Biology Reference
In-Depth Information
The cyclotides present in the butanolic frac-
tion can be readily subjected to HPLC.
Therefore, BuOH/H
2
O partitions of the
tannin-free 50% EtOH and ACN extracts
were carried out, and the butanolic and
aqueous fractions of each extract were ana-
lysed by HPLC with diode-array detection
(DAD). Upon analysing the fraction elution
pattern using an UV detector with a photo-
diode array, the butanolic fraction was cho-
sen to continue with the isolation of the
cyclotides because of its greater peptidic
content.
Owing to the cyclic nature of the back-
bone and the presence of the cystine knot,
which generates a tension in the molecule,
the hydrophobic amino acids are forced to
be exposed on the surface of the molecule
instead of locating themselves inside the
peptide structure (Craik
et al.
, 2001). This
feature would explain the behaviour of the
cyclotides during their isolation and purifi-
cation, such as the longer retention times
(rt), greater than 20 min, observed in
reversed-phase (RP)-HPLC and the capacity
to solubilize in the organic phase during the
BuOH/H
2
O partition.
contribution of the peptidic links, which
display a strong absortion at wavelengths
<230 nm. Disulfide bonds present a weak
absorbance at ~250 nm. The tyrosine and
tryptophan residues are those that contrib-
ute most to the UV absorption spectrum of a
protein (Wetlaufer, 1962).
The RP-HPLC analysis of the tannin-
free 50% EtOH and ACN extracts and their
butanolic and aqueous fractions allowed
the selection of the butanolic fraction cor-
responding to the 50% EtOH extract because
of its greater peptidic content. Under these
experimental conditions, the rt for these
peptides was greater than 20 min.
The butanolic fraction was subjected to
Superdex
TM
gel filtration chromatography
for peptides. The mass spectrometry (MS)-
matrix-assisted laser desorption/ionization
(MALDI) spectrum of this fraction showed
the presence of a series of compounds of
which the molecular weight corresponded
to the cyclotides (3120-3221 Da).
The isolation of the peptides by semi-
preparative RP-HPLC was then performed.
Briefly, a 21.38 mg/ml butanolic solution
of the peptides was subjected to a semi-
preparative C18 chromatography column.
An isocratic mobile phase of 25% ACN in
0.1% TFA (20 min) was employed followed
by a linear gradient to reach 60% ACN in
0.1% TFA. The fraction eluted with 25%
ACN in TFA 0.1% (90 mg) was kept for fla-
vonoid analysis. Nine fractions were pre-
pared by elution with the linear gradient.
The peptides started to elute after 20 min
with the increase in the percentage of the
organic modifier (ACN) and the concomi-
tant decrease in the polarity of the mobile
phase (Goranssön
et al
., 1999).
Analysis of the UV spectra of the
eluted substances revealed the presence
of a complex mixture of peptides. Of the
nine preparative peptidic fractions iso-
lated by RP-HPLC, the fraction displaying
a peak absorbance was selected. This frac-
tion was subjected to analytical chroma-
tography to assess its purity, employing a
mobile phase containing ACN, TFA and
i
PrOH and obtaining 0.2 mg. The fraction
purified by this procedure was analysed
by MS-MALDI, displaying a single peak
13.3.3
Fractionation and analysis
of the peptides
Isolation and purification of
the peptide hypa A
The elucidation of the primary structure of
a protein comprises the isolation, purifica-
tion and subsequent determination of the
amino-acid sequence of the purified protein
and the sequence of the fragments obtained
by chemical and enzymatic procedures. The
cyclotide-enriched fractions of
H. parvi-
florus
(tannin-free 50% EtOH and ACN
extracts and their corresponding butanolic
and aqueous fractions) were analysed by
HPLC with a UV detector with a photodiode
array. The UV detection was performed at
200-300 nm. The UV light absortion of pro-
teins in the range of 230-300 nm is due to
the tyrosine (l
max
= 274 nm), tryptophane
(l
max
= 280 nm) and phenylalanine (l
max
=
257 nm) aromatic rings, together with the
Search WWH ::
Custom Search