable in brine concentrations ranging from 7.5% NaCl, 3,700 ppm NaNO3 and 370 ppm NaNOZ up to 17% NaCl, 23,000 ppm NaNO3 and 2,300 ppm NaNO 2. Infections Caused by (_2_. Perfringens C. perfringens type A is the causative agent for two types of infections in man. One is food poisoning (enteritis) and the other gas gangrene. Food poisoning Several workers (Hall _e_t; a” 1963; Strong 31; a1" 1963; and Nakamura and Schulze, 1970) have wr itten reviews of food poisoning caused by C_. perfringens. They characterized the symptoms of food poisoning as being abdominal pain, diarrhea, occasional vomiting and nausea. They stated that these symptoms generally occur 8 to 12 hours after ingestion of the contaminated food and persist for 6 to 12 hours. They stated that recovery is rapid, and no deaths have been reported as a direct result of C_. per- f‘ri-ngens type A food poisoning. Hauschild (1971), in a review article, reported that: C_. perfringens type C is implicated in a necrotic enteritis of man commonly known as "pig-bel." He stated that it occurs in the highlands of New Guinea and coincides with the ritual of pig feasting, which is characterized by die‘Ltary changes and over—eating. He reported that the Symptoms of "pig-bel" are abdominal cramps, diarrhea, and aCUte inflammation of the small intestine with areas of necrosis and gangrene, particularly in the jejunum. He stated that "pig-bel" has a high mortality rate. Hauschild (1971) also reported that a subspecies of C. perfringens type C, originally thought to be a new type called F, was the cause of outbreaks of a necrotic enteritis called "darmbrand." He indicated that this rare disease was found after World War II in northern Germany. He stated that the causative agent of "darmbrand" was canned meat contaminated with C. perfringens type C spores. He indi- cated that factors involved in the onset of "darmbrand" include a sudden engorgement with rich food, and perhaps some predisposing dysfunction of the alimentary tract. He reported the symptoms of "darmbrand" included severe abdominal pain, vomiting, diarrhea, necrotic inflammation of the small intestine, particularly in the area of the jejunum, and a high mortality rate. The causative agent for food poisoning is not known with certainty. Hauschild and Thatcher (1968) stated that a-toxin is of little importance as a factor in food poison- ing. Duncan and Strong (1969) found a toxic factor in culture filtrates and cell extracts. The toxic factor was heat-labile, non-dialyzable, inactivated by pronase, but not by trypsin, lipase, or amylase, and was inactivated at a pH lower than 5.0 or higher than 12.0. They found lecithinase (a toxin) alone had no gastrointestinal effect. However, they were able to reproduce the enteritis by having volunteers ingest large quantities of viable cells. Hobbs (1965) determined that food poisoning strains of C, perfringens in Great Britain produce low levels of a-toxin, little or no e-toxin, and that they form heat resistant spores. Hall et 31. (1963) in the United States concluded that the ability to produce heat resistant spores is a doubtful criterion for classification of food poisoning strains because contamination can occur after cooking. Hobbs (1965) indicated that food poisoning out- breaks caused by g, perfringens are often traced to meat with a history of storage at warm temperatures. Earlier work by Hobbs (1957) showed that long, slow cooling and a lengthy warming period for meat dishes, as well as holding cooked meat dishes at serving temperatures of 39°C to 49°C for several hours contribute to food poisoning outbreaks. She found that holding meat at high temperatures (62°C to 94°C) will kill viable cells. Therefore, she recommended that cooked meat dishes be held either at refrigerator temperatures or at temperatures above 60°C. Gas gangrene Gas gangrene, the other infection caused by C, perfringens, causes marked structural changes in muscle (Robb-Smith, 1945; MacFarlane and MacLennan, 1945; MacLennan, 1962). Robb-Smith (1945) reported that gas gangrene results in disruption of the sarcolemma due to karyolysis and fragmentation of the muscle fibers. He observed that the myofibrils were preserved but there was an apparent disappearance of the Z line. He noted that the "reticulin membrane" was separated from the sarcolemma with the increased endomyseal space being occupied with edemal fluid. MacLennan (1962) reported that in gas gangrene the "reticulin fibrils" and collagen fibers were partially or completely destroyed, but elastin was unaffected. MacFarlane and MacLennan (1945) observed the gross appearance of infected muscle ranged from a friable, pinkish—grey mass to a blackish—green deliquescent mass. Ispolatovskaya (1971) reported that the only effective method of treatment for gas gangrene has been surgical intervention. He stated that no effective anti; toxin for gas gangrene in humans is known because of the complex action of the various toxins of C. perfringens. However, Ito (1970) reported development of a purified a-toxoid, which is 80 per cent effective against experi- mental gas gangrene in guinea pigs. MacLennan (1962) indicated that gas gangrene is not found in every wound contaminated with C. perfringens. He stated that the proper conditions must exist, the main requirements being a lowered oxidation reduction potential (Eh) at the site of contamination and a lowered pH. Hauschild and Thatcher (1968) demonstrated that food poisoning strains of C. perfringens are capable of producing gas gangrene in guinea pigs. They further 10 reported a correlation between virulence and the synthesis of a-toxin in strains causing gas gangrene. Toxins Produced by g, Perfringens The study of the various toxins of C, perfringens necessitates their separation and purification. Partial to complete separation has been accomplished by several workers, among them Oakley §t_al, (1946), Bidwell and van Heyningen (1948), Roth and Pillemer (1953, 1955), Stephen (1961), Thomson (1963), Habeeb (1964), Ikezawa et_al, (1964), Hauschild (1965), and Kameyama and Akama (1971). Alpha toxin MacLennan (1962) indicated that phospholipase C, or d—toxin, is produced by all types of C, perfringens. He stated that phospholipase C hydrolyses lecithin to phosphoryl choline and a diglyceride. He also reported that a-toxin is activated by Ca+2 and Mg+2 but is inhibited by phosphate, citrate, pyruvate and sodium dodecylsulfate. Ispolatovskaya (1971) indicated a-toxin also acts as an hemolysin. He further reported that a-toxin contains cystine, but is resistant to thiol poisoning and is, there- fore, not a sulfhydryl enzyme. He found zinc was bound to the enzyme and postulated that it was a metallo-enzyme. He determined that the pH optimum of the enzyme in borate 11 buffer was 7.4 to 7.6 and in bicarbonate-C02 buffer was 6.7. He further reported the molecular weight of phospho- lipase C was lO6,000:3,000. He postulated that the enzyme may exist in multiple forms or as iso—enzymes. Beta toxin Although little is known aboutE3-toxin, it causes changes in the cytoplasm and nuclei of guinea pig monocytes and is followed by lysis (Hauschild, 1971). Akama gt a1. (1968) purifiedfi3-toxin from 9, EEE‘ fringens type C. They determinedt3-toxin was immunologi- cally different from any of the toxins produced by g, per- fringens type A. Epsilon toxin Epsilon toxin causes damage to kidney cells and increased vascular permeability (Hauschild, 1971). Thomson (1963) purified e-toxin from C. perfringens type D. He reported that the toxin was produced as a prototoxin, which was readily converted into the active toxin by the action of proteolytic enzymes, especially by trypsin. He found that the prototoxin was converted into the active toxin in the absence of proteolytic enzyme, but the conversion was much slower. Thomson (1963) determined the sedimentation coefficient (8°20,w) of the prototoxin to be 2.48 S at a concentration of 6.96 mg/ml. Using the Archibald technique 12 of approach to equilibrium, he calculated a weight average molecular weight of 40,500. He determined the diffusion 7 cmz/sec and the partial coefficient was D = 6.76 x 10- specific volume to be V": 0.729 ml/g. He then concluded the molecule was asymmetrical and/or hydrated to a con- siderable extent. He also concluded from evidence obtained by ion exchange fractionation that the prototoxin was com— posed of a number of fractions differing slightly in charge. Thomson (1963) suggested that the data may be indicative of a stepwise degradation of prototoxin to active toxin. He explained that the isoelectric point decreases as the prototoxin degrades, which could be caused by stepwise removal of basic groups from the prototoxin molecule. Hauschild (1971) proposed that removal of the basic moieties from asparagine and glutamine in the prototoxin account for the high dicarboxylic acid content of e-toxin. Orlans gt 31. (1960) isolated e-prototoxin and e-toxin. Using mice, they found the lethal dose was 1.13 ug/kg of body weight. They determined that the 5°20,w of c-toxin was 2.8 S. The diffusion coefficient was D = 7.2 x 10'7 cmz/sec. Using these data and a partial Specific volume of V'= 0.74 ml/g, they calculated a molecular weight of 38,000i5,000. Orlans gt_al. (1960) determined that the e—proto- toxin and e-toxin were antigenically identical. They postulated that the toxin and prototoxin differed only by a small peptide at the active site and suggested that the 13 antigenic site differs from the active site. Habeeb (1964) isolated e-toxin from C, perfringens type D. He found the toxin was electrophoretically homo- geneous at pH 4.5, but at pH 8.6 he detected five components. He determined that these five components were antigenically identical. The toxin he isolated had an S° of 2.85 S. 20,w He obtained a molecular weight of 23,200 using sedimentation data and 25,100 using Archibald's method of approach to equilibrium. Hauschild (1965) prepared e-toxin by the method of Habeeb (1964) and separated the preparation into a major non-lethal fraction and a small portion containing all the e-toxin. He proposed that the disagreement in amino acid analyses and molecular weights between Habeeb (1964) and Thomson (1963) could be explained by the presence of a non— lethal contaminant in the former preparation. In order to explain overlapping of toxin and proto- toxin peaks and the increase of toxin during storage of prototoxin preparations, both Hauschild (1965) and Thomson (1963) proposed a gradual conversion of c-toxin from the non-toxic precursor to the toxic form. Theta toxin Roth and Pillemer (1955) reported that e-toxin is an oxygen-labile hemolysin. They stated that e-toxin is dermonecrotizing and reported that its lethality is due to its cardiotoxic action. 14 Roth and Pillemer (1955) determined the SOZO,W of e—toxin to be 6.5 S. They found the pH optimum was 6.75 to 6.8, and the temperature maximum was 37°C. Ispolatovskaya (1971) reported B-toxin does not require Ca+2 or Mg+2. Roth and Pillemer (1955) observed that the action of B-toxin was inhibited by hydrogen peroxide, potassium ferricyanide, iodoacetic acid and parachloromercuribenzoate. They reactivated the oxidized toxin using reducing agents, such as sodium sulfite and sodium hydrosulfite. They hypothesized that O-toxin is a protein containing disulfide linkages, which must be reduced to form the active toxin. Hauschild (1971) reported that no specific sub- strate is now known for e-toxin. He proposed that O-toxin may have a catalytic rather than an enzymatic role. Kappa toxin Oakley 33 El- (1946) determined that K-toxin has both collagenase and a gelatinase activity. Upon injection of K-toxin into guinea pig muscle, Oakley et_al. (1948) demonstrated that collagen was destroyed, but elastin was unaffected. They found that the K-toxin-treated collagen no longer stained red with van Gieson's stain, while elastin fibers remained intact. Oakley gt_§1. (1946) also observed that K-toxin destroyed the reticulum sur- rounding the muscle fibers and the liver trabeculae; this was true regardless of whether a—toxin was present 15 or not. They noted that a-toxin alone had no effect on the reticulum. Bidwell and van Heyningen (1948) prepared K-toxin relatively free from other toxins, but still containing small amounts of a- and G-toxins. They found K—toxin had a pH Optimum between 6.0 and 7.5. The K-toxin was stable in borate buffers at neutral and alkaline pH values, but was unstable at an acid pH or in phosphate buffers. These workers inactivated the K-toxin by heating to 80°C for 5 minutes. They found that the toxin was also inactivated by Cu+2, Hg+2, Mn+2, Co+2 and Fe+2'+3 salts, while Mg+2 and Zn+2 ions were ineffective. Later work by Bidwell (1949) showed heating to 50°C at a pH above 9.0 destroyed the activity against native collagen, while activity against "azocoll" was not lost under these conditions. Kameyama and Akama (1971) purified K-toxin free from neuraminidase, deoxyribonuclease, hyaluronidase and caseinolytic protease. Upon ultracentrifugation, they observed a single boundary with an 5°20,w of 5.19 S. They noted a single precipitan line with immunoelectro- phoresis and immuno double diffusion. They determined that maximum absorption occurred at 276-278 nm and minimum absorption at 252-254 nm. The molecular weight, which they calculated using Sephadex, was approximately 80,000. They determined that the pH optimum was 7.5 in borate buffer and 6.5 in phosphate buffer. Kameyama and Akama (1971) found that K-toxin was 16 most stable in borate buffer and least stable in phosphate buffer. They also found the toxin was heat stable to 40°C but unstable at 60°C. They observed that 30 ug of the purified toxin killed mice by causing extensive hemor- rhaging of the lungs. Levdikova (1966), as cited by Ispolatovskaya (1971), reported the molecular weight of collagenase isolated from C, perfringens to be 113,000:3,000. He found a high carboxylic acid content, which agrees with an isoelectric point of pH 5.0. He also reported that the enzyme had a high immunological specificity. The specific site of hydrolysis was shown to be in the non-polar regions of collagen with the general sequence (Gly-Pro-R)n, where Gly is glycine, R is any amino acid and Pro can be either proline or hydroxyproline. He determined that cleavage occurred between the general amino acid and glycine, i.e. [-Pro-R—4—Gly-Pro-R-1n, with the arrow indicating the site of cleavage. Lambda toxin Lambda toxin is a proteinase which does not attack collagen (Oakley §t_al., 1948). Bidwell (1950) isolated l-toxin from an extract of C, perfringens. She demonstrated that the enzyme was inactivated by heating to 60°C for 10 minutes. She also found A-toxin was inactivated by a pH lower than 5.0 or higher than 9.0. She determined the pH optimum to be between 6.0 and 7.5. She found the l7 enzyme attacked hide powder, gelatin, casein, hemoglobin and seracin, but not native collagen. The A-toxin was inhibited by Cu+2, Hg+2, Fe+2, cysteine and citrate. Iodoacetic acid and cyanide caused slight inhibition while Ca+2 and Mg+2 had no effect on activity. Mg_toxin Ispolatovskaya (1971) reported in a review that u-toxin, the so-called "spreading factor," has hyaluroni- dase activity, and alone is not lethal. He stated that u-toxin has a biological effect on polymerized muc0poly- saccharides, causing depolymerization of the ground sub- stance. He further reported that hyaluronidase promotes microbial growth in gas gangrene infections by liberating fermentable sugars. Other toxins Delta toxin is hemolytic for sheep, goat, cattle and swine red blood cells, but not for horse, rabbit and human red blood cells (Hauschild, 1971). Iota toxin is necrotic and lethal, acting by increasing the permeability of capillaries (Hauschild, 1971). Upsilon toxin is a deoxyribonuclease (Oakley and Warrack, 1951). 18 Changes in Muscle Caused by C, Perfringens C, perfringens toxins produce drastic changes in the structure of muscle. Using a light microscope, Robb-Smith (1945) studied the lesions in muscle at the spreading edges of a gas gangrene infection. He noted an increase in endomysial connective tissue space which became edematous. He observed that the separated fibers showed a greater affinity for eosin stain. He further noted that both the sarcoplasmic nuclei and the nuclei of connective tissues disappeared due to karyolysis. He observed that isolated collagen fibers were widely separated and.the fine reticular fibers between them disappeared as did the reticular membrane. The elastic fibers appeared to be unaffected. In more advanced lesions, Robb-Smith (1945) found ruptured, distorted muscle fibers, although the myofibrils were preserved. He observed that the reticular membrane was separated from the sarcolemma and the number of fibrils were reduced, but the elastic fibers remained intact. He reported that a culture filtrate from g, pggf fringens produced all the above changes ig_yit£g_with the exception of the edema. MacFarlane and MacLennan (1945) found similar changes in addition to a loss of normal muscle tone and elasticity. They also reported that the muscle fibers, though fragmented, were still recognizable. 19 Using an electron microscope, Strunk et a1. (1967) observed crude toxin from g, perfringens left the basement membrane intact with some loosening of its internal structure. They noted that destruction of the plasma membrane began with small discontinuities, which enlarged until the membrane disappeared. They observed that the sarcoplasmic reticulum dilated and vesiculated with formation of dense internal structures. They also observed that the mitochondria displayed enlarged’ prominent cristae, which later vesiculated. There was no observable change in the myofibrils initially, but further action of the toxin caused fragmentation and dis- tortion of the I band. They found that the width of the Z line increased and became less distinct until it dis— appeared. They determined that the sarcomere became dis- torted and unrecognizable as the A band filaments became disarrayed. Strunk et a1. (1967) reported that purified a-toxin alone produced the same changes as crude toxin, except for the alteration of the basement membrane, which they attributed to collagenase. They explained that the dissolution of the myofilaments was dependent upon changes in the Ca+2, HCO3‘1 and K+1 ion concentrations and pH changes. Other factors that they considered to be important were the exogenous and endogenous proteolytic enzymes inherent in muscle, which previously were excluded from the myofibrils by the cell membranes. l/ 20 Grossman et_al. (1967), using electron microscopy, observed that the predominant change caused by a-toxin alone was the appearance of electron-dense deposits on the inner and outer mitochondrial membranes. They found oxidative functions were depressed with a decrease in Krebs cycle intermediates. Bullen and Chushner (1962) theorized that the invasive properties of C, perfringens were not dependent upon a—toxin. They found inoculated muscle damaged by CaClz injection showed high levels of destruction even in the presence of large amounts of d—antitoxin. Hasegawa et 31. (1970) noted that C, perfringens caused proteolysis of urea soluble fractions of rabbit and porcine muscle. They also found C, perfringens destroyed aldolase, glyceraldehyde phosphate dehydrogenase, and lactic dehydrogenase in porcine muscle. Kameyama and Akama (1971) reported 0.7 ug of K-toxin produced hemorrhaging on rabbit skin. In work with guinea pigs, they suggested that the toxin attacks healthy connective tissue in the skin, where collagen fibers are the main component. This conclusion Was based upon pathological changes observed in the subcutaneous tissue of K-toxin-treated guinea pigs. They postulated that the primary site of K-toxin attack in the muscle tissue is the connective tissue supporting the muscle fiber. Kameyama and Akama (1971) confirmed the observa- tions of Strunk et a1. (1967) using a-toxin. They noted 21 differences in muscle destroyed by a-toxin from that des— troyed by K—toxin. Using a-toxin, they observed less prominent hemorrhaging and some of the network of con- nective tissue remained visible to the naked eye, which was not the case using K—toxin. MATERIALS AND METHODS Culturing and Growth of C_l. Perfringens g. perfringens requires a complete medium, including mo st of the common amino acids and vitamins for growth. For production of extracellular enzymes, the requirements are even more rigorous. g. perfringens can be grown on a Difco thioglycollate medium, but for increased toxin pro- duction a more enriched medium is necessary. Murata e_t; E- (1956) developed a reproducible Peptone medium, which was satisfactory for toxin production, but was not completely defined chemically. et a_]._. Later, Murata (1968) reported development of a completely defined Synthetic medium, which was capable of supporting growth and toxin production. Both the peptone medium and the SYnthetic amino acid medium were used in the present investigation . Stock culture Actively growing cultures in Difco thioglycollate lDroth were used as inoculum throughout this investigation. Frozen stock cultures of Clostridium perfringens (ATCC 12915 and ATCC 13124) were obtained from the culture collection at the Michigan State University Meat Laboratory. The 22 23 cultures were maintained anaerobically in Difco thiogly- collate broth at 37°C and transferred daily for a maximum of 30 days, at which time a new stock culture was intro- duced to decrease the possibility of mutations. Culturing for toxin production Culturing for toxin production was carried out using either a peptone or an amino acid medium. Peptone medium. A peptone medium supplemented with 0.247 g/l Difco Bacto beef‘was prepared according to the method of Murata gt g_l_. (1956) . The composition of the Peptone medium is shown in Appendix Table l. The peptone solution was adjusted to pH 7.6-7.8 and brought to 95% of the desired final volume prior to sterilization (15 min at 121°C) . A fructose solution (25% w/v) and an iron solution (70 mg FeSO4'7 H20/100 ml 0.1 N HCl) were autoclaved separately for 15 minutes at 121°C. The fructose solution (4% of final volume) and the iron solution (1% of final volume) were then added asepti- C3E3.Il_1y to the cooled peptone solution immediately prior to inoculation to give the complete peptone medium. Amino acid medium. The synthetic amino acid medium Was prepared according to the method of Murata g g]_._. (1968) . The composition of the amino acid and salt solution and the growth factor solution used in making up the medium is 0.4 A / H n o 0 st: a A S 0.2 A m 05 IO Enzyme Concentration (ml/Sml »reaction mixture) Figure 6. Effect of substrate concentration upon the azocoll reaction at different incubation times. 0 o o 15 min incubation o o o 30 min incubation A A A 45 min incubation A A A 60 min incubation 539 .cae ma ca a.o an woosnuomhm may omamu ou wouasqou weaned mo unseen umcu mm cocawmc ma hua>auom ofixucm no van: a « mma o.aa no.0 o.mn mo.o cam mm.m mma coaumcoauomum oomlm aoUIOam .m o.mm 0.0m m.o m.vv mm.o mama mh.m mma coaumcoauomuu omoaSaaouumdmo .m v.wv m.vm 5.0 a.vm me.o omma mm.oa ava coaumcoauomnm ooalm aouloam .v coaumuamaocum «.ma v.mm m.m hm.o 0m.m mama m.am mm enemasm adacoaad .m 5.0 m.av N.o mm.m mm.a mama v.v Nae c0aumuamaooum man an .N o ooa ooa mmv.o om.v ommv Nv.m mama oumuuaam ouduaso .a AcaOMV Amua>apom we Acaououm we Acamuoum oE\muacdo Aaa\mev Amuacdv Aas\muacsv Aaev ZOHaaonHMDm umm>00mm QamH> >9H>HBU¢ UHhHUmmm szfiomm MBH>HBU¢ «NBH>HBU¢ mzoao> mszzm Adfioa a¢2Hh NMDQNUQmm vmama 0094 mammcaumnom EdacaHUmOaO >h couscoum mmmmuoum c How wEmnom coaumoawancm a canoe 60 Bio-Gel P-lOO fractionation A typical elution pattern from the Bio-Gel P-lOO column is shown in Figure 7. The fractions of highest activity, tubes 12-16, were pooled as indicated by the shaded area and were used in subsequent steps. The pooled fraction was purified 3.66 fold with a 118% recovery of the enzyme in this step. The specific activity had increased from 6.57 to 24.1 units/mg protein. The majority of the enzyme activity was eluted just after the major protein peak, but prior to the diffuse low molecular weight proteins. This indicated that the enzyme has a molecular weight in the general range of 80,000 to 113,000 daltons, which is in agreement with values of 80,000 and 113,000 reported by Kameyama and Akama (1971) and Levdikova (1966), respectively. DEAE-cellulose fractionation A typical DEAE-cellulose elution pattern using a stepwise increase in salt concentration is shown in Figure 8. Fractions exhibiting the highest levels of activity, tubes 66-69, were pooled as shown by the shaded area and used for further purification. Chromatography on DEAR-cellulose resulted in a 1.84 fold purification with a recovery in this step of 83.5%. The specific activity increased from 24.1 to 44.3 units/mg protein. The gradient salt elution pattern from DEAE- cellulose is shown in Figure 9. The standard curve for 61 (Tm/5n) uorqexqueouoo ureqoxd OON 00? com com v madam powDmeQSm Ga poms manna mcoaaomum cmaoom «SNR ava>auom oahucm o o o coaumuucwocoo Gaououm o o o .coaumqoauomnm ooaim amouoam Amnsp\as mac «anssz mass ,on cm or o JRI- olllllOlllllY/IIIIO\\\\\O o “- \ ,/ \. ,l ./ l \ \\ \\\\ .N§§§§§§§§§§§§§§\‘ .\\\\ \\ .h masmam or me ON (Tm/sqrun) KarArqov emfizua 62 Athriov emfizug mmoum ecosqmmhsm Ca poms moiom mcoauomnm cmaoommmxxx. usooao mo coapmnucoocoo Ga cognac.» >ua>auom memucm o o o coaumuuqoocoo caououm o o o .coaumcoauomam mmOasaa001m¢mo mma3mmum .w musoam Amnsu\as was “massz mass o» cm on ov on ON Ow \ cow \\\\ .\\ OON . .\\\ ‘ §5 Q \\\\\\\\\\\\\\\\\\V 3\ com (Tm/81TH“) . \\\\ _uaz 22.3 82. 5.3 (Im/bn) u011911ueou03 ureqoxd 63 (w) uorqexqueouoa ISBN 3 U 2 rA m 8 V 0 a... T_. A TL- 1 K n U T: q S / m TL 0 F O N O ('0 coaumnusoocoo aomz I I I hua>apom oEMNcm o o o scaumuucmocoo cameoum o o o .coaamcoapomum ucoaowuo,mmoasaamolm¢mo Amndu\aE mm Hmnfidz once .m madmam oow CON ooa (rm/6n) uotqexiueouoa ureioxd 64 NaCl concentration is shown in Appendix Figure l. The majority of the enzymatic activity was eluted between 0.15 M and 0.23 M NaCl, which corresponds closely to the major protein peak. The specific activity of the pre— paration adsorbed on the column was 62 units/mg protein. The specific activity of eluate fractions 17-23 had increased to 114 units/mg protein, giving a 1.8 fold purification. Stepwise elution was used in the majority of this investigation because the abrupt change to 0.25 M NaCl concentrated most of the enzyme, which was eluted as a sharp peak. Bio-Gel P-200 fractionation A typical elution pattern from Bio-Gel P-200 is shown in Figure 10. While some activity was eluted with the initial protein, the majority of the activity came off the column with the second protein peak. Fractions 6, 7 and 8 were combined as indicated by the shaded area and used in the muscle incubation studies. The prepara- tion placed on the column had a specific activity of 59.7 units/mg protein. ‘The specific activity of the pooled eluate was 224 units/mg protein, giving a 3.7 fold purification. CM-cellulose fractionation Figure 11 shows the elution pattern from CM- cellulose. No protein values were reported because the 65 (Tm/sirun) AqrArqov emfizua ID or m— ON moacsum oaomse ca coma mio mcoauomum poaoomg >ua>auom mewncw o coahmupowocoo saouonm 0 OO O. .coawmcoauomum oomlm aowioam Awnsu\ae mac Hmnssz mass m. o. .IO/oH/Il/ \\ . ‘ ( 00F OON (Im/bn) uotqexqueouoa ureaord .oa enemas NaCl Concentration (M) Figure 11. 66 100 Quantity of Eluate (ml) CM-cellulose gradient fractionation. o o o enzyme activity 0 o 0 NaCl concentration 200 Enzyme Activity (units/ml) 67 high salt concentration of the eluent interfered with the Lowry protein determination (Lowry gt gt., 1951). The majority of activity was eluted between 0.05 and 0.5 M NaCl. The specific activity of the sample adsorbed on the column was 172 units/mg protein, while the specific activity of the active fractions of the eluate declined to 92 units/mg protein. Since 64.3% of the activity was lost by holding the enzyme preparation at pH 5.5, which was the pH of CM-cellulose elution, the reduction in specific activity during elution can be readily explained. Therefore, this step was not incorporated into the final enzyme purification scheme. Disc electrophoresis gt enzyme fractions Disc electrophoresis uSing 7% acrylamide gels was performed on the fractions from each of the six major steps utilized in purification. Densitometric tracings of these gels are shown in Figures 12-17. Relative intensity of the bands was expressed as a percentage of the total protein applied. This method of expressing the data was used to minimize errors from applying different quantities of protein and from day to day variations in the dye binding (Fazekis de St. Groth gt_gt., 1962). Upon disc gel electrophoresis, the culture filtrate (Figure 12) separated into 8 peaks with the majority (73.6%) of the protein occurring in the over- lapping peaks at RM 0.33 - 0.46. 68 Cl ORIGIN (- Figure 12. Disc gel electrophoresis pattern of culture filtrate. Peak A B C D E F G H I J RM 1.0 0.62 0.46 0.36 0.33 0.29 0.20 0.09 0.05 0.0 % protein - - 6.4 23.1 30.0 20.5 4.7 14.6 0.3 0.04 - - 69 Figure 13 shows the disc gel electrophoresis pattern of the resolubilized zinc chloride precipitate. The 2 major peaks (comprising 60.4% of the protein) migrated at RM 0.38 and 0.53, with 4 other peaks comprising the remainder. The resolubilized ammonium sulfate precipitate (Figure 14) had 2 major peaks (RM 0.37 and 0.48) con- taining 68% of the total protein upon disc gel electrophoresis. The remaining protein was distributed between 5 other peaks. Figure 15 shows the disc gel electrophoresis pattern of the pooled eluate from Bio-Gel P-lOO. Peaks with RM 0.39 and 0.43 contained 52% of the protein, while 8 smaller peaks contained the remainder of the protein. The pooled eluate from DEAE-cellulose, when rsubjected to disc gel electrophoresis (Figure 16), aseparated into 6 peaks. Two of these, RM 0.37 and 0.47, <:ontained 60% of the protein, while a third peak at FEM 0.34 contained 20% of the protein. The remaining Exrotein was found in three other peaks. Upon disc gel electr0phoresis, the purified enzyme preparation separated into 5 peaks (Figure 17) . Tflne peak with RM 0.38 contained 34% of the protein, that witfli RM 0.46, 25%, and that with RM 0.33, 20%. The two remaining peaks, RM 0.17 and 0.56 contained 9 and 12%, respectively. The presence of multiple peaks indicated that the:enzyme was not completely purified. Figure 13. 70 DYE > ORIGIN H Disc gel electrophoresis pattern of Zinc chloride precipitate. Peak A B C _ D E F RM 1.0 0.76 0.63 0.53 0.38 0.30 0.18 0.0 % protein --- 0.7 7.7 27.8 32.6 16.5 14. 71 > .2, ORHSWI I C) \J Figure 14. Disc gel electrophoresis pattern of ammonium sulfate precipitate. Peak A B C D E F G H I RM 1.0 0.70 0.64 0.48 0.37 0.29 0.18 0.06 0.0 % protein ——- 1.5 6.4 36.1 31.9 8.2 13.5 2.4 --- 72 U E ORIGIN L Figure 15. Disc gel electrophoresis pattern of Bio-Gel P-lOO fraction. Peak A B c D E F G H I J K L RM 1.0 0.66 0.62 0.58 0.56 0.43 0.39 0.30 0.19 0.13 0.07 0.0 s‘SPIQtein --- 3.2 3.8 7.2 9.7 31.1 20.8 10.7 9.4 0.7 3.4 --- 73 :30 gm ORIGIN DYE U Figure 16. Disc gel electrophoresis pattern of DEAR-cellulose fraction. Peak A B C D E F G H PM 1.0 0.62 0.47 0.37 0.34 0.18 0.07 0.0 %protein --- 5.2 32.9 26.9 19.6 11.3 4.2 --- 74 ORIGIN DYE Figure 17. Disc gel electrophoresis pattern of Bio-Gel P-200 fraction. Peak A B C D E F G RM 1.0 0.56 0.46 0.38' 0.33 0.17 0.0 % protein --- 12.0 25.3 34.2 19.8 8.7 --- 75 Attempts to identify the protein band(s) con- taining the enzyme activity were unsuccessful, as activity was diffusely apparent over all the major bands, indicating either diffusion of the activity prior to or during measurement, or the presence of several active components. Despite the obvious impurity of this enzyme preparation, it was used in the following studies upon muscle. Changes in Nitrogen and Protein of Incubated Muscle Muscle inoculated with Q. perfringens ATCC 12915 and the uninoculated control muscle were fractionated into the sarcoplasmic and myofibrillar extracts after incubation at 30°C. The extracts were then analyzed for total nitrogen and protein content. Measurement gt nitrogen py Kjehldahl anatysis The changes seen in the nitrogen content, as measured by Kjehldahl analysis, of the sarcoplasmic, myofibrillar and NPN fractions from the incubated muscle are shown in Figure 18. The standard curve for the Nesslerization reaction is shown in Appendix Figure 2. 12 10 Nitrogen Content (mg/g muscle) 76 2 4 Time (Days) 6 Figure 18. Nitrogen content of fractions from the incubated control and C. perfringens inoculated muscle 00 O. O O as measured by Kjehldahl analysis. control sarc0p1asmic extract Q. perfringens inoculated sarc0p1asmic extract control salt-soluble extract Q. perfringens inoculated salt-soluble extract control NPN Q. perfringens inoculated NPN 77 Changes tp the sarc0p1asmic extract The quantity of sarcoplasmic protein showed a decline with incubation in both control and inoculated muscle (Figure 18). Kronman and Winterbottom (1960) reported that aging of muscle resulted in a decreased extractability of water soluble proteins. Some of the decline in nitrogen content was a result of the decrease in extractable protein, since the control sarcoplasmic protein declined from 44.7 to 27.8 mg protein/g muscle. However, the inoculated sample declined considerably more than the control, decreasing from 42.8 to 10.8 mg protein/ g muscle. This is indicative of proteolysis by Q. perfringens. Changes tp the myofibrillar extract The myofibrillar (salt-soluble) proteins from inoculated and incubated muscle exhibited an increase in nitrogen content during the first 2 days of incubation and then declined to a point at or near the initial level as incubation was continued (Figure 18).' Because Kjehldahl determinations measure total nitrogen only, the source of nitrogen cannot be determined by that method alone. Thus, the increase seen in salt—soluble nitrogen may have come from either the sarcoplasmic fraction or the stromal proteins. The myofibrillar protein was obtained by sub— tracting the water-soluble extract from the salt-soluble 78 extract, while the remaining insoluble protein was assumed to be the stromal protein. Bendall and Wismer-Pedersen (1962) reported that holding muscle at elevated temperatures resulted in an increase in the protein content of the myofilaments. They postulated that this gain in protein was caused by a layer of sarcoplasmic protein being firmly bound to the surface of the myofilaments. McClain gt gt. (1965) reported the presence of both acid-soluble and salt-soluble collagen in bovine muscle. They reported an increase in the acid-soluble fraction and a decrease in the salt-soluble components during post-mortem aging. This supports the premise that not all collagen is insoluble, but varying portions may occur in the salt-soluble extract. The changes in salt-soluble nitrogen in the inoculated, incubated samples may be indicative of proteolysis of stromal, sarcoplasmic or myofibrillar proteins, as these cannot be differentiated using simple nitrogen measurements. Another factor, which must be considered, is the microbial protein produced. This PrOtein cannot be differentiated from muscle proteins Efifl; gg, which unquestionably explains some of the discrep- ancies in the various protein classes. 79 Changes tp non-protein nitrogen During incubation there was a consistent increase in the amount of NPN in both control and inoculated samples (Figure 18). The control sample increased from 6.63 to 8.03 mg N/g muscle, while the inoculated sample increased from 6.20 to 8.77 mg N/g muscle. Sharp (1963) found a similar increase in NPN from aseptic rabbit and bovine muscle stored at 37°C, which he stated was derived from the autolysis of sarcoplasmic proteins. Tarrant gt gt. (1971) also observed an increase in NPN upon incubation of muscle with t. ttggt. The fact that bacterial growth and the increase in NPN occurred simultaneously is contrary to the conclusions of Jay and Kontou (1967) and Lerke gt gt. (1967) who reported that bacteria are incapable of utilizing protein nitrogen. Measurement gt_protein py_Lowry and biuret methods Although the absolute values differ, both the Lowry method and the biuret method of measuring protein content show similar trends for the sarcoplasmic and salt-soluble fractions of muscle. The standard curves used for measurement of protein by the Lowry reaction and the biuret method are shown in Appendix Figures 3 and 4, respectively. 80 Electrophoretic Analysis of Sarcoplasmic Proteins After incubation at 37°C, the sarcoplasmic extracts from uninoculated muscle or from muscle inocu- lated with either Q. perfringens ATCC 13124, with purified enzyme solution, or with tris-HCl-CaCl2 buffer solution were subjected to electrophoretic analysis. Disc ggl electrophoresis Disc gel electrophoresis was performed using two concentrations of acrylamide. The relative mobilities of the major sarcoplasmic proteins and the areas of the protein peaks are shown in Tables 2 and 3. The results shown in Table 2 were calculated following electrophoresis in 7.5% acrylamide gels and after staining with amido black. Peak areas are expressed as percentages of total protein. Expression of concentrations relative to the total protein removes a source of error caused by variation in the staining conditions and/or in the concentrations of protein applied to the gels (Fazekas de St. Groth gt gt., 1963). The amount of protein was «determined using the method of Lowry gt gt. (1951). Sarcoplasmic protein was determined by subtracting NPN :from the total sarcoplasmic (water-soluble) extract. Some of the changes in electrophoretic patterns .are the result of incubation of the muscle at a relatively high temperature, while other differences resulted from incubation with either the enzyme or with .m.m u coauma>op cumccmum aadno>o«« .coruoa wnzoa ecu an cocafiuouoc ouo3 mcoaumuucmocoo caououme 81 m.om am so ma v m.mm m we mm o om N «.mv mm mm mm m ma a masses o.mm m me om m ea v e.mv oa om om oa o ma m m+oo spas o.mm oa mm om m m a aonucoo o.vm N an m oa ma a m ma 5 “.mm mm o aa mm m m ma a o.ve m am mm ma oa ea N mammcaumumm v.6v ea ma om ma 6 ea a ssaoanumoao o.oo «a am ma om ma a m.om mm mm ma v ma o m.mo n no em m ma m m.ao ma am oo o oa a o.~m ma om mg m a o aouucoo oaomos o as so.o oa.o mw.o om.o em.o ov.o mm.o oa.o oszeom ilauaaanoz o>auoaom aaeoe oneamsozH mo memo ezmseamme .axoaououm aouoe mo ucoouomc mange memo romam ocaem spas cocamum .ocaemawuom wm.h ca wamouocmouuooam nouns mcaouonm anmmamooumm mo mound room can muaaanoz o>aumamm N wanes 82 the viable organism. The total protein values given in Table 2 are in general agreement with the values reported earlier (Figure 18). The quantity of extractable protein in the control declined from 92 mg/g to approxi- mately 60 mg/g after 1 day at 37°C and then remained at about this level during further incubation. Bendall and Wismer—Pedersen (1962) reported that increasing the temperature of post-mortem muscle resulted in precipita- tion of an unextractable layer of sarcoplasmic protein upon the myofibrils. In the muscle treated with Q. perfringens, the quantity of extractable sarcoplasmic protein continued to decline slowly throughout incubation, reaching approximately 35 mg/g after 7 days. A continual decline in extractable sarcoplasmic protein was also evident in the muscle treated with purified enzyme or with Ca+2. The levels were comparable to those found with Q. perfringens treatment. The decline in total sarcoplasmic protein must be considered when interpreting changes in individual peaks after electrophoresis. In the samples inoculated with Q. perfringens, two new protein bands at RM values of 0.30 and 0.70 (Table 2) appeared after 4 days of incubation. The band at RM 0.70 was quite diffuse, which may be attributed to a series of low molecular weight and/or highly charged species. This band comprised 15% of the total protein. The peak at RM 0.30 contained 10% of the total protein. 83 Thus, the incubation of muscle with Q. perfringens was responsible for the formation of two new peaks, presumably protein breakdown products, which accounted for about one-fourth of the total extractable protein. A third peak at RM 0.37 appeared upon incubation (Table 2). This peak appeared in the control after 7 days of incubation, in the Ca+2 containing control after 2 days of incubation, but appeared after only 1 day of incubation with Q. perfringens. This indicates that +2 incubation with Q. perfripgens or the presence of Ca in the control accelerated formation of the band. The peak at RM 0.46 (Table 2) disappeared in the control after 7 days of incubation, and in the enzyme- treated sample and in the Ca+2-treated control after 4 days of incubation. On the other hand, this peak was present throughout incubation in the sample inoculated with Q. perfringens. In the control, the percentage of the band at RM 0.22 (Table 2) decreased from 43% at 0 days to 13% after 7 days of incubation. A more rapid decrease was seen in the sample inoculated with Q. perfringgns, which indicated that only part of the decline can be attri- buted to autolysis, while the remainder was due to protein degradation by Q. perfringens. The protein with RM 0.07 (Table 2) exhibited variable behavior. Since this peak represents material at the gel interface, slight variations in the gel 84 surface could alter this peak, and thus, the differences are of doubtful significance. Protein concentrations are also reported for peaks with RM values 0.55 and 0.16. The concentrations of these peaks varied slightly, but inconsistent and minor changes preclude any definite conclusions. The viable microorganism appeared to have a greater effect upon the sarcoplasmic proteins than the enzyme. The changes observed in the sarcoplasmic proteins prepared from the muscle inoculated with Q. perfringens cannot logically be attributed to the isolated enzyme, because the changes in the Q. perfringens-treated sample did not parallel those of the enzyme-treated sample. A factor, which may be involved in the changes occurring in the enzyme-treated sample and the corre- sponding control with tris-HCl-CaCl 2 2 buffer, is the (2 0.2 mM) in these samples. In 2 presence of added Ca+ several cases, it is apparent that addition of Ca+ ions may be at least partially responsible for alteration of the bands. The electrophoretic patterns of the sarcoplasmic proteins on 7% acrylamide gels, stained with coomassie blue are shown in Table 3. The differences between the 7% and 7.5% acrylamide gels can be attributed to differ- ences in pore size and the affinity of the various ‘9- .o.m u coauma>oc pumccmum aamum>o«« .conums mnzoa way an cocaeuouoc mums mcoaumuucoocoo caououm« 8‘5 N.om om _ a ma o N.mm mN mm oN o a N o.oo am AN oN oa N m a ossucm o.mm mo ma o Na o o.No am av NN m N N+oo spas o.mm NN AN oN ma m o a aouuooo o.om Na Na o N oa N N.om . mm km N o o.oo mm mm N o N mcommaumnwm v.oo mm Na oN m o a asaoauumoao o.oo om om a aa a N.om mm NN N a aa o m.mo oo om N m N N.ao oN oN NN ma N o a o.No NN aN as a a o aouucoo oaomos o\oe oo.o oa.o aN.o mN.o am.o om.o om.o oo.o *szeomo eases suaaanoz o>auoaom oneamDozH ezmzeamme no mean treaououo aouoe mo usoouomc mamma mama osam mammmeooo nuaa pocamum .ocaeoawuom No.5 ca mamouonmouuooam Houwm mcamuoum anmMamooumm mo nomad xmom can huaaaaoz o>aumamm m magma 86 sarc0p1asmic proteins for the coomassie blue or the amido black dyes. In the 7% acrylamide gels, adjacent bands, which initially were distinguishable, overlapped and became inseparable after a few days of incubation. Another notable difference in the 7% gels was the absence of the rapidly migrating peak in the sample inoculated with Q. perfringens after 4 and 7 days of incubation. This was probably due to the staining technique, since Fazekas de St. Groth gt gt. (1963) reported that insulin and small peptides gave inordinately low readings upon staining with coomassie blue. They reported that acid did not fix these substrates and that the dye-substrate complex was soluble in methanol. The protein band at RM 0.31 (Table 3) appeared only in the muscle incubated for 7 days with Q.' perfringens. This peak contained about 10% of the total protein. Although a small band at RM 0.39 appeared intermittently in all samples after incubation (Table 3), it never exceeded 6% of the total protein. The peak at RM 0.50 comprised 4% or less of the total protein. This band decreased or disappeared in all treatments upon subsequent incubation. Prior to incubation, a band appeared in the control at RM 0.21 (Table 3), which comprised about 40% of the total protein. Densitometer tracings for all the 87 samples indicated that incubation for 1 day resulted in splitting of this band into two peaks with RM values of 0.21 and 0.28. The splitting of the single band into two peaks is borne out by the fact that their combined areas comprised 35-40% of the total protein. After 2 days incubation, these two bands reformed a single diffuse band in all treatments. This diffuse peak disappeared from samples incubated for 7 days with Q. perfringens or for 4 days with the enzyme. Disappear- ance of this protein indicated proteolytic action by both Q. perfringens and the isolated enzyme. The fact that the isolated enzyme caused disappearance more rapidly than the organism suggests that the enzyme alone is responsible for breakdown of this protein. The peaks at RM 0.10 and RM 0.06 (Table 3) were differentiable prior to incubation, but they merged after incubation at various time periods. Merger occurred in the control at 2 days, in the enzyme- treated sample and Ca+2 containing control at 4 days. However, in the Q. pgrfringgns-inoculated sample, the peaks had already combined after only 1 day of incuba- tion. In the control sample, the relative concentration at.different time periods of the peak(s) with RM values ;between 0.10 and 0.06 varied from 50 to 60% of the total pnuotein. In the Q. perfringens-treated sample, the com- lained peak reached a maximum of about 70% of the total 88 protein at 7 days incubation. This peak increased even more reaching 84% in the sample incubated with enzyme for 4 days. The increased proportion of these protein peaks in the samples treated with Q. perfringens and the isolated enzyme appear to be due to the action of the enzyme in both samples. This premise is supported by the greater and more rapid increase in this peak in the sample incubated with enzyme than in the sample incubated with Q. perfringens. The peak at RM 0.64 (Table 3) did not change significantly with any of the treatments. This peak contained 5-13% of the total protein. Using this method of separation, there was some indication that a portion of the changes occurring as a result of incubation with Q. perfringens were caused by the action of the isolated enzyme. In two cases, the differences in densitometric tracings were similar for both the enzyme-treated and Q. perfringens-treated samples. SDS gel electrgphoresis The average molecular weights and the relative concentrations of sarcoplasmic proteins as determined by SDS gel electrophoresis are shown in Tables 4 and 5. Table 4 depicts the separation on gels with 0.27% cross linking while Table 5 shows the separation with 0.135% cross links . 89 .m.a u coauma>oc cumccmum aamno>0¢e .ponuofi >H3oa asp an cocafiumuoo ouo3 mcoaumuucmocoo caououmo N.vm mm mm mm m oa v N.mm ma Na em mm 0 ma N ¢.me n ma ma mm mm ma v a mahucm o.mm. mm 0m mm m ma v v.~v om ea mm mm m o N m+mO nua3 o.mm m ma ma om mm ha a aouucoo m.vm mm mm om n ma h n.mm am mm mm b w v o.vv mN m AN AN m N moomcaumuum v.we em ma 0m mm Na a adacauumoao 0.00 om aa ma om ha ma n N.om am ma am mm ma 0 v m.mo m mm ma am ma ea c m N.ao v om oa mm ma ha m a o.mm n ma ma mm ma ma 0 aouucoo oaomSE m\mE +ooo.hm ooo.on ooo.nm ooo.hv ooo.mm ooo.am ooo.ma «ZHmeomm A4908 wunoamz Hoadomaoz oomuo>¢ zowwmmmmMH Bzmzecmme sacaououm aouos mo ucoouomc mammm same ooaxcaq mmouo NAN.o spas mamouoemouuooam aoo mom “mums mcaououm anmMamooumm mo momma room can unmaoz Hoasooaoz v wands 90 With the higher level of cross linking (Table 4) major bands appeared at RM values corresponding to molecular weights of 15,000, 31,000, 39,000, 47,000, 57,000, 70,000 and 87,000. These points are shown with the standard marker proteins in Figure 19. The 31,000 dalton peak (Table 4) may correspond to the sarcoplasmic F-protein, identified by Scopes and Penny (1971) as having a molecular weight of 30,500. Porcine sarcoplasmic proteins, which have subunits in the molecular weight range 35,000 to 41,000, may contri- bute to the peak at molecular weight 39,000 (Table 4). Scopes and Penny (1971) reported that porcine sarcoplasmic proteins with subunit sizes in this weight range were glyceraldehyde phosphate dehydrogenase at 36,000, lactate dehydrogenase at 35,000 and creatine kinase at 41,000. Scopes and Penny (1971) found phosphoglycerate kinase had a molecular weight of 48,500, which may correspond to the peak with a molecular weight of 47,000 (Table 4). They also reported that porcine phospho- glucose isomerase had a subunit size of 54,000, pyruvate kinase had a subunit size of 57,000 and phosphogluco- mutase as having a molecular weight of 63,000. The protein peak with a molecular weight of 57,000 (Table 4) may correspond to one or more of these proteins. Other peaks shown in Table 4 were not identified. In the control sample, the 31,000 molecular weight peak showed little change during incubation, while the 200000 IOQOOO 91 PHOSPHORYLASE A 0 BOVINE SERUM ALBUMIN 6» a é’ M 50000 a OVALBUMIN U (I) 73' PEPSIN 2 CHYMOTRYPSINOGEN 20,000 10.000 0.2 0.4 0,6 RM Figure 19. Molecular weights of sarcoplasmic proteins determined by comparison with standard proteins using 0.27% cross-linked SDS gels. o o o marker proteins 0 o o sarcoplasmic proteins 92 peak from the sample incubated with Q. perfringens diminished. The relative concentration of this protein was also measurably reduced in both the enzyme-treated sample and the Ca+2-containing control during incubation. The relative concentration of the 57,000 molecular weight peak in the control sample (Table 4) remained fairly constant, while the sample treated with Q. perfringens exhibited a sharp decline, which resulted in complete loss of the peak after 4 days of incubation. This is indicative of breakdown by Q. perfringens. The enzyme-treated sample and the control with Ca+2 appeared to be quite similar in that the 57,000 dalton peak disappeared from both samples after the fourth day of incubation. It should be reemphasized that added Ca+2 ions may account for the difference between the control and the Ca+2-treated control. The peak with a molecular weight of 87,000 diminished in the control, until the band completely disappeared after 4 days incubation. This peak was not evident in the sample treated with Q. perfripgens at any time. This suggests that Q. pgrfringens destroyed or altered this protein during the first 24 hours period. In both the control with Ca+2 and in the enzyme-treated 93 sample, this peak was discernable after incubation for 1 day, but disappeared from both samples incubated for longer periods of time. The peak at a molecular weight of 15,000 was absent in the initial sample, but appeared in all samples after incubation for periods of 1 to 4 days. The levels increased in all treatments until the peak comprised 11 to 16% of the total protein. Minor treatment differences occurred in the peaks with molecular weights of 39,000, 47,000, and 70,000. Although changes in concentration are apparent, the trends appear to be similar in all treatments for each protein. In SDS gel electrophoresis with 0.135% cross linking (Table 5), peaks were apparent at RM values corresponding to molecular weights of 20,000, 29,000, 36,000, 44,000, 48,000, 63,000, 88,000, 106,000 and- 170,000. These points are plotted in relation to known marker proteins as shown in Figure 20. The peak with a molecular weight of 29,000 contained from 8 to 17% of the total protein. Complete disappearance of this peak occurred only in the control treated with Ca+2 ions. The peak with a molecular weight Of 48,000 also disappeared in the control containing Ca+2: but remained present in relatively constant amounts in all other treatments. 94 .m.m u coauma>op cumccwum aamuo>o*« .cozuofi muzoa or» means cocasuouoc mums mcoaumuucoocoo caououme N.om a MN N. am oN m a N.mm N aN ma am aN oa N «.mv mN ea NN mN m o a ossncm o.mm mN No oN o o o.No aN ma NN ON oa o N N+o0 ooa; o.mm a ea aa mN ma ha aa a aouuoou o.em oN o NN NN o a N n.om NN aa mN NN aa N o o.ov oN. m mN aN ma N N moomoauuuom o.oo 0N . aa MN ha Na ea a soaoauumoao o.oo ON a on mN ma a o.om NN Na AN aN aa N o m.mo oN oa mN oa aa m N N.ao o oN oa oN oN ea m a o.No a o m oa ma aN ma ea m o aouoooo oaomos m\ms ooo.oaa ooo.ooa ooo.oN ooo.mo ooo.oo ooo.oo ooo.om ooo.oN ooo.oN *szeomo aaeoe munmaos Hoaoooaoz omouo>4 oneamoozH ezmzeamme so mean sacaououm aouoe mo ucoouomc mamma mama ocaxoaq mmouo Nmma.o ooa; mamouoomouuooam aoo mam Hmumm mcaououm anmMamoouom mo mmou< room can unoaoa Headboaoz m wagon. 95 200,000 ' 100,000 PHOSPHORYLASE A OBOVINE SERUM ALBUMIN 50,000 OVALBUMIN Molecular Weight CHYMOTRYPSINOGEN . 20,000 10,000 0.2 0.4 0.6 0.8 RM Figure 20. Molecular weights of sarcoplasmic proteins determined by comparison with standard proteins using 0.135% cross-linked SDS gels. o o marker proteins 0 o o O sarcoplasmic proteins 96 Although peaks were apparent at 88,000 and 170,000 daltons in the unincubated (0 day) control (Table 5), they disappeared in all samples following 1 day of incubation. The disappearance of these proteins indicates that they were subject either to autolysis or else precipitated upon the myofibrils. The concentration of the protein with a molecular weight of 106,000 (Table 5) declined in the control sample, completely disappearing after 2 days of incubation. This protein was not present in the muscle incubated with Q. perfringens, which suggests that the microorganism hastened its disappearance. In the enzyme-treated and the control samples with Ca+2, the 106,000 molecular weight protein was present at very low levels (2% or less) at various times after incubation. The protein with a molecular weight of 20,000 was apparent at fairly constant levels in all cases, except it was absent from the control after 7 days of incubation and from the muscle incubated with enzyme after 2 days. The relatively low concentration of this band and its disappearance upon incubation of both the control and enzyme-treated sample makes explanation difficult. As seen previously with SDS gel electrophoresis using 0.27% cross linking (Table 4), there were no major treatment differences noted in the proteins at molecular weights of 36,000, 44,000 and 63,000. 97 Isoelectric focusing Few changes were noted with isoelectric focusing, since the isoelectric points appeared over the entire range of pH values used, making the distinction of individual proteins nearly impossible. In the Q. perfringens inoculated samples there appeared to be a decline in the proteins with isoelectric points in the pH range of 6.9-7.3 following 4 and 7 days of incubation. A slight decrease was also noted in the quantity of protein focused at alkaline pH values. Little difference could be detected between the enzyme and the Ca+2 containing control samples. The complexity of the crude muscle extracts was such that separation of the proteins was difficult to achieve. Therefore, isoelectric focusing could be of greater value in assessing changes in simpler mixtures or for purified protein fractions. Electrophoretic Analysis of Myofibrillar Proteins The myofibrillar extracts from uninoculated control muscle, from muscle inoculated and incubated at 37°C with either Q. perfringens (ATCC 13124), with enzyme solution, or with tris-HCl-CaCl solution were subjected 2 to electrophoretic analysis. 98 Disc gel electrophoresis tp urea Electrophoresis in urea took place using two methods of urea addition and two different protein stains. The proteins electrophoresed in the gels stained with amido black were dialyzed against 8 M urea, while those fi electrophoresed in gels stained with coomassie blue were brought to 8 M by the addition of solid ultrapure urea immediately prior to electrophoresis. During overnight dialysis, cyanate ion was reformed in dialyzed urea (Bechtel, 1971), which could lead to carbamylation of free amino groups, labile sulfhydryl groups, and disulfide bonds (Stark gt gt., 1960). When solid urea was added directly to the salt extract, the sample contained a high salt concentration, which may interfere with electrophoresis. The relative mobilities and relative concentra- tions of the myofibrillar proteins electrophoresed in the presence of urea are shown in Tables 6 and 7. Table 6 gives the analyses of the gels stained with amido black, while Table 7 shows the gels stained with coomassie blue. The relative mobilities determined using the two urea systems differed slightly, which can probably be ascribed to differences in handling and gel preparation, as well as to differences in the affinity for the dyes. Use of the relative mobilities determined by Rampton (1969) has allowed tentative identification of 99 some of the myofibrillar proteins. He identified the proteins and their respective mobilities as follows: myosin (0.00-0.15), actin (0.39-0.37), monomeric (oxidized) tropomyosin (0.34), reduced tropomyosin (0.51) and extra protein, fraction IA (0.90). The protein at RM 0.83-0.85 (Tables 6 and 7) corresponds to extra protein, fraction IA, which Rampton (1969) tentatively identified as troponin. The protein with RM 0.50-0.55 (Tables 6 and 7) appeared to be reduced tropomyosin, composed of two separate polypeptide subunits migrating together (Rampton, 1969), while oxidized (monomeric) tropomyosin migrated with RM 0.34— 0.35 (Tables 6 and 7). The protein with RM 0.42-0.46 (Tables 6 and 7) was assumed to be actin. Although myosin is found in the peak at the gel boundary (RM 0.04, Tables 6 and 7), much of the myosin in the myofibrillar fraction probably does not enter the gel structure and, therefore, cannot be analyzed. The concentration of extractable myofibrillar protein is shown in Table 6. These values differ slightly from those reported previously (Figure 18), which can be attributed to the higher incubation temperature, as well as to the different extraction procedure for myofibrillar proteins used in electro- phoresis. As already indicated, the myofibrillar fraction was extracted following the removal of the water-soluble protein. 100 .o.N u coauma>0c cumccmum aa0u0>o«« .pocuoa muzoa moans cocasuoumc caououme e.am ma NN oa ma vN m o e v.mN Na ma ma ha NN m m N v.oN ma oa vm ma 5a m v a ofihwcm o.ma oa 0N ma ma mN m N o o.mN aN NN oN NN o a N N+oo spas o.No ow mN Na ma o m a aouoooo o.om mo vN N aa o N a v.mN om Nm o o o . o N a o.ma om ea ha ea ma m N N mammGaHmuum m.Nm mN ma ma RN oa o a soaoauumoao o.mN ma ma ea ea mN o m a o.NN mN NN Na ma o m o o.am ma AN oa oN m m N o.om oN ma ma oN a a a o.Nm o o ma ma o on m m o aouoooo oaomos o\ma vo.o oa.o oa.o «N.o oN.o om.o No.o om.o mm.o zameomm .. .289 333% 9.33% 28.2803 Easing m0 mwfln :anpoum amuoe No 0.5308 mg 02mm xomam ocaac cuaz ocacamum can mamouonmouuooam “ovum mcaououm Hmaaaunamomz mo mmoum room can moauaaanoz o>au0a0m o canoe 101 In the control, the quantity of extractable myofibrillar protein declined from approximately 80 mg/g prior to incubation to 36 mg/g after 1 day at 37°C. The amount then slowly decreased during further incubation to about 25 mg/g. .In the case of muscle incubated with Q. perfringens, the extractable protein declined to 19 mg/g after 2 days with a subsequent increase to about 30 mg/g by 7 days. In the control containing Ca+2, the decline in extractable myofibrillar protein continued during incubation, reaching about 20 mg/g after 4 days (Table 6). In the muscle inoculated with enzyme, the extractable protein remained relatively constant during subsequent incubation with values near 30 mg/g. Upon incubation with Q. perfringens, the percent- age of troponin (RM 0.83, Table 6) declined, disappearing by 7 days. In all the other muscle samples, troponin remained at fairly constant levels throughout incubation. The concentration of reduced tropomyosin (RM 0.50, Table 6) remained relatively constant throughout incubation in all samples. On the other hand, oxidized tropomyosin (RM 0.34, Table 6) was present in the 0 time sample, but was not evident in any of the incubated samples. The actin peak (RM 0.42, Table 6) decreased during incubation of the control. Values declined from 36 to about 20% of the total protein. In the muscle incubated with Q. perfringens, the actin peak diminished 102 steadily until it comprised only 6% after 7 days. In the enzyme-treated sample and the control with Ca+2, the concentration of actin comprised approximately 20% of the total protein content at all incubation times. The protein migrating at RM 0.29 (Table 6) was evident in only two of the samples incubated at 37°C, namely, in the Q. perfringens-treated samples incubated for either 4 or 7 days. It seems likely that this peak is indicative of proteolytic breakdown, being composed of products of proteolysis different from those appearing in the unincubated control. The intact organism appears to be responsible for the presence of this band, whereas, the isolated enzyme does not produce this protein peak. Thus, the organism and the isolated enzyme appear to differ in regard to formation of this protein. The unidentified peak with RM 0.14 (Table 6), was not evident in the unincubated sample. In the incubated control, this peak became apparent after 7 days, while for the sample incubated with Q. perfringens, it appeared after 2 days. The peak was evident throughout incubation in the control with Ca+2 and in the muscle inoculated with enzyme. In the control, the relative concentration of the unidentified peak at RM 0.10 (Table 6) increased upon incubation from 5% to about 20% of the total protein. Upon incubation with Q. perfringens, the peak 103 at RM 0.10 increased similarly to the control at the end of 1 day, but it disappeared following 4 days of incubation. The peak was evident at levels of about 20% throughout incubation in the muscle treated with enzyme. It was absent in the control with Ca+2 ions at l and 2 days of incubation, but appeared at the 20% level after 4 days of incubation. The large peaks at RM 0.14 and 0.04 may mask a small peak at RM 0.10 (Table 6) since this small peak disappeared after 4 and 7 days of incubation with Q. perfringens, or after 1 and 2 days in the Ca+2-treated control. The changes in the peaks with RM values of 0.04 (myosin) and 0.24 (unidentified) are of doubtful significance. The large increase in the RM 0.04 peak upon incubation with Q. perfringens does suggest that this organism may solubilize the high molecular weight proteins which do not normally migrate into the gel. The gels stained with coomassie blue, shown in‘ Table 7, were less definitive than the gels stained with amido black. The gels did not destain as readily as those stained with amido black or non-urea containing gels stained with coomassie blue. It is postulated that urea in the gels in some manner affects the staining- destaining process. The protein migrating at RM 0.85 in the coomassie blue stained gels (Table 7) was assumed to be identical .N.o u ooaumaooo ouooaoom aaouooo.. .conuofi wusoa moan: cocaahouoc caououmg N.am NN Na om oa N N v.wN mN mN Nm oa oa N N.NN ea NN NN aa oa a assume N.Na Na Na NN aa o N e.wN NN Na ea mm m N N N+mu coax o.Nv N Na Na N oa Na a aouucoo N.om Na NN Na N aa aa N N «.mN Na NN Na N Na Na a o o.Na NN N Na NN ma N N mcomoaumumm N.NN N Na oN N Na N a soaoauumoao N.NN NN ma NN N N N N o.NN NN NN NN oa N o N.am Na NN N Na Na N aa N N o.NN Na NN ea NN N N N a N.NN NN Na om N N Na 0 aouuooo oaomoa N\Ns oo.o oa.o NN.o Nm.o No.0 NN.o NN.o NN.o «zameomm aaeoe . Nuaaanoz 0>auoamm oneamsozH ezmzeamme No memo Acamuoum amuoa mo unmouomv gmmm Mdmm 05am mammmEooo nuaz mcacamum can mawouonmouuooam Houmm mcaouonm Hoaaaunamoaz mo moons Mood cam moauaaanoz 0>auma0m h magma 105 to the peak at RM 0.83 in the amido black stained gels (Table 6). This was tentatively identified as troponin (Rampton, 1969). The relative concentration of this protein is greater in the samples stained with coomassie blue than in the samples stained With amido black. In the control, this peak declined after 1 day of incubation, and then remained relatively constant. In the sample treated with Q, perfringens, the RM 0.85 peak continued to decline during incubation, but the differ- ence was not significant. The enzyme-treated sample and the control with Ca+2 declined only slightly upon incubation. The decline in troponin upon incubation with Q. perfringens was also seen in the gels stained with amido black. This strongly suggests that the organism pgt gg is affecting the troponin peak. The concentration of the control peak which migrated with RM 0.63 (Table 7) did not change signifi- cantly during incubation. The concentration of this peak was higher in the Q. perfringens-treated sample, ranging from 11% to 16%. The relative concentration of this peak in the Ca+2-containing control and enzyme- treated samples remained rather constant during incubation. Identification of this peak was not definite. The reduced tropolyosin peak stained with coomassie blue (RM 0.55, Table 7) was apparent at low 106 levels in the control, except after 4 days of incubation. This peak was present at quite low levels in the sample incubated for 1 day with Q. perfringens, but was absent when incubated for longer periods. This peak was only apparent in the Ca+2-containing control after 1 day of incubation and was not present in the enzyme treated samples at any time. The protein migrating at RM 0.35 (Table 7), which was tentatively identified as oxidized tropomyosin, was apparent throughout incubation on gels stained with coomassie blue, which was not the case for gels stained with amido black (Table 6). The concentration of this protein in the control sample remained relatively constant except for a sharp increase at 4 days, which corresponded to the absence of reduced tropomyosin at this time. The concentration of oxidized tropomyosin in the muscle treated with Q. perfringens declined during incubation. The concentration of oxidized tropomyosin in the Ca+2 containing control and the enzyme-treated muscle remained relatively constant during incubation. Oxidized tropomyosin was consistently present in the samples treated with ultra pure urea, which were stained with coomassie blue (Table 7). The presence of small amounts of reduced tropomyosin in a few samples was probably caused by the formation of some cyanate 107 prior to electrophoresis, which would cause disruption of disulfide bonds (Stark gt gt., 1960). The protein (actin) with RM 0.46 (Table 7) declined in the control during the first 2 days of incubation with an increase to original levels after 7 days. This peak declined considerably in the muscle inoculated and incubated with Q. perfringens, decreasing from 50% to 11% of thetotal protein. A decline was also noted in the Ca+2-containing control, while the muscle treated with enzyme did not change during incuba- tion. Proteolysis of actin by Q. perfringens was indicated by these data. The unidentified protein with RM 0.25 (Table 7) was apparent in the control only at 2 days of incubation. This protein appeared in the muscle treated with Q. perfringens after 4 and 7 days of incubation. This suggests that Q. perfringens produced this protein by breaking down some other peak. In the control, the unidentified protein with RM 0.10 (Table 7) was found in relatively constant amounts through the second day of incubation, after which time it was absent. This peak decreased after 1 or 2 days incubation with Q. perfringens, but further incubation caused an increase in concentration. The peak was . . . + present only at 2 days of incubation 1n the Ca 2- containing control and was absent at all times upon incubation with enzyme. 108 The low mobility peak (myosin, RM 0.04) was not apparent in the unincubated control (Table 7). This may indicate that myosin was too large to enter the gel pores (Florini and Brivio, 1969). The peak appeared upon incubation for 1 day, with the concentration increasing as incubation was continued. In the Q. perfringens- treated sample, myosin did not appear until the second day of incubation, while further incubation did not change the quantity greatly. In the control with Ca+2, the concentration of this peak increased throughout incubation, while in the sample treated with enzyme the peak did not change significantly during incubation. In the samples incubated for 4 or 7 days with Q. perfringens, there was an increase in the concentration of the three slowest migrating peaks, RM 0.25 (unidentified), RM 0.10 (unidentified) and RM 0.04 (myosin). This increase implies proteolysis of large molecules which previously did not enter the gel. Results show there was an increased quantity of salt— soluble proteins after incubation with Q. perfripgens for 4 days. It is assumed that the proteolysis of collagenous material is the source of the increase, since the action of Q. perfringens against collagen is well known (Oakley gt gt., 1946; Bidwell and van Heyningen, 1948; Kameyama and Akama, 1971; Hasegawa gt gt., 1971). 109 Since the myofibrillar proteins are not com- pletely characterized and identified upon disc gel electrophoresis with urea, the changes in individual proteins cannot always be reported with certainty. As proteolytic breakdown products are introduced into the system, the changes in myofibrillar proteins may be masked. SDS gel electrophoresis The average molecular weights of the myofibrillar proteins and the salt soluble proteolytic breakdown products as determined by SDS gel electrophoresis are shown in Figure 21. Major protein peaks were observed with molecular weights of 17,000, 21,000, 25,000, 31,000, 35,000, 46,000, 70,000, 92,000, 110,000, 140,000 and 200,000. The latter 3 molecular weights are extrapolated values, based upon an assumed molecular weight of 200,000 for the experimental myosin peak (Hay gt gt., 1973). Tentative identification of some of the proteins was made on the basis of reported molecular weight values. Figure 22 shows the densitometric tracing of the unincubated myofibrillar fraction with tentative peak identification. The DTNB light chain of myosin, troponin C and another light myosin chain appear to have migrated together at an average molecular weight of 19,000. Reported molecular weights for the myosin light 110 200,000 MYOSIN F-PROTEIN 100.000 PH OSPHORY LASE A d-ACTININ BOVINE SERUM ALBUMIN 50,000 TROPONIN T, TROPOMYOSIN PEPSIN Molecular Weight TROPONIN I, AM MYOSIN LIGHT CHAIN CHYMOTRYPS'NOGEN 20,000 'OPONIN C, DTNB-LIGHT MYOSIN CHAIN MYOGLOBIN ‘I 0,000 RM Figure 21. Molecular weights of myofibrillar proteins determined by comparison with standard proteins using 0.135% cross-linked SDS gels. o o o marker proteins 0 o o myofibrillar proteins Figure 22. .< 111 Densitometric tracing of the unincubated myofibrillar fraction electrophoresed upon a 0.135% cross-linked SDS gel with tentative peak identification. (A) (B) (C) (E) (G) (I) (K) (Y) DTNB light chain of myosin and troponin C, A-l light chain of myosin and troponin I, unidentified, (D) tropomyosin, troponin T, (F) actin and M-protein unidentified, (H) unidentified, a-actinin and M-protein, (J) F-protein, C-protein and M-protein, (L) myosin, marker dye band (Z) gel origin 112 chains are 16,000 and 14,000 (Starr and Offer, 1971), 18,000 (Sender, 1971), 18,000 and 16,000 (Offer g g” 1973) and 16,000, 17,500 and 22,000 (Scopes and Penny, 1971). Wilkinson 22.2l- (1972) reported troponin C had a molecular weight of 18,000. Troponin I and the A-l light chain of myosin are probably inseparable upon SDS gel electrophoresis, both migrating at an average molecular weight of 24,500 (Figure 22). The A-l light chain of myosin has been reported to have a molecular weight of 24,000 by Sender (1971), 27,000 by Starr and Offer (1971), or 25,000 by Offer gt gt. (1973). Wilkinson gt gt. (1972) reported troponin I had a molecular weight of 23,000 while Offer gt gt. (1973) reported a value of 24,000. The protein migrating at 31,000 daltons (Figure 22) is probably identical to an unidentified peak observed in chicken (Hay gt gt., 1973) and rabbit muscle (Offer gt gt., 1973). No other 30,000 molecular weight peaks have been observed in the myofibrillar fraction of muscle. Tropomyosin migrated with an average molecular weight of 35,000 (Figure 22). Tropomyosin has been reported to have a molecular weight of 34,000-35,000 by Scopes and Penny (1971), 36,000 by Hay gt gt. (1973) and 32,000 by Offer gt gt. (1973). The peak at 37,000 daltons (Figure 22) was tentatively identified as troponin T. Offer gt gt. 113 (1973) reported a molecular weight of 35,000 for troponin T, while Sender (1971) reported 38,000 and Wilkinson gt gt. (1972) found a value of 37,000. The peak with an average molecular weight of 44,000 was tentatively identified as actin (Figure 22). Scopes and Penny (1971) have reported a molecular weight of 41,500 for actin, Hay gt gt. (1973) recorded a molecular weight of 49,000, Sender (1971) reported 44,000 and Offer gt gt. (1973) 41,700. Offer gt gt. (1973) also have reported that an M-protein component may comigrate with actin. Hay gt_gt. (1973) speculated that the 44,000 dalton component, which was lost upon aging of chicken muscle may be M-protein. Two small peaks with molecular weights 67,000 and 75,000 were unidentified. Scopes and Penny (1971) report unidentified bands at 55,000 and 75,000 daltons. The protein with a molecular weight of 95,000 has been tentatively identified as a-actinin (Figure 22). Molecular weight values for a-actinin have been reported as 90,000 (Scopes and Penny, 1971; Offer gt gt., 1973), 115,000 (Hay gt_gt., 1973) and 102,000 (Sender, 1971). Offer gt gt. (1973) also reported that a component of M-line protein comigrates with a-actinin. An unidentified peak, possibly F-protein, was seen at a molecular weight of 120,000 (Figure 22). Scopes and Penny (1971) reported an unidentified peak at 114 105,000 daltons. Starr and Offer (1971) reported F- protein to have a molecular weight of 110,000. C-protein was tentatively identified at an average molecular weight of 145,000 (Figure 22). Offer gt gt. (1973) noted the molecular weight of C-protein was 140,000. They also reported that a component of M-line protein was found at 140,000 daltons. Myosin heavy chain protein was tentatively identified at approximately 200,000 daltons. The molecular weight of the heavy chain has been reported to be 200,000 by Offer gt gt. (1973), 207,000 by Sender (1971) and 210,000 by Hay gt gt. (1973). In the present study, some non-migrating proteins were noted at the gel origin, which is in agreement with the results of Sender (1971). The relative peak areas of the salt-soluble proteins upon SDS gel electrophoresis are shown in Table 8. Some proteins are reported together, such as tropomyosin and troponin T, because they were not clearly separable on the densitometric tracings. A low molecular weight component at approximately 15,000 appeared at all time periods in the samples incubated with Q. perfringens or with enzyme (Table 8). 2 This peak also appeared in the control with Ca+ after 1 day of incubation, but at lower levels than for the corresponding enzyme-treated or the Q. perfringens- treated samples. This peak is probably a proteolytic breakdown product. :11.5 .a.a I coauoa>oo unaccouo aamuo>o.. .conuos anaoa moans coaaauouoc caououme v.aN N Na Na NN NN N N N v e.NN N N N Na NN Na aa N N N N.NN a N N ea NN Na Na oa N a osNucm N.Na a N Na NN NN N v N e e.NN e N N Na eN oa aa N N N+No aha: o.Ne a N N N Na NN Na aa N e a aonuooo N.oN N N N 0N NN ea N N N N e.NN N e N NN eN Na Na N N N e o.Na N N N N N oN NN N N N N N mcomoauuumm N.NN aa oa N N Na NN N N N N a suaoauumoao N.NN N ea N N Na NN N N N N o.NN N N N N aN NN N N N e N.aN N N N Na NN N aa N N o.NN Na N N Na NN N Na N a v.NN NN N N N N ea Na N N N o aonueoo oaomsa Nxma ooo.ooN ooo.Nea ooo.oNa ooo.NN ooo.oN ooo.NN ooo.NN ooo.aN ooN.NN ooo.NN ooo.NN «ZHMBOMQ acfioa armada Haaoooaoz 0UNHO>¢ ZOMW4MWWMH BZMZB¢MMB Nixcamuoum amuoe «0 unwoummc Name MNNN mamouonmouuooam amu mom an coumudmom mcaououm uNaaaunamomz no mmwu¢ xmom cam munmamz Hmasooaoz oomuo>< m canoe 116 The band at 19,000 daltons containing troponin C and the DTNB-light chain of myosin (Table 8) was com- pletely removed from the Q. perfringens-treated sample after 7 days of incubation, but remained in fairly high concentrations in all other samples, indicating proteolysis by Q. perfringens. The relative concentration of the band at 24,500 daltons, which tentatively was identified as containing trOponin I and the A-l light chain of myosin, was reduced in the sample incubated for 1 day with Q. perfringens and then remained at rather low levels during further incubation. The control sample had a maximum level of this protein atil day of incubation, after which time it gradually declined to pre-incubation levels. The enzyme-treated sample and the control with Ca+2 ions also had maximum levels of this band after 1 day incuba— tion, which then gradually declined. The reduction upon incubation with Q. perfringens in the peaks containing troponin (19,000 and 24,500 daltons) is consistent with the reduction in the concentration of extra protein fraction IA (troponin) as measured by disc gel electrophoresis in urea (Tables 6 and 7). The unidentified 31,000 molecular weight component remained fairly constant in all samples throughout incubation (Table 8) except for an increase on the fourth day of incubation with enzyme. 117 The tropomyosin-troponin T band (35,000) decreased in the sample incubated with Q. perfripgens (Table 8). The relative concentration of this peak in the control sample increased during the first day of incubation, and then remained rather constant. The control containing Ca+2 increased throughout incubation, while the enzyme-treated sample remained relatively constant. The decrease seen upon growth of Q. perfringens is significant in light of previous reductions noted in troponin containing peaks (Tables 6, 7 and 8). When comparisons are made between the tropomyosin peaks obtained upon SDS gel electrophoresis and disc gel electrophoresis with urea (Tables 6 and 7), some differences were noted. In the gels stained with amido black (Table 6), tropomyosin migrated in the reduced form (RM 0.50). In the coomassie blue stained gels (Table 7), tropomyosin was seen at RM 0.55 (reduced) as well as RM 0.35 (oxidized). If the reduced and oxidized tropomyosin were combined, the relative amount in the Q. perfringens treated sample was less than in the control sample, indicating proteolytic breakdown of tropomyosin. Another interesting feature was the presence of an unidentified peak at RM 0.63 (Table 7) in the coomassie blue-stained gels, which was not apparent in the amido black-stained gels (Table 6). It may be that this peak (RM 0.63, Table 7) migrated with or 118 overlapped tropomyosin (RM 0.50) in the amido black- stained gels (Table 6) and thereby concealed changes in the tropomyosin peak. If this were indeed the case, any breakdown of tropomyosin would be hidden. Never- the-less, both the SDS gels (Table 8) and the coomassie blue-stained urea gels (Table 7) suggested that tropo- myosin was degraded by Q. perfringens. Upon SDS gel electrophoresis, the actin peak (44,000 daltons, Table 8) remained relatively constant in the control sample throughout incubation. The muscle incubated with Q. perfringens showed a consistent but slight increase in this peak as incubation proceeded. The Ca+2-containing control also exhibited a slight increase, while the muscle treated with enzyme remained relatively constant. This is contrary to evidence obtained upon electrophoresis in urea (Tables 6 and 7) which indicated a decline in the percentage of actin in samples treated with Q. perfringens. The small unidentified peaks with average weights of. 70,000 upon SDS gel electrophoresis (Table 8) decreased in the control sample during the first 4 days incubation, but increased at 7 days incubation. The relative concentration of this peak in the muscle incubated with Q. perfringens increased throughout incubation, probably because of the formation of proteolytic breakdown products. Both the control with Ca+2 and the sample treated with enzyme had similar ‘ 119 concentrations of this peak, and both exhibited a slight increase at 4 days of incubation. Upon SDS gel electrophoresis, the a-actinin peak (95,000, Table 8) was discernible in only a few samples after incubation. The fact that a-actinin was not present in the control after incubation is contrary to the results of Hay gt gt. (1973),who reported that a-actinin was unchanged in chicken muscle up to 168 hours post- mortem. The 120,000 molecular weight peak (Table 8) was seen Luxni SDS gel electrophoresis of the incubated control only after 7 days incubation, which probably indicates autolysis. This peak was seen in the sample incubated with Q. perfringens in steadily diminishing amounts as incubation continued. The concentration of the peak tentatively identified as C-protein (145,000, Table 8) remained relatively constant in the control, with a slight rise at 7 days incubation. The relative concentration of C-protein decreased slowly in muscle incubated with Q. perfringens. In the enzyme-treated and Ca+2- containing control samples, this protein remained at a relatively constant concentration, with the enzyme- treated sample showing only a slight decline upon 4 days incubation. The myosin peak (200,000) is the most difficult to analyze. The concentration of this peak (Table 8) 120 diminished greatly during incubation. Because of the large size of myosin and the difficulty in subjecting it to electrophoresis, the fate of myosin cannot be completely determined. It may be rapidly degraded by the high temperature of incubation, rendered unextract- able, or aggregated to such an extent that it does not enter the gels. Isoelectric focusing tp urea As found with the sarcoplasmic proteins, changes in the patterns of the myofibrillar proteins seen upon isoelectric focusing are difficult to interpret. In general, incubation appeared to decrease the proportion of proteins with isoelectric points above 6.5. This trend was observed in the Q. perfringens-treated sample at 1 day of incubation. At 2 days incubation, the majority of the proteins from the sample incubated with Q. perfringens focused in the pH range 4.5-6.2. After 7 days incubation, however, the control sample retained some proteins having isoelectric points above pH 6.5. After 7 days incubation with Q. perfringens, two peaks reappeared at pH values 8.7 and 6.6. Other peaks with isoelectric points of 6.2 and 5.3-5.8 were observed after 7 days incubation in both the Q. perfringens- treated sample and the control. Both the enzyme- treated sample and the Ca+2-containing control had iso- electric focusing patterns similar to the control sample. 121 General Observations on Electrophoresis While many differences can be seen between one particular electrOphoresis pattern and another, the changes attributable to the treatments are difficult to discern because of the complex nature of the protein system. During incubation many of the proteins changed concurrently, and the net effect was not detectable using these relatively crude separation techniques. However, it is possible that these changes are dependent upon each other and that they might not occur inmore simplified systems. Until more definitive methods of separating and identifying the proteins in the inoculated and incubated samples are developed, the effect of these treatments upon individual proteins will remain diffi- cult to assess. Electron Microscopy Electron micrographs of samples from control and Q. perfringens-inoculated muscle were examined after incubation for both 1 and 4 days. After 7 days incuba- tion, the muscle was degraded to such an extent that it was impossible to prepare satisfactory electron micro- graphs. The myofibrils were classified on the basis of Z line width as either red fibers or white and inter- mediate fibers (Gauthier, 1970). Dutson (1971) 122 determined that the width of the Z line in porcine red fibers was about 12.0 pm, in intermediate fibers about 7.75 um and in white fibers approximately 6.25 pm. Since the difference in the width of the Z lines for intermediate and white fibers is relatively small and the amount of disruption was relatively large in this study, no attempt to differentiate between the two was made. Changes tp the ultrastructure gt incubated muscle A typical electron micrograph of a white or intermediate fiber from control muscle incubated for 1 day is shown in Figure 23. The width of the Z lines in this fiber was about 7.5 um. The ultrastructure remained relatively intact, with no noticeable disruption. The Z and M lines are intact and the thin filaments (I band) are clearly visible and are attached to the Z line. A typical electron micrograph of red fibers from the control muscle incubated for 1 day is shown in Figure 24. The Z lines in this micrograph were from 10.0 to 12.5 um in width. The red fibers appeared to be intact, with the primary features of the sarcomere being clearly evident. The intact structures seen in both the red and white fibers are in contrast to the report of Henderson gt gt. (1970), who reported that Z lines in prerigor porcine muscle became amorphous and disrupted within 4 hours at 37°C. Figure 23. 123 Electron micrograph of white or intermediate fiber from control muscle incubated for 1 day at 37°C. M = M line, Z = Z line, I = I hand, H = H zone. 26,563 X. 124 Figure 24. Electron micrograph showing red fibers from control muscle after incubation for 1 day at 37°C. M = M line, Z = 2 line, I = I hand, H = H zone. 36,000 X. 125 Figure 25 shows white or intermediate fibers after incubation for 1 day with Q. perfripgens. The width of the Z lines was about 6.4 um. The electron micrograph shows some disruption of the myofibrillar structure. The thin filaments had broken loose from the Z line in several places, although the Z line itself, while diffuse and amorphous, was still recognizable. The characteristic structure of the M line had disappeared in some sarcomeres. Henderson gt gt. (1970) reported the loss of 60% of the M lines in prerigor porcine muscle stored for 24 hours at 37°C. An electron micrograph of red fibers from muscle incubated with Q. perfringens for 1 day is shown in Figure 26. The Z line showed some disruption, while its width varied from 12 to 13.5 um. The attachment between the thin filaments and the Z line was severed in several places. The M-line had become quite amorphous, with almost complete disappearance of the H zone. After 4 days incubation, the red fibers in the control muscle remained relatively intact (Figure 27). The Z line was 13.6 um wide and was quite distinct. The thin filaments remained connected to the Z line in the majority of the sarcomeres. The M line was amorphous and had lost its distinct structure. There appeared to be some overlapping of the thin filaments, which resulted in a slightly darkened area in the region of the H zone, while the characteristic H zone had completely disappeared. Figure 25. 126 Electron micrograph of white or intermediate fibers after incubation for 1 day with Q. perfringens at 37°C. M = M line, Z = Z line, I = I band, H = H zone, ++ indicate disruption in thin filaments. 56,250 X. ' Figure 26. 127 < -0... O I Electron micrograph showing red fibers from muscle incubated for 1 day at 37°C with g. erfrin ens. M = M line, Z = Z line, I = I band, ++ indicate disruption in thin filaments. 34,230 X. 128 Figure 27. Electron micrograph of red fiber from muscle incubated for 4 days at 37°C. Z = Z line, H = H zone, I = I band. 22,770 X. . 129 In the electron micrograph (Figure 28) of red fibers (Z line = 13.0 pm) from muscle incubated for 4 days with Q. perfringens, the characteristic M line structure and the M line material was definitely absent. The Z line was recognizable, but showed some disruption and breakage in several Spots. The thin filaments appeared to be quite degraded and were no longer attached to the Z line. This is in agreement with the observations of Strunk et a1. (1967), who determined that one of the first effects of Q. perfringens toxins upon the myofibrils was the fragmentation and distortion of the I band. In contrast, Dutson et 31. (1971) showed that g, fragi disrupted the thick filaments (myosin) in the A band. They noted that the material in the H zone and the Z line had disappeared, but observed that the thin filaments (actin) were still distinct. Thus, 9. perfringens appears to have distinctly different effects than g. fragi upon the ultrastructure of muscle. 9. perfringens causes degradation of the thin filaments, whereas, the thin filaments are not greatly affected by g. fragi. The disruption of the thin filaments is in agreement with the electrophoretic evidence (Tables 6, 7 and 8) that troponin is destroyed or altered by Q. perfringens, since troponin is a constituent of the thin filament (Maruyama and Ebashi, 1970). 130 Figure 28. Electron micrograph showing red muscle fibers incubated at 37°C for 4 days with Q. perfringens. Z = Z line, M = M line, I = I band, ++ indicate areas of disruption in thin filaments. 23,040 X. 131 Q. perfringens also completely removed the M line from red fibers after 4 days incubation (Figure 28). This was in contrast to the sample incubated for 1 day with Q. perfringens, in which the M line from red fibers was disrupted, but could still be seen. It was noted that treatment with enzyme or Ca+2 ions increased the disruption of the Z lines, but it was difficult to assess the changes because of mechanical disruption caused by incorporation of the enzyme or the Ca+2 solution into the muscle sample. Disruption of the Z lines has also been reported upon addition of l m M Ca+2 to intact muscle by Busch §t_al. (1972). Tarrant et a1. (1973) also noted a loss of the Z line after incubation of muscle with an enzyme isolated from g. fragi. There was some indication that Q. perfringens degraded white or intermediate fibers more readily than red fibers. After 4 days incubation it was not possible to definitely identify white or intermediate fibers in the incubated muscle, but the red fibers were still readily discernible. A comparison of red and white fibers from inoculated muscle after 1 day of incubation with Q. perfringens shows that the thin filaments from white fibers underwent a greater amount of disruption. After 7 days incubation, samples inoculated with enzyme or with Q. perfringens had an acrid, lachrymatory odor. When these samples were embedded prior to electron 132 microscopy, the epon blocks did not polymerize properly, making it impossible to obtain good sections. Appearance 9: Q. perfringens An electron micrograph showing a longitudinal section of Q. perfringens growing in muscle is shown in Figure 29. The organism measured about 220 um in length and approximately 95 pm in width and was quite typical of other organisms observed. The outer membrane appeared to be smooth with no indication of bleb-like evaginations. This is in contrast to g. fragi, which was reported to develop surface blebs upon growth in muscle (Dutson, 1971). Importance of Spoilage Studies Some effects of growth of Q. perfringens upon porcine muscle were determined in this study. Results indicated that proteolysis of muscle does indeed occur during microbial growth, but did not indicate whether proteolysis is the cause of spoilage or merely occurs during the spoilage process. Never-the-less, protein degradation does occur simultaneously with microbial spoilage and likely contributes to the development of the off-odors and flavors characteristic of spoiled meat. Prevention of spoilage is of major economic importance to both the meat industry and the consumer. When the mechanism by which spoilage occurs can be 133 determined, it will be possible to use this knowledge in blocking spoilage. The development of new preserva- tion techniques which interfere with biochemical deterioration would be of great benefit in increasing the shelf life of meat and meat products, thereby improving meat quality. 134 Figure 29. Electron micrograph showing a longitudinal section of Q. perfringens grown in muscle for 1 day at 37°C. 53,760 X. )v. 1. a‘ SUMMARY Aseptic porcine muscle samples were inoculated either with viable cultures on. perfringens (ATCC 13124 or 12915) or with an enzyme isolated from the culture filtrate of the same organism grown in peptone medium. The enzyme was prepared by Zn C12 precipitation, disodium phosphate extraction and ammonium sulfate precipitation, followed by gel filtration and ion exchange chromato- graphy. After incubation at 30 or 37°C, the muscle samples were separated into sarcoplasmic, myofibrillar or non-protein nitrogen fractions. A11 fractions were analyzed for nitrogen. The sarcoplasmic and myofibrillar extracts were subjected to disc gel electrophoresis, SDS gel electrophoresis and isoelectric focusing. Incubation with Q. perfringens resulted in an increase in non-protein nitrogen, which implied that proteolysis had occurred with formation of small decomposition products. Proteolytic breakdown of the sarcoplasmic protein was also indicated by a decrease in sarcoplasmic nitrogen upon incubation with Q. perfringens and by the reduction in size of certain protein peaks upon disc gel or SDS gel electrophoresis. Growth of 135 136 Q. perfringens also resulted in the formation of new peaks in the gels of the water-soluble fraction. New salt-soluble proteins also appeared in the gels of the myofibrillar extract after incubation with Q. perfringens, indicating formation of other pro- teolytic breakdown products. In the myofibrillar extract, growth of Q. perfringens caused a consistent reduction in the level of troponin as well as some evidence for degradation of actin and tropomyosin. Electron microscopy revealed destruction of the thin filaments in muscle during incubation with Q. perfringens, which provides supporting evidence for the breakdown of troponin. In this study, the intact organism was shown to be capable of degrading both sarcoplasmic and myofibrillar proteins from porcine muscle. Although the enzyme isolated from cultures of Q. perfringens caused major breakdown in the sarcoplasmic fraction, it caused little proteolysis of the myofibrillar fraction. Since the enzyme and the intact organism did not always cause proteolysis of the same peaks, it is suggested that Q. perfringens produces more than one enzyme capable of degrading muscle. BIBLIOGRAPHY BIBLIOGRAPHY Akama, K., S. Otoni and S. Kameyama. 1968. Purification of B toxin of Clostridium perfringens type C. Jap. J. Med. Sci. 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APPENDIX 144 APPENDIX TABLE 1 Composition of Peptone Medium Salt Solution I NaZHPO4°7 H20 KH2PO4 Salt Solution II ZDSO4 ° 7 H20 Vitamin Mixture Ca-d-pantothenate Nicotinic acid Thiamine hydrochloride Pyradoxamine hydrochloride Riboflavin Biotin Adenine sulfate Uracil Peptone Medium Difco proteose peptone Difco Bacto beef Salt Solution I Salt Solution II Vitamin Mixture Thioglycolic acid g/100 ml 11.4 2.8 0.4 14.2 10 0.1 340 250 amount/1,900 ml 100 g 494 mg 100 ml 20 ml 100 ml 0.2 ml 145 APPENDIX TABLE 2 Composition of Amino Acid Medium Amino Acids and Salts mg/850 ml DL-Alanine 500 L-Arginine 10,000 L-Asparagine 500 L-Cystine 50 L—Glutamic acid 750 Glycine 500 L-Histidine 250 DL-Isoleucine 500 L-Leucine 350 L-Lysine 250 DL-Methionine 500 L-Proline 150 DL—Phenylalanine 1,000 DL-Serine 500 DL-Threonine 500 L-Tryptophan 250 L-Tyrosine 100 DL—Valine 250 Na HPO4 2,300 Na 1 3,000 KCl 800 MgSO4-7 H20 200 CaC12-2 H20 74 ZnSO4-7 H20 14 Growth Factor Mixture mg/l Glutamine 5,000 Ca-d-pantothenate 20 Nicotinic acid 20 Thiamine hydrochloride 20 Pyradoxamine hydrochloride 14.2 Riboflavin 10 Biotin 0.1 Adenine sulfate 340 Uracil 250 146 APPENDIX TABLE 3 Formulation of 7.5% Polyacrylamide Gel Electrophoresis System Separating Gel (7.5% Acrylamide, 0.18% BIS, pH 8.9) Stock solutions amount/100 m1 a) Tris 36.3 g N,N,N',N'-Tetramethy1ethylene- diamine (TEMED) 0.23 ml 1 N HCl to yield pH 8.8-9.0 b) Acrylamide 30 g N,N'—Methylenebisacry1amide (BIS) 735 mg c) Ammonium persulfate 140 mg Working solution - 1 part (a):1 part (b):2 parts (c) Stacking Gel (2.5% Acrylamide, 0.625% BIS) Stock solutions amountgloo m1 a) Tris 5.98 g TEMED 0.46 ml 1 N HCl to yield pH 6.7 b) Acrylamide 10 g BIS 2.5 g c) Riboflavin 4.0 mg d) Sucrose 40 9 Working solution - 1 part (a):2 parts (b):l part (c): 4 parts (d) Buffer Solution (0.04 M Tris, 0.2 M Glycine, pH 8.3) g/l Tris 3.0 14.4 Glycine 147 APPENDIX TABLE 4 Formulation of 6.25% Polyacrylamide Gel Electrophoresis System with Urea Separating Gel (6.25% Acrylamide, 0.1% BIS) amount/25 m1 Acrylamide 1.625 g BIS 0.025 g TEMED 0.01 ml Riboflavin (4 mg/100 ml) 2.5 ml Tris 1.21 g Urea 10.5 g HCl (2 N) 0.8 ml Stacking Gel (5% Acrylamide, 0.1% BIS) amount/10 m1 Acrylamide 0.5 g BIS 0.01 g TEMED 0.004 ml Riboflavin (4 mg/100 ml 1.0 m1 Tris 0.075 g Urea 4.2 g HCl (2 N) 0.3 m1 Buffer Solution (0.005 M Tris, 0.04 M Glycine, pH 8.3) 9_/_1 Tris 0.6 Glycine 2.88 148 _4) Conductivity (umho x 10 02 04 06 Concentration NaCl (M) (18 10 Appendix Figure 1. Conductivity of NaCl solutions. 0 o<3NaC1 in 0.02 M Tris—HCl 0 o oNaCl in 0.02 M Acetate 149 1.5 g 1.0 C1 (0 .Q )4 0 § 0 O 0.5 2 4 6 8 Quantity of Nitrogen (ugN/ml of solution) Appendix Figure 2. Standard curve for determining nitrogen using Nessler's reagent. 0 O 0 Absorbance at 420 nm 0 O O Absorbance at 500 nm 150 14 Q8 05 Absorbance at 660 nm 0A 200 400 600 800 Quantity of Bovine Serum Albumin (ug/ml) Appendix Figure 3. Standard curve for determining protein using Lowry's method. 151 1.6 3 m 1.2 4.! (U Q) U C 3 08 H . O 5 0.4 2 4 6 8 10 Quantity of Bovine Serum Albumin (mg/ml) Appendix Figure 4. Standard curve for determining protein using the biuret method. IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII mu(LIIMIMI)“wwwwflwwlHm