Carboxylesterase and Glutathione-S-Transferase Quantification ... 97
Preparation of Enzyme Homogenate
Fifth instar larvae C. medinalis were used for en-
zyme preparation. Larval midguts were excised
with replicated samples and were homogenized
in 500 μl homogenization buffer (50 mM sodium
phosphate buffer, pH 7.4). After centrifugation at
10,000 rpm for 20 min, the clear supernatant was
collected and used as enzyme sources for analy-
sis. All the operations were carried out on ice and
centrifugation was carried out at 4 °C to minimize
losses of enzyme activity. The protein content of
enzyme extract was estimated by Coomassie Bril-
liant Blue G-250 dye binding method using bovine
serum albumin as the standard (Bradford 1976 ).
Carboxylesterase Assay
Carboxylesterase activity was determined fol-
lowing van Asperen ( 1962 ) with necessary modi-
fications and α-naphthyl acetate as a substrate. A
0.3 mM substrate solution of 1-naphthyl acetate
was prepared in acetone. The assay mixture con-
tained 15 μl of enzyme preparation, 0.5 ml of
50 mM sodium phosphate buffer pH 7.4, and
800 μl of 0.3 mM substrate solution. The mixture
was incubated at 30 °C for 30 min. Finally, 200 μl
of 0.1 % tetrazotizedo-dianisidine (Fast blue B)
in 3.5 % Sodium Dodecyl Sulphate (SDS) was
added and incubated for 20 min at room tempera-
ture in the dark. The α-naphthol formation was
measured at 590 nm. Enzyme activity was calcu-
lated from α-naphthol standard curve.
Esterase Isozyme Studies
Native Polyacrylamide Gel Electrophoresis
(PAGE) with 10 % resolving gel was performed
to separate esterase isozymes. Qualitative chang-
es in the esterase-banding pattern were performed
using F 2 generation larvae reared after surviving
the insecticidal bioassay with monocrotophos. On
the native PAGE, 5 μg protein concentrations of
the midgut homogenate per well were loaded and
run at a constant voltage of 90 for 45 min. Gels
were stained briefly for esterase activity with
freshly prepared 0.05 % (w/v) α-napthyl acetate
and 0.1 % (w/v) fast blue B in 50 mM phosphate
buffer pH 7.4. For inhibition studies, gels were
cut into strips and incubated in 10−^4 and 10−^6 M
serine sulphate each and 10−^4 M DDVP indi-
vidually in 50 mM phosphate buffer pH 7.4 for
30 min at 28 °C with occasional shaking. Control
gels were incubated for 30 min in buffer alone.
All the gel strips were stained and incubated for
30 min in α-napthyl acetate substrate diazonium
mixture for confirming the esterase activity.Glutathione-S-Transferase Activity
Estimation
Glutathione-S-transferase assay was performed
using reduced glutathione (50 mM), midgut ho-
mogenate supernatant (10,000 × g) from the F 2
generation larvae reared after surviving the insec-
ticidal bioassay with monocrotophos, chlorodini-
tro benzene (CDNB) 50 mM, sodium phosphate
buffer (pH 6.5, 100 mM), and Ethylenediamine-
tetraacetic acid (EDTA) (1 mM). The assay mix-
ture contained 50 μl of 50 mM CDNB, 150 μl of
reduced glutathione, and 2.77 ml of 100mM, pH
6.5 phosphate buffer containing 1mM of EDTA.
To the above assay 30 μl of enzyme (midgut ho-
mogenate) was added and the contents shaken
gently and incubated for 2–3 min at 25 °C then
the contents were transferred to 4 ml cuvettes and
absorbance was recorded for 6–7 min at 340 nm.
Based on the increase in absorbance over 5 min
the enzyme activity was calculated in μmol/min/
mg/protein.Results and Discussion
Bioassay results for the two selected C. medina-
lis populations, DRR and ICRISAT, Patencheru
revealed LC 50 of 60 ppm for ICRISAT C. medi-
nalis population and showed twofold resistance
ratio over the DRR population which showed
LC 5030 ppm against monocrotophos (Table 1 ).
Qualitative and quantitative changes of
CarE and GST’s for DRR population in
α-napthyl acetate and CDNB revealed a