Biology Reference
In-Depth Information
isoelectric points, this method allows calculation of the peroxidase
activity. After electrophoresis, the gel is equilibrated in buffer at
adequate pH and water insoluble peroxidase substrates are applied.
After conversion of the substrate in the presence of H
2
O
2
the col-
ored reaction product precipitates inside the gel. After digitaliza-
tion, intensity of the bands can be estimated using ImageJ
(
http://rsbweb.nih.gov/ij/
)
. Quantifi cation of the colored spots
is possible if non-saturating protein concentrations are used and
both biological and technical replicates have been done in ade-
quate amounts [
18
].
High resolution clear native electrophoresis (hrCNE) is a
native PAGE that separates proteins according to their native
molecular mass [
33
]. The ionic detergent deoxycholate is used to
introduce a negative charge to the solubilized proteins before elec-
trophoresis. Compared to blue native PAGE (BN-PAGE) the
advantage of hrCNE is that it is colorless.
Modifi ed SDS-PAGE (modSDS-PAGE) was used to visualize
plasma membrane-bound peroxidases [
31
]. The method is based
on the protocol of Thomas et al. [
34
]. In contrast to SDS-PAGE,
modSDS-PAGE works with a reduced concentration of SDS and
without reducing agents like dithiothreitol (DTT) or mercapto-
ethanol in the sample buffer. Although modSDS-PAGE is not a
native method
heme
-containing peroxidases can be visualized by
staining with tetramethylbenzidine (TMB) and H
2
O
2
[
31
].
Staining is fi nished within two minutes, because the prosthetic
heme
group is still present but the enzyme is not active any more.
This method allows estimation of molecular masses and abundance
of the isoenzymes [
21
]. ModSDS-PAGE can be used either as a
fi rst dimension or as a second dimension after native gel-based or
gel-free separation methods. Gradient gels may be used to receive
a higher resolution of isoperoxidases.
Originally TMB was used for staining of the peroxidase activity
of
heme
-containing cytochrome P-450 [
34
]. It was shown that
TMB also stains copper-containing proteins [
35
]. Thus TMB will
react with all
heme
- and copper-containing proteins in a sample.
Among these proteins are class I and class III peroxidases. Besides
TMB staining, class III peroxidases can be detected by diamino-
benzidine (DAB) or more specifi cally by phenolic substrates that
precipitate after conversion in aqueous solutions [
36
-
38
]. Guaiacol
(2-methoxyphenol) and
-chloro-naphthol are used for visualiza-
tion of the activity of class III peroxidases. DAB precipitates as
an intense brown reaction product,
α
-chloro-naphthol produces
violet bands and the product of guaiacol is orange [
32
]. For
BN-PAGE DAB and guaiacol are the best appropriate peroxidase
stains due to their complementary contrast to Coomassie Blue
[
39
], whereas TMB and
α
-chloro-naphthol can be used addition-
ally for native IEF-PAGE, hrCNE or modSDS-PAGE [
10
,
31
,
32
,
40
]. After visualization with TMB, peroxidase spots can be picked,
α
