Electrophoresis of acid dyes
By JENNIFER M. BYRNE
(From the Cytological Laboratory, Department of Zoology, University
Museum, Oxford)
Summary
Electrophoresis experiments were performed to find out whether the hydroxyphthalein dyes which have been found to behave as basic rather than as acid dyes in
their staining of phospholipid showed any signs of an amphoteric character.
It was found that it is precisely those acid dyes that stain phospholipids that show
least anionic movement in electrophoresis at neutrality.
Introduction
D U R I N G the course of work on the uptake of dyes by extracted phospholipids
and cerebrosides (Byrne, 1962) it was found that basic dyes were without
exception taken up by phospholipids, while the typical acid dyes were not
taken up. However, the weakly acidic dyes, aurantia, eosin Y, eosin SS, and
erythrosin B were taken up by all the phospholipids, although to a smaller
extent than the basic dyes. These dyes do not colour cerebroside.
A reaction between the dye ions and the amino-groups of the phospholipid
might conceivably occur, but if this is so it is difficult to see why cerebroside
is not also coloured. Can these normally acidic dyes be amphoteric and react
with the phosphoric acid radicle of the phospholipids to give the observed
colouring? Mann (1902) noted that eosin in some respects approaches basic
dyes.
Electrophoresis experiments were carried out with the acid dyes used in
the absorption experiments to find out whether the anomalous acid dyes
showed any signs of an amphoteric character.
Material and methods
The dyes used are listed in fig. 1. Eosin B, fluorescein, and phloxine were
added to the dyes used in the original experiments as three further dyes of
the hydroxy-phthalein (xanthene) group. The ability of these dyes to stain
phospholipid and cerebroside was tested as described before (Byrne, 1962).
Spirit-soluble eosin (ethyl eosin) could not be used as it is only slightly
soluble in water, and a spot of the saturated solution could not be seen on
chromatography paper.
The dyes were made up as 1 % solutions and diluted 1:1 with buffer. The
buffer used was a phosphate (M/ys Na2HPO4/M/7-5 KH2PO4) buffer adjusted to pH 7-1, since the dye uptake experiments had been done in neutral
aqueous solution.
The electrophoresis tank used was a Shandon horizontal paper electrophoresis tank no. PE/1160, taking three paper strips 5 cm wide and 15 cm
long. The paper used was Whatman no. 1 chromatography paper.
[Quart. J. micr. Sci., Vol. 104, pt. 4, pp. 441-3, 1963.]
Byrne—Electrophoresis of acid dyes
442
acid
fuchsin
light
green
methyl
blue
o
o
<
>o
orange G
xylidine
picric
— —=—
O
acid
isamine ( = pyrrol)
blue
<
o
O
azocarmine
phenol
red
o
O
fluorescein
cresol
red
methyl
O
o
red
o
orange
o
aurantia
phloxine
'
o
eosin B
o
eosin Y
ode
erythrosin
chlorazol
trypan
(
black
blue
Ei
<
O I 2 3 4 S 6 7 8 9
10 II 12
FIG. I. The movement of acid dyes during electrophoresis at neutrality.
The dyes were put on as spots about 3 mm in diameter. It was not possible
to run all 19 dyes at once, so one dye (xylidine red) was run every time to
ensure a direct comparison between all the dyes despite slight instrumental
variations on different runs. The instrument was run for 10 h at 40 V with a
current of 1-5 mA (o-i mA per cm width of paper).
Results
Eosin B and phloxine both stain phospholipids strongly, although the
background solution is not exhausted (compare with eosin Y, eosin SS, and
erythrosin B (Byrne 1963)). Fluorescein does not stain phospholipids. None
of the dyes stains cerebroside.
Byrne—Electrophoresis of acid dyes
443
The movement of the dyes during electrophoresis at neutrality can be seen
in fig. 1. All the dyes are more or less anionic. Methyl blue and isamine
( = pyrrol) blue stain cellulose and so leave long trails of dye instead of moving
as spots. Chlorazol black E and trypan blue are highly polymerized dyes and
are consequently virtually immobile despite the electric field. Of the dyes
that are readily diffusible, methyl orange and aurantia move only slightly, while
phloxine, eosin B, eosin Y, and erythrosin hardly leave the base line.
Discussion
These results show that at neutrality it is precisely those acid dyes which
stain phospholipids that show least movement towards the anode in electrophoresis (excluding chlorazol black E and trypan blue, whose lack of movement
is correlated with their degree of polymerization). It is significant that
fluorescein, although of the same group of dyes as the eosins, does not stain
phospholipids and is definitely anionic.
The experiment shows that at neutrality the hydroxy-phthaleins, with the
exception of fluorescein, are either hardly dissociated or carry both positive
and negative charges. This could be resolved by running the dyes at an acidic
pH, but unfortunately a buffer was not found in which the dyes would remain
in solution at a definitely acidic pH. However, Stoward (in preparation)
suggests that the second possibility is correct, and that at neutrality dyes of the
eosin group carry both positive and negative charges.
I wish to express my thanks to Dr. J. R. Baker, F.R.S., and to Dr. P. J.
Stoward for the help and valuable advice given during the course of this work,
and to Professor J. W. S. Pringle, F.R.S., for accommodating me in his
Department. This work was carried out during the tenure of a Medical
Research Council Scholarship.
References
BYRNE, J. M., 1962. Quart. J. micr. Sci., 103, 47.
MANN, G., 1902. Physiological histology. Oxford (Clarendon Press).
STOWARD, P. J., in preparation.