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Figure 1.
Figure 1 Purification of AaV-SP-I and AaV-SP-II from A. acutus
venom. (a) 1.5 g of crude venom was dissolved in 50 ml buffer
A (20 mM Tris-HCl pH 8.2) and left for 2 h at 277 K. The
insoluble materials were removed by centrifugation (4500g) at
277 K for 20 min. The supernatant was then applied to a
DEAE-Sepharose column (1.6 × 40 cm) pre-equilibrated with
buffer A. The effluent was monitored at 280 nm and adjusted to
a flow rate of 134 ml h-1. After the first peak was washed
out, the column was sequentially eluted with a linear gradient
made by mixing 400 ml of buffer A with an equal volume of
buffer B (20 mM Tris-HCl pH 8.2 containing 0.25 M NaCl) and
0.5 M NaCl solution. The effluent was collected for 3 min per
glass tube and investigated for arginine esterase activity.
Three major peaks with arginine esterase activity were observed;
the first peak with arginine esterase activity was pooled,
ultra-filtered and desalted to a volume of 20 ml. The
absorbance at 280 nm is indicated by a solid line, the salt
gradient by a dashed line, arginine esterase activity by squares
and the collected fractions by a solid bar. (b) The protein
fraction pooled from previous chromatography was loaded onto a
CM-Sepharose column (1.8 × 20 cm) pre-equilibrated with a
solution containing 50 mM sodium acetate buffered at pH 5.0 and
then eluted with same solution at a flow rate of 94 ml h-1.
The first two elution peaks (indicated by a solid bar)
possessing arginine esterase activity were collected,
ultra-filtered and desalted to a volume of 10 ml for further
purification. The other materials bound on the column were
eluted with the solution of 0.4 M NaCl (shown by an arrow). (c)
The protein fraction pooled from the second step was applied to
another DEAE-Sepharose column (1.0 × 20 cm) pre-equilibrated
with buffer A. Using a flow rate of 74 ml h-1, the column was
eluted with a linear gradient made by mixing 100 ml of buffer A
with an equal volume of buffer C (20 mM Tris-HCl pH 8.2
containing 0.10 M NaCl). Two protein peaks were found to
possess arginine esterase activity (indicated by solid bars);
the major (on the right) and minor (on the left) components were
designated AaV-SP-I and AaV-SP-II, respectively. Inset in (c):
SDS-PAGE of AaV-SP-I (lanes 1 and 2) and AaV-SP-II (lanes 4 and
5). Lanes 1 and 5 are under non-reducing conditions. Lanes 2 and
4 are under reducing conditions containing  -mercaptoethanol.
Lane 3: markers for molecular-weight estimation.
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