Recently the use of proteolytic enzymes to tenderize meats has increased tremendously. The enzyme papain has been used extensively in commercial meat tenderizers, but many other enzymes also possess the prerequisite proteolytic activity for meat tenderization. Because of the wide variety of proteolytic enzymes from plant, animal, and microbiological sources, which appear suitable and have been suggested as meat tenderizers, a comparison is desirable.As an integral part of the investigation on papain and meat tenderization being conducted in this laboratory, the relative merits of different proteolytic enzymesb were investigated by digesting rehydrated, ground, freeze-dried biceps fentoris muscle of beef with the various enzymes and then fractionating the component protein fractions of meat by extraction and precipitation techniques. I n order to evaluate the extent and mechanism of digestion, the soluble-nitrogen fraction was separated into solubleprotein nitrogen and non-protein nitrogen, and the insoluble-protein fraction was separated into collagen and elastin. The degree of enzymatic digestion of each fraction and sub-fraction was determined by Kjeldahl nitrogen determinations or hydroxyproline determinations.Very little has been written on the relative activities of different proteolytic enzymes on meat. Essentially, the only information on this subject comes from Gottschall and Kies (5) and Hay, Harrison, and Vail (6), who have reported investigations on meat digestion and tenderization by papain and a commercial papain-constituted meat tenderizer, respectively.Despite the recent investigations by Lampitt, Baker, and Brown (10, 11) on the relative extractive ability of dilute hydrochloric acid and sodium hydroxide solutions in extracting soluble meat proteins, it appeared desirable to extend their investigation to include two additional reagents, 0.5% potassium chloride and 20% urea, which are also used for extracting soluble meat proteins. Soluble proteins have been extracted with urea solutions by Neuman and Logan (15) and Beck and Schormuller ( 3 ) , while 0.5% potassium chloride solution has been used as an extraction medium by investigators at this laboratory in dehydrated meat studies. The sodium hydroxide extraction procedure is essentially that introduced by Lowry, Gilligam, and Katersky (12) and subsequently employed by Prudent (16), Husaini, Deatherage, Kunkle, and Draudt (7, 8) and Baker, Lampitt, and Brown (10, 11). I n comparing extractions with hydrochloric acid and sodium hydroxide, the last group of investigators found sodium hydroxide ' Supported in part by a grant from Adolph's Foundation.The enzymes used in this investigation are papain, bromelin, ficin, trypsin, pepsin, Rhozyme P-11, Protease 15, Rhozyme A-4, and a commercial meat tenderizer containing papain. 21 7

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The use of meat tenderizers containing the proteolytic enzyme papain has grown to large proportions. However, there have been no critical studies of the mechanism of meat tenderization by papain treatment, or of the factors influencing the effectiveness of papain tenderization of meat. Histological investigation could yield much valuable information concerning the mechanism of meat tenderization with papain ; however, such studies are lacking.I n one of the first papers on the tenderization of meat with papain, Gottschall and Kies ( 3 ) observed a penetration of 2.5 mm. by soaking raw meat in papain solution for 20 minutes at 23°C. Penetration studies led them to conclude that there is no advantage in pretreatment because enzyme digestion takes place during the cooking process. Nevertheless, empirical precooking holding periods, usually of the order of 1 hour per each inch of thickness of the meat, are recommended commercially. For example, this precooking holding period has been used in the studies of Hay, Harrison, and Vail ( 5 ) . Because of the controversial aspect of the effect of the precooking holding period, a more critical evaluation of -enzyme penetration and hydrolytic activity at room temperature and of the effect of this precooking holding period seems appropriate.The mechanism of meat tenderization by papain at the molecular level can be described by the relative hydrolysis of the muscle-fiber protein, mainly actomyosin, and by hydrolysis of the connective tissue, mainly collagen and elastin. Studies of the hydrolysis of various beef protein fractions by papain at 60°C. have been recently reported (6).It was the purpose of this investigation to define by appropriate physical, chemical, and histological methods the effect of time of papain application prior to cooking and the effect of temperature on protein hydrolysis by papain. MATERIALS AND METHODS Histological examination.For histological studies of enzyme action, a semitendinosus muscle from U.S.D.A. Choice grade of beef was used. The meat was cut into % inchthick slices by parallel cuts along the length of the muscle fibers, and adjacent discs, 1 inch in diameter, were cut from these slices with a cork borer. Some discs were left raw, some cooked in a pressure cooker (10 p.s.i. for 20 min.), and some were coated on the upper flat surface with powdered papain (S. B. Penick and Co.) before pressure cooking as above. Several pieces of meat were cut so that the ends of the muscle fibers were exposed. Papain powder was placed on this exposed surface and samples were taken initially, after 3 hr. a t room temperature and after % hr. at room temperature and 10 min. of broiling. a Supported in part by a grant from Adolph's Foundation.3 75

Serum levels of several commonly measured enzymes are abnormal in patients with end-stage renal disease (ESRD). In the present study we measured serum total LDH activity and LDH isoenzymes in a group of 25 ESRD patients shortly before, during, and immediately after hemodialysis. Baseline serum total LDH activity in the ESRD group was midly but significantly higher than that found in the normal control group. The observed elevation was associated with an isomorphic pattern. Single passage of blood through the extracorporeal apparatus led to a rise in total LDH, LDH-3, LDH-2 and LDH-4, a pattern consistent with release from the platelets. A steady increase was noted in total LDH, LDH-5 and LDH-1 of arterial blood during hemodialysis. This was thought to be due to ultrafiltration-induced hemoconcentration, enzyme release from the formed elements within the extracorporeal circuit and complement-mediated leukocyte activation and pulmonary leukostasis. These observations should be taken into consideration with regards to interpretation of LDH and LDH isoenzyme values and proper time of blood sampling in ESRD patients.

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