Identification of Red Snapper (Lutjanus campechanus) using Electrophoretic Techniques TUNG-SHI HUANG, MARTY R. MARSHALL and CHENG-I WEI ABSTRACT Isoelectric focusing (IEF), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and two-dimensional (2-D) gel electro- phoresis were employed to produce protein profiles for species identifi- cation of red snapperand 11 other fish species.Comparing the distinctive patterns of water-soluble sarcoplasmic proteins for each species on IEF and SDS gels, red snapper could be identified. IEF gels of ampholyte mixture of 20% pH 3-10 and 80% pH 4-6.5 resolved better than gels with ampholyte ranging from pH 3-10, 4-6.5, 6-8, or 5-6 for species identification. The 10 and 12.5% SDS-PAGE gels produced more dis- tinctive protein profiles for identification than 7.5 and 15% gels. Thus, these techniques could be applied to identify fish species. Key Words: red snapper, protein profiles, electrophoresis INTRODUCTION RED SNAPPER (Lutjunus campechanus) is one of the most im- portant commercial fish in the snapper-grouper fishery in the Northern Gulf of Mexico (FAO Species Catalogue, 1985). It is a popular eating fish with relatively high market value. Because of their solitary habits and territorial behavior, red snapper are not caught in huge quantities. The yield of red snapper around Florida waters from about 1984 to 1994 has become nearly steady and is estimated to be 1,360-1,814 metric tons annually. The high value of red snapper and the lack of predicted in- creasesin this resource lead to illegal sale of other red snapper- like fish and/or the substitution of less valuable species in seafood markets. Illegal substitutions have occurred for red snapper and other valuable snappers and groupers and to a lesser extent, for mackerel and swordfish. The FDA (1980) issued a ‘Compliance Policy Guide’ (7108.21) to resolve concerns for selling Pacific coas! rockfish (family Scorpaenidue) as red snap- per. The development of analytical methods to identify fish spe- cies is, therefore, important in preventing willful or unintentional substitution of lower valued fish species for high valued fish in the marketplace. Electrophoretic methods such as starch gel-zone electropho- resis, acrylamide disc electrophoresis, thin layer polyacrylamide gel isoelectric focusing and cellulose acetate strip have been accepted as official methods by the Association of Official An- alytical Chemists (AOAC, 1990) to differentiate seafood species or seafood products. Lundstrom (1980) and An et al. (1988) utilized various electrophoretic support matrixes to successfully separatefish muscle proteins for identification. Sodium dodecyl sulfate (SDS) has been incorporated into the gel to improve resolution of electrophoretic protein patterns (Lundstrom 1979, 1980; Melvin, 1987; An et al., 1988). Isoelectric focusing (IEF) has been extensively used for iden- tification of seafood speciesbecause it provides reliable and re- producible resolved protein patterns for differentiating closely related species(Lundstrom, 1983a, 1983b; An et al., 1989). Urea has also been incorporated to enhance protein separation and resolution (Keung et al., 1985; An et al., 1989). The Florida Department of Agriculture and Consumer Services used thin layer IEF to identify retail snapper fillets (Hsieh et al., 1989). The authors are affiliated with the Food Science & Human Nutri- tion Dept., Univ. of Florida, Gainesville, Florida 3261 I-0370. Ad- dress inquiries to Dr. C. I. Wei. Our objective was to investigate the feasibility of using SDS- polyacrylamide gel electrophoresis (SDS-PAGE), IEF, and two- dimensional (2-D) gel electrophoresisto prepare protein profiles for red snapper identification compared with other closely related fish speciesthat may be substituted illegally for red snapper. MATERIALS & METHODS Fish samples Authentic samples of red snapper (Lutjanus campechanus, RS), ver- milion snapper (Rhomboplites aurorubens, VS), gray snapper (Lutjunus griseus, GS), hogfish (Lachnoluimus muximus, HF), lane snapper (Lu- tjunus synagris, LS), mutton snapper (Lutjanus analis, MS), and yellow- tail snapper (Ocyurus chrysurus, YS) were obtained from the Food Laboratory, Florida Department of Agriculture and Consumer Service, Tallahassee, FL. Red snapperobtained from the Texas and Florida coasts off the Gulf of Mexico were provided by Dr. W: S. Otwell, University of Florida. Onespot snapper (Lufjunus monostigma, OS), blackspot snap- per (Lutjanus jiilvzjfamma, BS), and Madras snapper (Lutjanus lutjanus, LL) were obtained from Taiwan and shipped in dry ice to the laboratory at the Food Science and Human Nutrition Dept., Univ. of Florida. VS, GS, HF, pink porgy (Pugrus pngrus, PG), and white grunt (Haemulon plumieri, WG) were purchased from a local seafood store. Each species was represented by at least two fish. Fish filets were prepared after they were scaled, gutted, and headed. The filets (white muscle only) were then cut into small pieces of about 2Og, put in Whirlpak@ bags, and stored at -33°C until needed. Protein extraction and sample preparation The outer layers of defrosted fish samples were removed. Only the center part of each sample, about 6g, was homogenized with 3 volumes of water (w/v) at 24°C for 1 min using a Polytron (setting 6.2, Brink- mann Instruments, Westbury, NY). The water contained 0.1 mM phen- ylmethylsulfonyl fluoride (PMSF), 10 mM EDTA and 0.01% sodium azide to inhibit proteases and microbial growth. After homogenized sam- ples were centrifuged at 26,900 X g for 20 min at 5”C, the supematants were collected and protein concentrations determined by Lowry method (Lowry et al., 1951). Following protein adjustment to 5 mg/mL with water, the supematantswere added with sucrosegranules to contain 6% sucrose. The sample preparations were then placed into small vials in 100 pL aliquots, and stored at -70°C. Isoelectric focusing electrophoresis (IEF) A gel mixture containing 4% (w/v) acrylamide (containing 5.3% cross-linker N’N’-bis-methylene-acrylamide [Bis]), 2% (w/v) Triton X- 100 and 9.2 M urea was mixed for 5 min at 37°C. Following addition of ampholyte (Pharmacia, Piscataway, NJ) mixture containing 20%, pH 3-10 and 80%, pH 4-6.5 (Wei et al., 1990) to a final concentration of 2% (v/v), the gel mixture was degassedfor 3 min. After adding fresh ammonium persulfate (0.02%, v/v) and N,N,N’,N’-tetramethylethylene- diamine (TEMED, 0.14%, v/v), the mixture was poured into 16 X 20 cm slab gel plates (0.75 mm thick) assembledwith a comb. The gel was then allowed to polymerize for 2 hr. Following removal of the comb from the gel, lysis buffer containing 9.5 M urea, 2% Triton X-100 and 2% (v/v) ampholyte (PH 4-6.5) was overlaid on the gel for 1 hr. The gel was prefocused at 200 V for 15 min, then increasedto 300 V for 30 min and 400 V for a final 30 min using 0.01 M phosphoric acid as the anode solution and 0.02 M sodium hydroxide as the cathode solution. After prefocusing, protein samples (100 pg/well) were applied to the gel and overlaid with an aqueous solution containing 2% Triton X-100 and 2% ampholyte (pH 4-6.5). The gel plate was then reassembled in the electrophoresis unit; and after fresh cathode solution was added to the chamber, proteins were focused at 24°C for 17 hr at 400 V with circu- lating tap water. After the IEF run, gels were fixed in a fixative (4% sulfosalicylic acid and 12.5% trichloroacetic acid) for 6 hr, stained with Volume 60, No. 2, 1995-JOURNAL OF FOOD SCIENCE-279