A novel 8.7 kDa protease inhibitor from chan seeds (Hyptis ... - UAM

84 C. Aguirre et al. / Comparative Biochemistry and Physiology Part B 138 (2004) 81–89
Fig. 1. Zymogram of the crude extract in native PAGE. A:
Crude extract of chan (H. suaveolens) seeds, B: Amaranth
trypsin inhibitor used as positive control.
(1993) using casein as substrate. For protease
activity on X-ray film, we used the method
described by Machhandra and Manavendra (1994).
2.3.2. Protease inhibitor activity determination
Inhibitor activity against trypsin and trypsin-like
enzymes of P. truncatus was assayed by monitoring
the initial rate of hydrolysis of BAEE at 253
nm in 0.15 M Tris–HCl, CaCl2
0.05 M, pH 8.1
after 3 min of incubation (Schwertz and Takenaka,
1955). Inhibitor activity against proteases of T.
castaneum, C. maculatus, A. obtectus and S. zeamais
was assayed by monitoring the hydrolysis of
casein at 280 nm in 0.04 M succinic acid, 0.06 M
NaCl, pH 6.5 after 3 min of incubation (Kakade
et al., 1969). At pH 2.5, it was assayed by
monitoring the hydrolysis of hemoglobin at 280
nm in 0.2 M citric acid, 0.1 M NaCl, after 3 min
of incubation (Lenney, 1975). One unit of proteolytic
activity was defined as an increase in 0.01
absorbance units under the assay conditions
described. Inhibitor activity was defined as the
difference between the proteolytic activity measured
in the absence and in the presence of the
inhibitor. Inhibition Units (IU) were calculated as
follows:
IUymls
Enzyme ODyŽ EnzymeqInhibitor.
OD
0.01=Sample
Ž ml.
Samples of the inhibitor obtained from the
purification procedure were screened for inhibitory
Fig. 2. Gel filtration chromatography after (NH ) SO precip-
4 2 4
itation. Fractions were monitored for absorbance at 220 nm
(line) and trypsin inhibitor activity (diamonds).
activity against trypsin, chymotrypsin, papain and
elastase.
3. Results
3.1. Purification
When subjected to electrophoresis and stained
for inhibitory activity, crude extract of Chan flour
contained a single protein inhibitor (Fig. 1). Proteins
were precipitated and separated by gel filtration
(Fig. 2). The peak containing the inhibitory
activity was collected and resolved by ion
Fig. 3. Ion exchange chromatography of the fraction obtained
after gel filtration. Fractions were monitored for absorbance at
220 nm (line), trypsin inhibitor activity (diamonds) and NaCl
gradient (broken line).