buffer A and collected in 4 ml fractions. The eluate was tested for − SH groups with Ellman’s
reagent. − SH positive fractions were pooled and brought to 80 saturation with the addition of
solid ammonium sulfate. The suspension was cen- trifuged and dialyzed against buffer A. The di-
alysate was concentrated by lyophilization and chromatographed on a Sephadex G-50 column
2 × 52 cm equilibrated in buffer B 10 mM am- monium acetate pH 7.0. Four millilitre fractions
were collected and examined for the presence of −
SH groups and Cu. Cu content in the fractions was estimated by atomic absorption spectrophoto-
metry. − SH positive and Cu-containing fractions were pooled and lyophilized and were referred to
as the Cu-binding material. For comparison of treated and untreated extracts, untreated cells were
homogenized and the supernatant lyophilized. The product was redissolved in buffer B for chro-
matography on Sephadex G-50 as described above.
2
.
12
. Statistical analysis The results were subjected to statistical analysis
by Students’ t-test and the significance levels were determined at both P B 0.05 and P B 0.01.
3. Results
Growth was found to be adversely affected by copper treatment. There was an extension of lag
phase and decrease of 15, 30 and 50 in growth rate, respectively in the presence of 50, 100 and
200 mM copper Fig. 1. Copper content in the cells increased with time Table 1. Total thiol
content in 200, 400 mM and 1 mM copper-treated cells remained almost constant whereas non-
protein thiol content decreased Fig. 2.
GSH content in the cells after 24, 48 and 72 h of copper treatment decreased progressively in all the
concentrations tested. The decrease in GSH con- tent was drastic in the first 24 h of copper treat-
ment and gradual after 24 h Table 2. This may be due to enhanced utilization of GSH and de-
creased rate of synthesis.
Levels of H
2
O
2
in different stages of treatment were estimated and were found to decrease after
an initial period of increase Table 3. H
2
O
2
level increased in 500 mM and 1 mM copper-treated
Fig. 1. Growth curves of Cu-exposed and unexposed cells of S. bijugatus. Growth was expressed as the increase in Chl a
content with time. There was a 52 decrease in Chl a content in 100 and 200 mM Cu-treated cells after 33 and 37 days of
treatment.
cells after 6, 12 and 24 h of treatment and there- after decreased steadily. H
2
O
2
levels also decreased when treated with exogenous antioxidants like
mannitol MNL, sodium benzoate SB, butylated hydroxytoluene BHT and glutathione GSH
Table 3.
The observed decrease in the level of endoge- nous GSH was taken as a marker of metal-binding
peptide synthesis. Copper-binding peptides were isolated from cells treated with 200 mM CuSO
4
for 3 days. Identity was checked with the presence of
sulfhydryl groups and copper Figs. 3 and 4. Bound copper content was estimated by atomic
absorption spectrophotometry.
The activity of g-GCS, which has an important role in GSH synthesis, showed only a moderate
Table 1 Copper content in cells mggm dry wt after treatment with
different concentrations of copper for 0, 24, 48 and 72 h.
Duration h Concentration
48 24
72 Copper treatment
38.4 28.16
33.28 39.68
Control 39.67
100 mM 92.16
72.18 58.88
200 mM 84.48
147.9 180.8
65.28
Fig. 2. Total thiol TT and non-protein thiol NPT content in Cu-treated cells in mmolesmg chlorophyll. The values are
means 9 SD of 3 experiments with replicated measurements.
synthesis of GSH from constituent aminoacids was inhibited to 50 by GSH concentrations of 30
m M [17].
The activity of GSSG-R showed a significant decrease of 44, 51 and 69, respectively in cells
treated with 50, 100 and 200 mM CuSO
4
Fig. 5. The rate of decrease in GSSG-R activity was more
in 50 mM treatment than in the other treatments. This reflects the decrease in GSH content, which
may be due to its oxidation to GSSG by reaction with Cu ions.
The activity of GSH-PX increased significantly by 26, 68 and 123, respectively in 50, 100 and
200 mM CuSO
4
treatments Fig. 5. GSH-PX has only recently been reported in plants, especially in
algae. Due care was taken not to expose GSH to air and GSSG-reductase was included in the reac-
tion mixture, hence it can be safely assumed that the observed peroxidation of GSH was due to the
activity of GSH-PX.
The activity of GST showed an increase of 17, 35 and 78, respectively in 50, 100 and 200 mM
CuSO
4
treatments Fig. 5. During Cu stress pro- tective mechanisms of the cell are employed, lead-
ing to activation of protective enzymes like GST.
4. Discussion
