Parvalbumins of the sciaenidae: Purification, characterization, and potential use in the immunoidentification of red drum (Sciaenops ocellata)
Robison, Buena Chambers, III
Doctor of Philosophy
Parvalbumins are low molecular weight, water soluble sarcoplasmic proteins that are found in abundance in the fast twitch muscles of fish and amphibia. They are further characterized by a high affinity for calcium, acidic nature, polymorphism, unusual amino acid composition, unique ultraviolet absorption spectrum and high antigenicity. Parvalbumins are believed to serve in the rapid removal of calcium ions from troponin C during the relaxation phase of fast twitch muscle. Parvalbumins are species-specific for each species of fish in terms of these physico-chemical characteristics. In view of this specificity and of the protein's considerable antigenicity, it was proposed to employ the parvalbumin from red drum (Sciaenops ocellata) as the basis for an immunological field test to identify the red drum species from muscle samples. The ability to identify red drum from only a sample of muscle tissue would allow game law enforcement personnel to screen contraband fish fillets on site. A field test as described would greatly strengthen enforcement efforts of parks and wildlife personnel. The major component parvalbumins were isolated from red drum and spotted seatrout (Cynoscion nebulosus) by gel filtration and ion-exchange chromatography, and antisera were produced in two rabbits against the red drum parvalbumin. This antisera was then examined for cross-reactivity against the parvalbumin from spotted seatrout and against the myogens of the following species of fish: sand seatrout (Cynoscion arenarius), black drum (Pogonias cromis), southern kingfish (Menticirrhus americanus), freshwater drum (Aplodinotus grunniens), vermillion snapper (Rhomboplites aurorubens), sheepshead (Archosargus probatocephalus) and southern flounder (Paralichthys lethostigma). The antisera were examined by double diffusion in gels, by interfacial ring testing, and by enzyme-linked immunosorbent assay (ELISA). In double diffusion in gels, the anti-red drum parvalbumin antiserum reacted specifically with its homologous antigen when the antiserum used was the five-week post primary inoculation antiserum. As the antiserum matured, following secondary and tertiary inoculations with antigen, the specificity lessened and the antiserum reacted in varying degrees with the heterologous antigens. With ELISA, reactivity of the antiserum was demonstrated against all antigens tested, regardless of the stage of the antiserum.