Physicochemical Changes in Pacific Whiting Muscle Proteinsduring Iced Storage SOOTTAWAT BENJAKUL, THOMAS A. SEYMOUR, MICHAEL T. MORRISSEY, and HAEJUNG AN ABSTRACT No changes in actomyosin Ca -, Mg -, or Mg -Ca -ATPase activities were observed during iced storage of Pacific whiting fillets, but Mg2+EGTA-ATPase increased with a loss of Ca2+-sensitivity. Surface hydrophobicity of actomyosin increased substantially within 2 days, but not total sulfhydryl (SH) content. During longer storage, the SH content decreased gradually, but surface hydrophobicity remained constant. Autolytic degradation products increased in fish muscle with storage time. Myosin heavy chain (MHC) was degraded by 45% within 8 days, but no noticeable difference was observed in actin. Results indicated that autolysis may be the main cause of physicochemical changes in Pacific whiting muscle proteins during iced storage. 2+ 2+ 2+ 2+ Key Words: Pacific whiting, muscle proteins, actomyosin, ATPase INTRODUCTION INTEGRITY OF MYOFIBRILLAR PROTEINS is of prime importance for surimi production (An et al., 1996; Lin and Park, 1996). High-strength surimi gels cannot be produced from denatured myosin, and consequently, much study has been directed toward preventing myosin from denaturation. Fish, particularly those with high proteinase activities, have been recommended to be held below 47C and processed into surimi within 24 hr of capture (Peters et al., 1996). Fish that are not processed within a short time, or kept at low temperatures result in deterioration of final surimi quality. Among post-harvest changes, degradation of fish muscle caused by endogenous proteases is a primary cause of quality losses during cold storage or handling (Haard et al., 1994). The degradation of muscle structure results from many proteases, such as cathepsin B or L (Yamashita and Konagaya, 1991), cathepsin D (Jiang et al., 1990), alkaline proteases (Folco et al., 1984) and calcium-dependent proteases (Koohmaraie et al., 1988a,b). The effects of individual proteases on degradation are difficult to estimate during iced storage. Proteolytic activity in Pacific whiting has been induced by the infection of Myxosporidea, and the level correlated highly with the number of white pseudocysts in muscle (Patashinik et al., 1982; Kabata and Whitaker, 1985; Morrissey et al., 1995). More than 50% of Pacific whiting samples had notable textural defects due to increased proteolytic activity after 3 days in ice (Morrissey et al., 1992). Myofibrillar ATPase activities have been widely used as a measure of actomyosin integrity (Roura et al., 1990). Factors that cause denaturation or degradation of the protein can affect ATPase activities. Such activities have been widely used to monitor postmortem changes during iced or frozen storage (MacDonald and Lanier, 1994; Kamel et al., 1991; Seki et al., 1979). Myofibrillar proteins are susceptible to degradation by lysosomal enzymes and calcium-activated neutral proteinases (Ouali and Valin, 1981). Therefore, degradation of myofibrillar proteins can be indirectly measured by changes in ATPase activity. This makes measure of ATPase activity useful for quality Authors Seymour, Morrissey, and An are affiliated with the Oregon State Univ. Seafood Laboratory, 250-36th St., Astoria, OR 97103-2499, and author Benjakul is with the Dept. of Food Technology, Fac. of Agro-Industry, Prince of Songkla Univ., Songkhla, Thailand. Address inquiries to Dr. Haejung An. assessment of muscle (Kamal et al., 1991; Ko et al., 1991; Tachibana et al., 1993; MacDonald and Lanier, 1994). Biochemical changes in muscle proteins, i.e., actomyosin and myofibrils, have been reported for some fish (Roura et al., 1990, 1992; Roura and Crupkin, 1995) and bivalves (Paredi et al., 1990). Information regarding changes during commercial storage of Pacific whiting is scarce. Our objective was to study biochemical and physicochemical properties of Pacific whiting myofibrillar proteins during iced storage. MATERIALS & METHODS Reagents Ammonium molybdate, 1-anilinonaphthalene-8-sulfonic acid (ANS), 4-methylaminophenol (hemisulfate salt), 5,5'-dithio-bis(2-nitrobenzoic acid), ethylene glycol-bis(b-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA), CaCl2, adenosine 5'-triphosphate (disodium salt) were purchased from Sigma Chemical Co. (St. Louis, MO). MgCl2 was purchased from J.T. Baker Chemical Co. (Phillipsburg, NJ). Sample preparation Pacific whiting (Merluccius productus) were caught commercially off the Oregon coast, stored in refrigerated seawater, and off-loaded within 12–16 hr capture. The fish were transported in ice to OSU Seafood Laboratory, immediately washed, filleted, and packed in polyethelene bags. The packed fillets were stored in ice and removed at 0, 2, 4, 6 and 8 days storage for analyses. All experiments were carried out in duplicate. Preparation of actomyosin Actomyosin was prepared according to the method of MacDonald and Lanier (1994). Pacific whiting muscle (4g) was homogenized in 40 mL chilled (47C) 0.6M KCl, pH 7.0 for 4 min using a Polytron (Brinkmann Instruments, Westbury, NY). The beaker containing the sample was placed in ice and each 20 sec of blending was followed by a 20 sec rest interval to avoid overheating during extraction. The extract was centrifuged at 5,000 3 g for 30 min at 07C. Three volumes of chilled deionized water were added to precipitate actomyosin. Actomyosin was collected by centrifuging at 5,000 3 g for 20 min at 07C, and the pellet was dissolved by stirring for 30 min at 07C in an equal volume of chilled 1.2M KCl, pH 7.0. Undissolved material was removed from the preparation by centrifugation at 5,000 3 g for 20 min at 07C. Actomyosin ATPase activity and protein ATPase activity was determined using modified methods of MacDonald and Lanier (1994) and Roura and Crupkin (1995). The prepared actomyosin was diluted to 2.5–4 mg/mL with 0.6M KCl, pH 7.0, and 1 mL of the diluted solution was added to 0.6 mL of 0.5M Tris-maleate, pH 7.0. To that mixture one of the following solutions was then added for each ATPase activity assay to a total volume of 9.5 mL: 10 mM CaCl2 for Ca2+-ATPase; 2 mM MgCl2 for Mg2+-ATPase; 0.1 mM CaCl2 and 2 mM MgCl2 for Mg2+-Ca2+-ATPase; and 2 mM MgCl2 and 0.5 mM EGTA for Mg2+-EGTA-ATPase. To each assay solution, 0.5 mL of 20 mM ATP was added to initiate the reaction. The reaction was conducted for exactly 8 min at 257C and terminated by adding 5 mL chilled 15% (w/v) trichloroacetic acid. The reaction mixture was centrifuged at 3,500 3 g for 5 min, and the inorganic phosphate liberated in the supernatant was measured by the method of Fiske and Subbarow (1925). Specific activity was expressed as µmoles inorganic phosphate (Pi) released/mg protein/min. A blank solution was prepared by adding chilled trichlo- Volume 62, No. 4, 1997—JOURNAL OF FOOD SCIENCE—729 WHITING MUSCLE CHANGES DURING ICED STORAGE . . . Fig. 1—ATPase activities of actomyosin from Pacific whiting stored in ice. One unit of activity was defined as that releasing 1 µmole Pi/mg protein/min. Fig. 2—Ca2+ sensitivity of actomyosin extracted from Pacific whiting muscle stored in ice. roacetic acid prior to addition of ATP. Ca2+ sensitivity was calculated according to Seki and Narita (1980) as follows: et al. (1996a). Actomyosin (1 mL, 0.4%) was added to 9 mL 0.2M TrisHCl buffer, pH 6.8, containing 8M urea, 2% SDS, and 10 mM EDTA. A 4 mL-aliquot of the mixture was taken, and 0.4 mL of 0.1% DTNB solution was added and incubated at 407C for 25 min. Absorbance was measured at 412 nm with a spectrophotometer (Beckman Instrument, Inc., Redmond, WA). A blank was prepared by replacing the sample with 0.6M KCl, pH 7.0. SH content was calculated from the absorbance using the molar extinction of 13,600 M21 cm21 and was expressed as mol/105g protein. ~ Ca2+ sensitivity 5 1 2 ! Mg2+-EGTA-ATPase activity 3 100 Mg2+-Ca2+-ATPase activity Electrophoretic analysis of protein degradation Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) was carried out according to the method of Laemmli (1970) using 4% stacking gel and 10% separating gel. Sample (3g) was homogenized with 5% (w/v) SDS in a final volume of 30 mL. The homogenate was incubated at 857C for 1 hr to dissolve total proteins. The supernatant was collected after centrifuging at 3,500 3 g for 5 min at ambient temperature. Protein (40 µg) was applied on the gel. Proteins were stained in 0.125% Coomassie brilliant blue R-250 and destained in 25% ethanol and 10% acetic acid. High molecular weight standards (Sigma Chemical Co, St. Louis, MO) included rabbit muscle myosin (205,000), E. coli b-galactosidase (116,000), rabbit muscle phosphorylase b (97,000), bovine serum albumin (66,000), ovalbumin (45,000) and bovine erythrocytes carbonic anhydrase (29,000). Band area was quantitated by scanning gels with a HP DeskScan II (Hewlett-Packard Co., Minneapolis, MN) and analyzing the image with software (National Institutes of Health Image 1.54, Washington, DC). pH and protein determination Pacific whiting muscle was homogenized in 10 volumes water (w/v), and pH was measured using a pH meter (Corning Science Products, Corning, NY). Protein concentration was measured by the method of Lowry et al. (1951) using bovine serum albumin as standard. Hydrophobicity Protein surface hydrophobicity was determined by the method of LiChen et al. (1985) as modified by Roura et al. (1992). Prepared actomyosin in 10 mM phosphate buffer, pH 6.0 containing 0.6M NaCl was diluted to 0.1, 0.3, 0.5, 0.9% (w/v) protein using the same buffer. The diluted protein (2 mL) was stabilized by incubation at 207C for 10 min and added with 10 µL of 8 mM ANS in 0.1M phosphate buffer, pH 7.0. The relative fluorescence intensity of ANS-protein conjugates was measured using an Aminco Bowman spectrofluorometer (American Instrument Co., Silver Spring, MD) at excitation wavelength 374 nm and emission wavelength 485 nm. For standards, 2 mL methanol was added directly to 10 µL ANS, and readings were adjusted to 80% of full scale as suggested by Roura et al. (1992). Protein hydrophobicity was calculated from initial slopes of plots of relative fluorescence intensity vs. protein concentration (%, w/v) using linear regression analysis. The initial slope was referred to as SoANS. Total SH analysis Total SH content was measured using 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) according to Ellman (1959) as modified by Sompongse Measurement of autolytic degradation product Fish muscle (3g) was homogenized in 27 mL of 5% (w/v) trichloroacetic acid. The homogenate was kept on ice for 1 hr and centrifuged at 5,000 3 g for 5 min. Tyrosine in the supernatant was measured as an index of autolytic degradation products according to the method of Morrissey et al. (1993) and expressed as µmol tyrosine/g muscle. Statistical analyses Data were analyzed by analysis of variance (ANOVA). Mean difference was determined using the least significant difference (LSD) multiple range test (Statgraphics Version 6.0, Manugistics Inc., Rockville, MD). Significance of difference was established at p≤0.05. RESULTS & DISCUSSION ATPase activity No changes in Ca2+-ATPase, Mg2+-Ca2+-ATPase or Mg2+ATPase were observed, but Mg2+-EGTA-ATPase activity gradually increased during iced storage (p,0.05) (Fig. 1). Ca2+ATPase activity is a good indicator of the integrity of the myosin molecule (Roura and Crupkin, 1995). Mg2+ and Mg2+-Ca2+-ATPase activities are indicative of the integrity of the actin-myosin complex in the presence of endogenous or exogenous Ca2+ ions, respectively. Mg2+-EGTA ATPase activity indicates the integrity of the tropomyosin-troponin complex (Ouali and Valin, 1981; Ebasi and Endo, 1968; Watabe et al., 1989). Increased myofibrillar Mg2+-ATPase activity can enhance ATP consumption, resulting in accelerated rigor mortis (Hwang et al., 1991; Sikorski et al., 1990; Watabe et al., 1989). All ATPase activities including Ca2+-, Mg2+- and EDTA-ATPase of ordinary and dark muscles began to decrease when pH declined to 6.4. The decrease in fish myofibrillar ATPase activities was reported to be a direct function of pH (Kamal et al., 1991). In our results, the pH of Pacific whiting muscle did not change and ranged 7.2–7.4 during 8 days storage. Mg2+-ATPase activity in the presence of EGTA increased during storage with a concomitant loss of Ca2+ sensitivity (Fig. 2). This result confirmed the change in Mg2+-EGTA-ATPase activity of carp myofibrillar proteins reported during iced storage (Seki and Narita, 1980). Mg2+-EGTA-ATPase activity of myofi- 730—JOURNAL OF FOOD SCIENCE—Volume 62, No. 4, 1997 Fig. 3—Surface hydrophobicity of actomyosin from Pacific whiting stored in ice. SoANS designates the slope of relative fluorescence intensity vs protein concentration (%). Fig. 4—Total sulfhydryl group content of actomyosin from Pacific whiting stored in ice as related to storage time. Total SH was calculated from absorbance at 412 nm using the molar extinction coefficient of 13,600 M21cm21 for 2-nitro-5-thiobenzoic acid. brils was reported to increase by treatment with lysosomal proteases (Ouali and Valin, 1981). Also, Ca2+-activated neutral proteinases increased in activity by releasing a-actinin from Zlines, which was found to be an activator of ATPase in desensitized actomyosin (Ouali and Valin, 1981). Therefore, we hypothesized that proteinases contributed to the changes in Mg2+-EGTA-ATPase activity observed in Pacific whiting during iced storage. Ca21-sensitivity Ca2+-sensitivity of Pacific whiting actomyosin decreased with an increase in storage time (Fig. 2). Ca2+-sensitivity was reported to be a good indicator of Ca2+ regulation of myofibrillar proteins (Roura and Crupkin, 1995) and was dependent upon the affinity of the troponin molecule for Ca2+ ion (Ebashi et al., 1968). Thus, reduction of the Ca2+ sensitivity of myofibrils would be indicative of proteolytic degradation of tropomyosin or troponin or both (Okitani et al., 1980). Removal of troponin, the Ca2+-receptive protein of the contractile system, has resulted in a decrease in Ca2+-binding capacity (Ebashi et al., 1968). The decrease in both Ca2+-binding capacity and Ca2+ sensitivity was shown to be caused by proteolysis (Tokiwa and Matsumiya, 1969). Therefore, we hypothesized that the observed decrease in Ca2+-sensitivity we observed was related to proteinase activity, particularly that of cathepsins, in Pacific whiting muscle. Conformational changes of actomyosin Changes in actomyosin surface hydrophobicity during iced storage of Pacific whiting showed that SoANS increased 56% after 2 days storage and remained constant during the next 6 days (Fig. 3). This confirmed results of Roura et al. (1992) that the surface hydrophobicity of hake actomyosin increased during iced storage, particularly during the first 3 days. ANS, a fluorescence probe, has been found to bind to the hydrophobic amino acids containing an aromatic ring, i.e., phenylalanine and tryptophan, when conformational changes occur in the protein (Roura et al., 1992, Kato and Nakai, 1980). Multilangi et al. (1996) reported that SoANS of heat-denatured whey protein isolate increased with enzymatic hydrolysis and the magnitude of increase was related to the type of enzyme. Hydrophobic interactions between amino acids and oxidation of SH residues were reported to affect surface hydrophobicity (Hill et al., 1982). Increased surface hydrophobicity indicates an exposure of the interior of the molecule due to denaturation or degradation (Multilangi et al., 1996). Such changes usually result in decreased water-holding ability, less succulence of intact flesh or lower gelling ability in surimi. Fig. 5—Autolytic degradation products in Pacific whiting muscle during iced storage as indicated by TCA-soluble tyrosine. Total SH content Total SH content of actomyosin increased slightly after 2 days storage followed by a gradual continued decrease up to 8 days (Fig. 4). This confirmed results of Sompongse et al. (1996b) that the total SH group content of carp actomyosin decreased constantly, especially after 3 days storage in ice. A decrease in total SH group was reported to be due to formation of disulfide bonds through oxidation of SH groups or disulfide interchanges (Hayakawa and Nakai, 1985). Oxidation of thiol groups of myosin has been shown to reduce Ca2+ sensitivity and modified actinmyosin interactions (Seki et al., 1979). Chan et al. (1995) reported that myosin contained 42 SH groups. Two types of SH groups on the myosin head portion (SH1 and SH2) have been reported to be involved in ATPase activities of myosin (Kielley and Bradley, 1956; Sekine et al., 1962; Yamaguchi and Sekine, 1966). Another SH group (SHa) found later was localized in the light meromyosin region of myosin molecule and was responsible for Mg2+-ATPase activity (Yamashita and Horigome, 1977; Horigome and Yamashita, 1977). Sompongse et al. (1996b) reported that SHa was responsible for oxidation of MHC and its dimer formation resulting in an increase in Mg2+-EGTA-ATPase activity of carp actomyosin during iced storage. Our results indicated that the Mg2+-EGTA-ATPase in Pacific whiting also increased during storage. Therefore, the decrease in total SH content was probably due to oxidation of SHa groups. Volume 62, No. 4, 1997—JOURNAL OF FOOD SCIENCE—731 WHITING MUSCLE CHANGES DURING ICED STORAGE . . . Fig. 7—Changes in MHC and actin contents of Pacific whiting muscle during iced storage. chum salmon than did cathepsin B against myofibrillar proteins (Yamashita and Konagaya, 1991). Calpain II, Ca2+-activated neutral proteinase, may also contribute to autolytic degradation. Calpain II was reported to be important in post-mortem tenderization of tilapia muscle (Jiang et al., 1991). After death, the Ca2+ ion equilibrium fails, which was reported to permit free ions to come to equilibration through the tissue, resulting in activation of the Ca2+-requiring proteinases in muscle cells (Etherington, 1984). CONCLUSION PROTEIN DENATURATION and degradation of muscle proteins became obvious within 8 days iced storage. Tropomyosin-troponin complex was more affected by iced storage than actin-myosin interactions in muscles of Pacific whiting. These changes were due to proteolytic activity in the muscle. These results emphasize the importance of rapid and proper post harvest handling to ensure quality of Pacific whiting. Fig. 6—SDS-PAGE patterns of Pacific whiting proteins during iced storage. Forty µg protein were applied on 10% polyacrylamide gel. Numbers designate storage days. M, high molecular weight standard; MHC, myosin heavy chain. Proteolytic degradation Autolytic degradation products were detected throughout storage (Fig. 5). The tyrosine level detected at day 0 indicated the endogenous tyrosine in muscle plus the degradation products accumulated during post-harvest handling. An et al. (1994) reported that among the Pacific whiting proteins, MHC was the most extensively hydrolyzed, followed by troponin-T and a- and b-tropomyosin. Degradation of myofibrils has occurred at 07C by cathepsins and serine proteinases, though the hydrolysis rate was low (Tokiwa and Matsumiya, 1969; Busconi et al., 1989). Microbial proteases may also be a potential source of proteolytic degradation. Protease from Pseudomonas marinoglutinosa was reported to hydrolyze actomyosin at 0–27C and the optimal pH was above 7.0 (Venugopal et al., 1983). The proteolytic degradation patterns of muscle proteins analyzed by SDS-PAGE showed that MHC was hydrolyzed continuously throughout storage (Fig. 6). MHC decreased to 45% of the original content within 8 days storage. No notable changes in actin were observed on SDS-PAGE (Fig. 6 and 7). Fish muscle contains several proteinases capable of causing autolytic degradation of muscle. An et al. (1994) reported that cathepsin B was the most active cysteine protease found in Pacific whiting fillets at ,377C followed by cathepsin L and H. However, cathepsin L exhibited more hydrolytic activity in REFERENCES An, H., Peters, M.Y., and Seymour, T.A. 1996. Roles of endogenous enzymes on surimi gelation. Trends Food Sci. Technol. 7: 321–327. An, H., Weerasinghe, V., Seymour, T.A., and Morrissey, M.T. 1994. 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Hydrolytic action of salmon cathepsins B and L to muscle structural proteins in respect of muscle softening. Nippon Suisan Gakkaishi 57: 1917–1922. Ms received 10/21/96; revised 2/3/97; accepted 2/12/97. This work was partially supported by Grant No. NA36RG0451 (Project No. R/SF-1) from the National Oceanic and Atmospheric Administration to the Oregon State University Sea Grant College Program and by appropriation made by the Oregon State legislature and by Grant No. 96-35500-3340 from the U.S. Dept. of Agriculture-Cooperative State Research, Education, and Extension Service. The views expressed herein are those of the authors and do not necessarily reflect the views of NOAA or any of its subagencies. Volume 62, No. 4, 1997—JOURNAL OF FOOD SCIENCE—733