Taxonomy of Finfish (1+2)

6.2.9 Sources and Extraction of Proteins

For electrophoretic separation, proteins must be in solution.  These can be body fluids such as plasma, serum, milt, haemolymph or aqueous extracts of tissue proteins.  Extracts are often made from muscle, liver, eyes, etc in distilled water, 10% sucrose solution or specific extraction buffer.  Crustaceans can be sampled non-lethally by removing a walking leg.  With larvae or other small organisms, it is necessary to use the whole animal.  The pH and ionic concentration of the buffer used, permits differential extraction of proteins.  

After removal from the animal, proteins begin to denature rapidly and so the tissue must be used immediately or stored deep-frozen.  Storage at –196^oC in liquid nitrogen or at –80^ooC in ultra low freezers retains 90% of initial activity indefinitely and at –40^oC most tissues will remain usable for a year or more, but at –18C denaturation is relatively rapid and will produce altered electrophoretic pattern.  

Sarcoplasmic proteins (water-soluble proteins)

The soluble proteins of the sarcoplasm, located within the sarcolemma are referred to as sarcoplasmic proteins.  Among them, some albumins and so called myogens; to which belong most of the glycolytic enzymes are the real water-soluble proteins.  (The other fractions of sarcoplasmic proteins are soluble in low salt concentrations).  The genetic differences between species are more pronounced in this than in other group of proteins, as they are responsible for widely divergent enzymatic transformations in the muscle cell.  Hence, the separation patterns of profiles obtained on electrophoresis or isoelectric focusing (IEF) can be used for the unequivocal identification of the species.

Myofibrillar proteins (salt-soluble proteins)

They are salt soluble proteins present in the myofibrils of the muscle fibre.   Of the different myofibril proteins, myosin and tropomyosin find application in fish species identification by electrophoresis.  Fish myosin, similar to myosin of other vertebrates, is a hexameric protein consisting of two identical heavy chains and four light chains, of which two of them are identical.  Electrophoretic pattern of heavy chains from different species are similar whereas that of the light chains is different for different species.  Hence, an electropherogram of myosin light chain isolated from fish muscle is used for species identification.  Electrophoresis of most of the fish muscle tropomyosin gives a single band whose electrophoretic mobility is different for different species.  Tropomyosin is a heat stable protein that can be extracted from heat-treated fish products, thus useful in identifying the species of fish of the product by studying the SDS-electrophoretic pattern of tropomyosin. 

Eye-lens Proteins

The soluble proteins of the eye-lens have great value in taxonomic studies, because they are synthesized only one cell type present in the eye as a single layer.  Three saline soluble eye lens proteins are distinguishable by electrophoretic and immunological techniques.  There are alpha, beta and gamma crystallines in order of decreasing electrophoretic mobilities, each of which constitutes a family of similar, but no identical proteins.  Protein with alpha-crystallin characteristics have been found in all vertebrate species and regarded as a classical organ-specific protein.  The beta- and gamma- crystallin patterns are species-specific and can be used to resolve taxonomic disputes using ultra-thin IEF technique.

Allozymes

Isozymes are functionally similar and separable forms of enzymes encoded by one or more loci. Isozyme products of different alleles at the same locus are termed as “allozymes”. The most important quality of allozyme data is the codominant nature of inheritance of gene products and thus genetic interpretation (genotype) of the phenotype is facilitated because all products are normally visible and not masked by dominance of one over another. Other advantages include function of most of the proteins are known and, extensive database is available for many fish species. Allozyme electrophoresis has been used in defining genetic markers for stock identification on the basis of differences in allelic frequencies between stocks in many species. Using allozyme markers, it is possible to determine whether a population is a random mating one with equilibrium genotypes frequencies or sample comprises of an assembly of genetically distinct units. Their allele frequencies primarily respond to mutation, gene flow and drift. One of the limitations of enzyme variants as genetic markers is the low level polymorphism observed in some species and populations. The extensive allozymes studies undertaken on f1sh stocks have not only proven valuable for estimating population divergence, but also have focused attention on the underlying evolutionary forces that promote differentiation.