Biology Reference
In-Depth Information
12.4.1 Protein Electrophoresis
The term
isozyme
is a general designation for multiple forms of a single
enzyme. Isozymes will catalyze the same reaction, but they may differ in prop-
erties such as the pH or substrate concentration at which they best function.
Isozymes are complex proteins made up of paired polypeptide subunits; their
subunits may be coded for by different loci. For example, protein Z could be
a tetramer made up of two polypeptides, A and B. Five isozymes of protein Z
could exist and be symbolized: AAAA, AAAB, AABB, ABBB, and BBBB. Isozymes
may have different isoelectric points and be separated by gel electrophoresis.
The term
allozyme
refers to variant proteins produced by
allelic
forms of
the
same locus
. Thus, A is now A'. A different mutation of A could produce A”.
Allozymes are a
subset
of isozymes; allozymes may differ by net charge or size
so they can be separated by electrophoresis.
The process of analyzing isozymes or allozymes can be divided: extraction of
proteins, separation, staining, interpretation, and application. Proteins are more
difficult to handle than DNA because they are more susceptible to degradation.
Proteins must be frozen and stored at
−
70°C but, even at those temperatures,
some proteins can degrade within months.
Proteins are separated in an electric field on a gel. In gels with a single pH
the proteins move through the gel at a continuous rate, but in gels with a pH
gradient, they move until they reach their isoelectric point and then stop. The
resultant electrophoretic bands are visualized by appropriate staining (
May
1992, Murphy et al. 1996
). If a general protein-detection system is used, only
those proteins present in large quantities are detected, but more-specific stains
can be used. Specific stains and buffer recipes are available for
>
50 enzymes
(
May 1992
). The banding phenotypes observed on the gels can be interpreted in
terms of genes and their alleles (
Pasteur et al. 1988, May 1992
).
Protein-coding genes are often codominant, with both alleles being expressed
in heterozygous organisms. This makes it possible to relate a particular pheno-
type to a given genotype, if we assume that isozyme data reflect changes in the
encoding DNA sequence. To interpret the banding patterns, the number of sub-
units in the enzyme and the distribution of enzymes in particular cells or tissues
should be known (
May 1992
).
Analyses of isozymes remain a cost-efficient and useful method for decipher-
ing the systematics, population genetics, and evolution of insects (
Table 12.3
).
Protein electrophoresis can be conducted using starch (horizontal or vertical
