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corresponding to the cyclotide hypa A for
which the structure could be determined.
theoretical molecular weight of the deriva-
tive in which six cysteines reacted with
4-VP (3780 Da = 3143 + 6 [H] + 631
[4VP:105.14 × 6]).
It is known that the cyclization of the N
terminus (e.g. due to the presence of a cyclized
glutamine or glutamate) does not allow the
determination of the protein sequence. For
this reason the cyclic PEC derivative was
treated with endoproteinase Glu-C to obtain a
linear peptide. Endoproteinase Glu-C is a ser-
ine protease that cleaves the peptidic links of
the C terminus bearing glutamic acid. This
proteinase has a better specificity in ammo-
nium bicarbonate pH 7.8. Because the hypa
A pepide contains only one glutamic acid (E)
its cyclic PEC derivative was readily digested
with endoproteinase Glu-C, rendering a single
linear product that needed no further treatment
before sequencing.
The linear peptide obtained by this
procedure was isolated by RP-HPLC. The
molecular weight observed for the deriva-
tized peptide (3798 Da) was the same as the
calculated molecular weight (3798 Da =
3780 + 18). The complete sequence of this
derivative was determined automatically
by Edman's degradation. The N terminus
Edman's method is the most frequently used
procedure for the sequencing of proteins
(Matsudaira, 1993). Finally, the sequence
of the hypa A cyclotide was determined
(Broussalis
et al
., 2001).
Determination of the primary
structure of the hypa
A peptide
To assess the primary structure, the total
amino-acid content of the cyclic peptide
was determined after the hydrolysis with
6N HCl at 110°C for 24 h (Penke
et al
.,
1974). The cysteine was determined as
cysteic acid following the methodology
described by Moore (1963). The tyrosine
(Y) was protected from the HCl action by
using phenols as radical scavengers. The
amino acids released during the hydrolysis
were identified and quantified by C18
RP-HPLC employing ninhydrine as detec-
tion reagent.
To analyse the sequence, the peptide
was reduced with DTE in a buffer contain-
ing EDTA and guanidine-HCl. The guani-
dine acts as a denaturing agent but the
disulfide bonds are not cleaved by this
agent. The treatment of the peptide with
DTE reduces the cystines with the subse-
quent cleavage of the disulfide bonds and
transforming them into cysteine residues.
The addition of 4-vinylpiridine (4-VP) to
the peptide solution prevents formation of
new disulfide bonds and leads to the forma-
tion of the S-(b-4-piridylethyl) cysteine
(PEC) derivative, which is more stable dur-
ing the sequencing process and more easily
detected (PEC −l
max
= 254 nm), and has a UV
spectrum that is different from 4-VP (l
max
=
254 nm; Anders, 2002).
Craik
et al.
(1999) have assayed a wide
range of proteases on the cyclotides, finding
that the cystine knot (CCK) is resistant to
enzymatic cleavage. The enzymatic diges-
tion of the cyclotide employing proteases is
therefore only possible after the removal of
the cystine knot by the reduction of the
disulfide bonds. Thus, the proteolytic diges-
tion of the CCK required reduction,
S-alkylation and subsequent cleavage of
the cyclic backbone. The PEC derivative
was desalted and isolated by gel filtration
chromatography. The empirical molecular
weight was 3780 Da, a similar value to the
Verification of the sequence of
the hypa A peptide
The combination of HPLC and MS has
proved to be an efficient method for the
analysis of peptides and proteins. During
the past years, both MS and MS/MS have
become commonly used methods for the
identification and characterization, includ-
ing the determination of the molecular
weight, of these compounds. These spec-
troscopic techniques give information on
both the structure and the sequence of
the peptide. However, some issues must be
borne in mind for this methodology to be
successful: the peptides and their fragments
must have a suitable molecular size and
charge to carry out the analysis, which is
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