Use of Protein
Electrophoresis to detect Allozyme
Hemoglobin A versus S
mutations that result in
replacement of one amino acid by another with a different
can lead to slight changes in the overall charges of the
allelic variants (in DNA) give rise to protein variants called allozymes that
slightly in electrical charge. Protein
electrophoresis [left] is used to detect allozyme
extracts are introduced
solid support medium (a gel)
in the sample wells at
Origin on the right-hand side of the gel. An electrical field is
applied: many proteins have a net negative charge and will
the Origin at the cathode ("black"
"negative") end towards
the anode ("red" = "positive") end of the
positions of the protein products are detected either directly
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
as a single "fast" band, and
the HbS tetramer as a
Hemoglobin from a heterozygous
(with both alleles)
comprises both forms
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].
Critique the following
of Hemoglobin shows two alleles, F
for the Hb gene."