Páginas: 5 (1059 palavras) Publicado: 10 de dezembro de 2012
Column chromatography
The cardiolipin
phospholipids were dissolved in 30 ml of eluent (2-propanol-
cyclohexane-water 50:43:7 (v/v/v)) and isocratically
eluted over a 135 x 3 cm column, packed with Polygosil
60-63100 (Marchery-Nagel and Co.), with a flow rate of
10 ml/min. The column was packed and equilibrated in
the same eluent system. The eluate was collected in fractions
of about 200ml; the content of lipid was determined
by HPTLC. Cardiolipin was the first component in the
eluate. Fractions 5-10, containing cardiolipin and some
contaminants with a high Rf value, were collected. Following
fractions, containing other acidic phospholipids, were
discarded. The yield was about 2.5 g.
About 300 mg of the cardiolipin was
dissolved in 3 ml of eluent(2-propanol-cyclohexanewater
45:50:5 (v/v/v)) and eluted over a 250 X 22 mm
HPLC column, packed with Lichrosorb Si 60-5 (Merck),
with a flow rate of about 17 ml/min. The eluate was detected
with a Melz differential refractometer (LCD 201).
The fractions that contain pure cardiolipin (elution time:
10-20 min) were pooled and evaporated to dryness. After
each elution the column material was rinsed with 500 ml
ofmethanol and re-equilibrated with the eluent until the
refractive index of the eluate was constant. The total yield
was 1.5-2.1 g.
Conversion of cardiolipin into the sodium salt form
During the chromatographic steps, a small part of the
cardiolipin was converted from the calcium salt form into
other undefined salt forms (see Table 1). That is why it
was considered necessary that theHPLC-purified cardiolipin
was first converted quantitatively into the calcium
salt, in order to enable a more efficient and quantitative
conversion into the sodium salt.
The HPLC-purified cardiolipin (2 g) was dissolved in
100 ml of chloroform, whereafter 200 ml of methanol and
100 ml of 0.1 M CaClZ were added. The mixture was
shaken for 5 min and phase separation was induced by
adding 50 ml ofchloroform and 50 ml of 0.1 M CaClZ.
The chloroform phase (150 ml) was separated, 300 ml of
methanol and 150 ml of a 0.1 M EDTA, 0.1 M NaCl, and
0.05 M Tris-HC1 (pH 8.2) buffer solution were added,
and the mixture was shaken for 5 min. Then 100 ml of
chloroform and 100 ml of buffer solution were added and
the chloroform phase was separated, washed twice with 10
mM NaCl, and subsequentlyevaporated to dryness. The
sodium cardiolipin was dissolved in benzene and kept
under argon at -8OOC in well-sealed glass bottles.

In former isolation procedures (I), the total lipid fraction
of beef heart was obtained by extraction with chloroform-
methanol 2:l in a two-phase system without addition
of an aqueous buffer. An advantage of a simple one-phase
Bligh andDyer extraction, as we are using, is the optimal
contact between the organic solute molecules and the
amphipathic molecules in the tissue, because they are all
in the same solvent phase (9). In addition, the pH of the
system and the salt form of the acidic phospholipids (i.e.,
cardiolipin) can be more easily controlledb y varying the
composition of the buffer solution.
The yield of theextraction is about 20 g of dry lipid
material per kg of moist tissue. The extract contained
sphingomyelin, phosphatidylcholine, phosphatidylserine,
phosphatidylethanolamine, cardiolipin, cholesterol, cholesteryl
ester, free fatty acids, and pigments (Fig. 1). Most
of the latter four components and some of the phosphatidylcholine
were removed in the acetone precipitation
(Fig. 1). Precipitationof the Caz* salts of the acidic phospholipids
in 4% aqueous methanol resultedin the r e m d
of some of the phosphatidylethanolamine and most of
the phosphatidylcholine (Fig. 1). Lower or higher water
content of the solvent in this step caused, respectively,
incomplete precipitation of the cardiolipin or an inferior
purification. The total yield of the phospholipid fraction
after both...
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