O in bread wheat Triticum


Glutathione determination



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Ascorbate-glutathione cycle for scavenging H2O2 in bread wheat (Triticum aestivum L.) genotypes during drought stress and following recovery

Glutathione determination: The amount of 
reduced 
glutathione 
was 
determined 
spectrophotometrically at 412 nm using Glutathione 
Assay Kit (Sigma-Aldrich). Leaf samples of 0.1g 
were taken from 3 variants of plants - control
drought-exposed and rehydrated plants, and ground 
in liquid nitrogen. After adding 5% sulfosalicylic 
acid, the mixture was stirred, kept at 2-8°C for 10 
min and centrifuged at 10, 000g. The obtained 
supernatant was used for the reaction. The content 
of the reaction mixture was as follows: 95 mM 
potassium phosphate buffer (pH 7.0), 0.95 mM 
EDTA, 0.038 mg/ml (48µM) NADPH, 0.031 
mg/ml DTNB, 0.115 units/ml glutathione reductase 
and 0.24% 5-sulfosalicylic acid. To construct the 
calibration curve, solutions (0.5 - 20 µg/ml) of GSH 
of various concentrations were prepared in 0.4 M 
Tris-HCl buffer (pH 8.5).
Ascorbic acid determination: In an acidic 
environment, ascorbic acid reduces potassium 
hexacyanoferrite 
(Fe
+3

to 
potassium 
hexacyanoferrate (Fe 
+2
). Samples were taken from 
3 variants of plants and frozen in liquid nitrogen. 
Then 1 ml of citrate buffer (pH 3.69) was added to 
samples of 0.5g, stirred and centrifuged for 5 min at 
12,500g. The reaction mixture was prepared as 
follows: 25 µl K
3
[Fe(CN)
6
] (1%) and 25 µl 2% NaF 
were added to 500 µl or 0.5 ml of the plant extract. 
After keeping for 5 min, 1.9 ml of dH
2
O, 50 µl 
FeCl
3
were added, kept for 5-7 min and optical 
density was measured spectrophotometrically at 
680 nm (Matei et al., 2012). 
Extraction of the enzyme. 0.5g of the leaf 
sample was ground in liquid N
2
. 100 mM Na-
phosphate (pH 7.8) buffer containing 1 mM EDTA, 
2 mM FMSF, 1% PVP and 0.1% Triton X-100 was 
used as a homogenization medium. Then the 
sample was centrifuged at 4
0
C, for 20 min, at 
15,000 g and the obtained supernatant was used for 
the APX and GR assays. 

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