Taxonomy of Finfish (1+2)

6.2.1 Introduction

Each fish species is chemically composed of different proteins at varying levels, so techniques that separate proteins may help to identify different species. Of these techniques, electrophoresis is the most important one. Many biological molecules such as proteins are made up of amino acids with electrically charged side chains. Basic amino acids such as arginine, histidine and lysine are positively charged while the acidic amino acids such as aspartic acid and glutamic acid carry negative charges. Thus, virtually all proteins have a net charge depending on the relative proportions of amino acids, unless they are at their “iso-electric point” (pI), the definite pH at which the net charge of the protein molecule is zero. The basis of electrophoretic separation is that proteins of different net charge and different molecular size will migrate at different rates within an electric field and it is a very useful technique for the separation of cellular proteins and DNA.

The term electrophoresis comes from the Greek, and means, “transport by electricity” and has been known since the end of 19 th century. In 1807, a Russian Physicist, Alexander Reuss observed a novel phenomenon - when electricity was passed through a glass tube containing water and clay, colloidal particles moved towards the positive electrode. The term electrophoresis describes the migration of a charged particle under the influence of an electric field. Charged molecules are having either positive or negative charge. At a given pH, the biological molecules exist in solution as electrically charged particles. Under the influence of an electric field, these charged particles will migrate either to the cathode or to the anode, depending on the nature of their net charge.

The theory of movement of a particle in electrophoresis is as follows: When a potential difference (voltage) is applied across the electrodes, it generates a potential gradient, E , which is the applied voltage ( V ), divided by the distance ( d) , between the electrodes. The force that drives a charged molecule towards an electrode is the product of potential gradient, and the charge of q coulombs on the particle. However, the frictional force that retards the movement of a charged molecule is function of hydro-dynamic size of the molecule, shape of the molecule, the pore size of the medium in which electrophoresis is taking place and the viscosity of the buffer.

The velocity (v ) of charged molecule in an electric field-

v = Eq

      F

where, F = frictional coefficient, which depends upon the mass and shape of the molecule; E = electric field (V/ cm); q = the net charge on molecule; and v = velocity of the molecule.

Most of the large molecules possess both anionic (basic positively charged) and cationic (acidic-negatively charged) groupings as part of their structure and hence are termed as “amphoteric molecules” or “Zwitterions”. The actual charge of protein molecule is the result of the sum of all single charges. Because dissociation of the different acidic and basic groups takes place at different hydrogen ion concentrations of the medium, pH greatly influences the total charge of the molecule. At lower pH, they migrate to the negative pole (cathode) and at higher pH to the positive pole (anode). Ionic strength also affects the migration, low ionic strength permits high rate of migration. The choice of buffer strength may be seen to be crucial, since it determines the amount of electrical power that can be applied to the system. The rate of migration will also depend upon the charge density (the ratio of charge to mass) of the proteins concerned; the higher the ratio of charge to mass, the faster the molecule will migrate.