Use of protein electrophoresis to detect allozyme variation

    Substitution mutations that result in the replacement of one amino acid by another with a different electrical charge result in slight modification of the net charge of the protein. These protein variants are called allozymes because they are encoded by different alleles at an enzyme gene locus. [Allozymes should not be confused with isozymes, which are different forms of the same enzyme encoded at different gene loci].

    Allozyme variation is detected by means of protein electrophoresis.  Tissue extracts are introduced into a solid support medium (a gel, typically starch or cellulose acetate) at a cut or slot near one end, called the origin. An electrical field is applied with the negative (cathodal) end at the origin, and the positive (anodal) end opposite. Because many proteins have a net negative charge, they migrate away from the cathodal origin towards the anode at the other end of the field. [Positively charged protein move towards the cathode]. The rate of migration depends on the ratio of charge to mass of the protein. After several hours, the positions of the allozymes may be detected either directly by staining, or by a coupled enzymatic reaction that links the enzyme substrate to a coloured dye. The result is an electropherogram.

    In the examples below, the cathodal end is at the top and the anodal end at the bottom. In the first example, an analysis of the monomeric (single-subunit) enzyme Alcohol Dehydrogenase (ADH), three distinct bands are seen in lanes 1, 2, & 3 with slow, medium, or fast mobilities (measured from the cathodal end).. These phenotypic enzyme patterns indicate that the individuals have homozygous genotypes for one of three alleles, which we call s, m, or f, respectively. These genotypes may be designated ADHss, ADHmm, and ADHff, respectively. The next three individuals show the patterns expected from the three possible heterozygous genotypes, each of which has two bands: ADHsm, ADHsf, and ADHmf, in lanes 4, 5, & 6, respectively.

monomeric allozyme

      In the second example, analysis of the dimeric (two-subunit) allozyme Transcendentalase (TRN), three bands are again seen in lanes 1, 2, & 3  with slow, medium, or fast mobilities, corresponding to homozygotes of the alleles s, m, or f : TRNss, TRNmm, and TRNff, respectively. As before, the two alleles in each of the heterozygous genotypes encode alternative protein subunits, but in forming the dimeric protein these subunits combine in the ratio 1 : 2 :1 . The heterozygous TRNsm, TRNsf, and TRNmf  therefore produce three-banded phenotypes, recognizable by the mobilities of the products of the homozygotes, with an intermediate band with twice the protein product in the heterozygote.

monomeric allozyme

    IMPORTANT: Be sure to distinguish between the observed allozyme phenotypes on the gel, and the inferred allele genotypes in the DNA.

All text material 2012 by Steven M. Carr