system calibrated
with phenylthiohydantoin
PTH-amino acid standards prior to each se- quencing run.
2
.
5
. Electrospray ionization mass spectrometry Prior to electrospray ionization mass spectrome-
try ESMS, protein fractions from RP-HPLC were concentrated under vacuum in a SpeedVac
concentrator and reconstituted with milli-Q H
2
O. ESMS was carried out using 1 – 100 pmol protein
in 2 – 4 ml of 50 CH
3
CN containing 0.1 formic acid. Samples were infused at a flow rate of 0.2
mlmin into a Perkin-Elmer Sciex API-300 triple quadrupole mass spectrometer fitted with a micro-
ionspray ion source and calibrated to an accuracy equivalent to
9 0.01 using singly charged
polypropylene glycol ions. Mass spectra, typi- cally 30 – 100 scans, were recorded in the first
quadropole Q1 scan mode over the mass-to- charge range mz 200 – 3000 Da per unit charge,
using a constant peak width at half peak height of 0.6 Da per unit charge. Mass-to-charge ratio
data were processed by signal-averaging, manual mass determination and transformation using PE-
Sciex Biomultiview software.
3. Results
3
.
1
. Purification of C. fistula PI A PI was purified from C. fistula seeds by two
very similar procedures, both successively involv- ing methanol extraction of seeds to yield a crude
cell wall fraction, extraction of the PI from the cell wall fraction at high ionic strength, elution from
carboxymethylcellulose CM52 at high ionic strength, gel filtration on an Ultrogel AcA 44
column and RP-HPLC on a C8 column.
Stepwise gradient elution from CM52 yielded one peak of trypsin inhibiting activity eluting be-
tween 0.1 and 0.7 M NaCl in 10 mM glycine pH 9.5. Subsequent gel filtration yielded two peaks of
protein, one eluting at the void volume and the second corresponding to low molecular weight
material. Trypsin inhibitory activity is associated with both peaks Fig. 1.
RP-HPLC of the low molecular weight material from gel filtration in procedure 1 resolved one
major peak of material eluting at 33 – 34 CH
3
CN in 0.1 TFA Fig. 2A. SDS PAGE of this mate-
rial subsequently shown to be a defensin revealed only one major entity M
r
6000 Da data not shown. The high molecular weight material from
gel filtration was not further analyzed. As outlined in Section 2, a second abbreviated
procedure was used to isolate the C. fistula de- fensin. This second procedure was the same as the
first but eliminated overnight imbibing in H
2
O, extensive washing of the crude cell wall fraction in
H
2
O and a second gradient elution from CM52. Further, in this second procedure the cell wall
fraction was extracted in 2 M NaCl – 10 mM glycine at pH 9.5. This second procedure again
yielded a protease inhibitory defensin peak on RP-HPLC peak 4, Fig. 2B, but this final step
also resolved three further less hydrophobic com- ponents peaks 1 – 3, Fig. 2B to be described
further below.
Peaks 1 and 2 are not protease inhibitory at 5 and 3.8 mM, respectively, but peak 3 inhibits
trypsin with an IC
50
concentration for 50 inhibi- tion value of 0.7 mM. The C. fistula 5459 Da
defensin peak 4, Fig. 2B inhibits bovine TPCK- treated trypsin with an IC
50
of 2 mM. This defensin at 4.4 mM does not inhibit chymotrypsin.
3
.
2
. Analysis of C. fistula basic proteins by mass spectrometry
C. fistula seed defensin isolated by procedure 1 Fig. 2A was analyzed by ESMS in the conditions
described in Section 2. The average molecular mass of the defensin in the fully oxidized form no
reductants having been added during the isolation procedure is 5458.6 9 0.8 Da Fig. 3A. The
5458.6 Da component was effectively the only major entity present Fig. 3A. Similar ESMS
analysis of the protease inhibitory peak 4 from RP-HPLC of material from procedure 2 Fig. 2B
revealed one major component with an average molecular mass of 5458.8 9 0.8 Da, i.e. the same
mass within experimental error as the protein iso- lated by procedure 1.
Analysis of peaks 1 – 3 from the RP-HPLC step of procedure 2 Fig. 2B by ESMS revealed major
proteins with
average molecular
masses of
9377.9 9 1.2, 5143.6 9 0.8 Fig. 3B and 7117.1 9 1.0 Da, respectively Table 1. Complete Edman
sequencing of the major protein peak from proce- dure 1 Fig. 2A peak 4 from procedure 2 Fig.
2B identified this entity as a defensin Fig. 4 and N-terminal sequencing of peaks 1 and 2 from the
RP-HPLC step of procedure 2 Fig. 2B identified these components as LTP and defensin homo-
logues, respectively Figs. 4 – 6. The minor 9177 and 9539 Da components in peak 1 can be related
to the major 9378 Da component by differential processing as shown in Table 1. The minor 4928,
5015 and 5305 Da components in peak 2 can be similarly related to the major 5144 Da component
Table 1.
3
.
3
. Edman sequencing of
5459
and
5144
Da C. fistula defensins
N-terminal sequencing of the 5459 Da defensin by Edman degradation yielded a sequence of 50
amino acids Fig. 4. The amino acid residues in positions 3, 14, 23, 27, 38, 44, 46 and 50 are
inferred to be cysteines in these positions for the following reasons. The PTH derivative of cysteine
deriving from sequential Edman degradation is unstable [26] and hence no PTH-cysteine signals
are expected. However the PTH-cysteine-derived PTH-dehydroalanine was detected in positions 3,
14, 23 and 27, indicating cysteines in these posi- tions. The cysteines in plant seed defensins g-
thionins are highly conserved in position Fig. 5 and the cycle positions of major signal absences
positions 3, 14, 23 and possible signal carry-over from the preceding residue positions 27, 38, 44, 46
and 50 Fig. 4 exactly correspond with the eight cysteine locations found in other seed defensins
Fig. 5. The calculated mass of the fully reduced polypeptide from the deduced sequence Fig. 4 is
5467.37 Da. Given eight cysteines involved in four disulphide linkages, the calculated mass of the
fully oxidized protein is 5467.37 − 8.06 Da = 5459.31 Da, which agrees within experimental er-
ror with the observed average molecular mass of 5458.6 9 0.8 Da for the fully oxidized defensin
Fig. 3A; Table 1.
The deduced sequence of the 5459 Da defensin as aligned in Fig. 5 has 52 identity and 62
similarity with the corresponding sequence of a defensin from Vigna unguiculata sequence 3, Fig.
Fig. 3. Electrospray ionization mass spectrometry ESMS of the purified defensins. Mass spectra mass transforms of: A protease inhibitory 5459 Da defensin from procedure 1 peak 1, Fig. 2A; B protease non-inhibitory 5144 Da defensin from
procedure 2 peak 2, Fig. 2B.
Table 1 Electrospray ionization mass spectrometry of purified Cassia
fistula basic proteins
a
Mass Da Comment
Component [CH
3
CN] Procedure
1
Major peak 33–34
5458.8 9 0.8 Defensin
Procedure
2
9377.9 9 1.2 28–29
X+ILS Peak 1
major 9538.8
X+ILSY minor
9177.0 X = LTP
minor 5143.6 9 0.8
29–30 Peak 2
Z+SEKQ major
5305.4 Z
minor +
SEKQY 5015.0
Z+S minor
4927.6 Z
= defensin minor
7117.1 9 1.0 31
Peak 3 major
9378.2 minor
9537.2 minor
9811.2 minor
5458.8 9 0.8
32–33 Defensin
Peak 4 major
a
Possible interpretations of the mass differences between the LTP homologues and defensins in peaks 1 and 2, respec-
tively, are given based on inclusion of the following particular amino acids average molecular mass Da contributions in
parenthesis: I or L 113.16, E 129.12, K 128.17, Q 128.13, S 87.08 and Y 163.18.
residues than the legume defensins an exception being V. faba fabatin sequence 6, Fig. 5 in the
site corresponding to a position between C. fistula residues 42 and 43 Fig. 5.
Edman sequencing of the 5144 Da peak 2 mate- rial eluting from the C8 column at 29 – 30
CH
3
CN in 0.1 TFA Fig. 2B, Table 1 yielded a sequence showing evident homology with the 5459
defensin Fig. 4 and with other plant defensins Fig. 5. The absence of a major PTH-amino acid
signal in positions 3, 14, 23 and 27 is consistent with cysteines in positions 3, 14, 23 and 27, as is
the presence of a PTH-dehydroalanine in these cycles. The cysteines in positions corresponding to
positions 38 and 43 in the 5144 Da protein are highly conserved in the plant defensins Fig. 5
and accordingly the PTH-threonine and PTH-ala- nine in cycles 38 and 43 may be due to carry-over
from the threonine and alanine in positions 37 and 42, respectively Fig. 4. No Edman sequencing
was carried out for cycle 50 Fig. 4 but a C-termi- nal cysteine is highly conserved in plant defensins
Fig. 5. All of the inferred cysteines align with the corresponding highly conserved cysteines found in
other defensins Figs. 4 and 5 and the mass of the deduced sequence is the same within experimental
error as the observed mass. Thus the calculated average molecular mass for the fully reduced de-
duced sequence Fig. 4 is 5152.13 Da. Subtracting 8.06 Da for the formation of four disulphides
yields a calculated oxidized mass of 5144.07 Da as compared to the observed mass of 5143.6 9 0.8
Da.
The 5144 Da defensin exhibits 44 identity and 50 similarity with the 5459 Da defensin se-
quences 1 and 2, Fig. 5. However while the 5459 defensin is a trypsin inhibitor IC
50
2 mM, the 5144 Da defensin does not inhibit trypsin at 3.8
mM. The 5144 Da is similar to the 5459 Da C. fistula defensin and other legume defensins in not
having the additional residues present in Cruci- ferae defensins in a position between residues 41
and 43 Fig. 5. Further, other legume and non- legume defensins variously lack residues present in
both C. fistula defensins in positions 16 – 22 Fig. 5.
3
.
4
. Edman sequencing-based identification of a C. fistula LTP
Edman sequencing of the 9378 Da peak 1 mate- rial yielded a 20 residue N-terminal amino acid
5 and 52 identity and 64 similarity to the corresponding sequence of the Pisum sati6um
pI230 defensin sequence 4, Fig. 5. However the C. fistula 5459 Da defensin has much less homol-
ogy to some other legume defensins, namely the Pisum sati6um pI39 sequence 5, Fig. 5 and Vicia
faba fabatin sequence 6, Fig. 5, and to a variety of other plant defensins Fig. 5. It should be
noted that the pI230 and pI39 sequences Fig. 5 derive from translation of cDNAs [27,28]. C.
fistula defensin has extra residues in positions 20 – 23 compared to most of the other plant defensins.
Conversely, in general the non-legume plant de- fensins an exception being Sorghum bicolor de-
fensin SIa1 sequence 19, Fig. 5 have more
sequence sequence 1, Fig. 6 that is clearly related to the N-terminal sequences of plant LTPs Fig.
6. Thus the C. fistula 9378 Da protein N-terminal amino acid sequence has 60 – 65 identity and
70 – 75 similarity with the corresponding se- quences of LTPs from Daucus carota, Nicotiana
tabacum, Spinacea oleracea and Petunia hybrida LTP Pet 2 Fig. 6, sequences 3, 4, 5 and 18,
respectively. In contrast, there is a much less homology with LTPs from Triticum aesti6um and
LTPs Pet 1, Pet 3 and Pet 4 from Petunia hybrida see sequences 13 – 20, Fig. 6. The 9378 Da
protein does not inhibit trypsin, in keeping with the observations that, while plant LTPs exhibit
some sequence homology to PIs [46], there are no published reports of LTPs actually shown to in-
hibit proteases.
3
.
5
. Edman sequencing of the peak
3
protease inhibitory fraction
Peak 3 from RP-HPLC of C. fistula seed basic cell wall proteins Fig. 2B contains trypsin in-
hibitory activity IC
50
0.7 mM. ESMS established that the major component is a 7117 Da entity but
there are also minor components present of about 9378, 9537 and 9811 Da Table 1 that have
masses similar to the masses of LTP homologues resolved in peak 1 Table 1. Edman sequencing of
the peak 3 material yielded a major 20 amino acid N-terminal
sequence, namely
VN – SPVALS- PLLGAITYSSLSPK noting that – in cy-
cle 3 indicates that no significant PTH-amino acid signal was observed. Minor sequence components
were also detected. No homology of the major amino acid sequence with sequences of other
proteins was detected from database analysis.
4. Discussion
