Use of Protein
Electrophoresis to detect Allozyme variation:
Hemoglobin A versus S
mutations that result in
replacement of one amino acid by another with a different electrical
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
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
for the Hb gene."