Use of Protein Electrophoresis to detect Allozyme variation:
Hemoglobin A versus S

        Substitution mutations that result in the replacement of one amino acid by another with a different electrical charge can lead to slight changes in the overall charges of the protein. These allelic variants (in DNA) give rise to protein variants called allozymes that differ slightly in electrical charge. Protein electrophoresis [left] is used to detect allozyme variation. Tissue extracts are introduced into a solid support medium (a gel) in the sample wells at the Origin on the right-hand side of the gel. An electrical field is applied: many proteins have a net negative charge and will migrate from the Origin at the cathode ("black," "negative") end towards the anode ("red", "positive") end of the field. The positions of the protein products are detected either directly by staining, or by coupled enzymatic reactions.

    In the case of Sickle Cell hemoglobin [right], replacement of a negatively-charged glu in the standard HbA beta-globin by a neutral val in HbS results in a protein with a slightly reduced negative charge. In homozygous individuals, the HbA tetramer electrophoreses as a single "fast" band, and the HbS tetramer as a single "slow" band. Hemoglobin from a heterozygous individual (with both alleles) comprises both forms of the tetramer, and therefore runs as two bands.
   The gel would therefore be "scored" (from top to bottom) as FS, SS, and FF, indicating the presence of S & A, S, and A hemoglobin, respectively. [Note that an SS individual has sickle-cell anemia, whereas an AS heterozygote is said to show sickle-cell trait].

    Homework: Critique the following statement: "Electrophoresis of Hemoglobin shows two alleles, F and S, for the Hb gene."

All text material © 2009 by Steven M. Carr