2.1. Plant materials
Eight day old maize seedlings (var BARI Hybrid Maize 7) were used to purify the most active GST.
2.2. Extraction of soluble protein
Maize seedlings (except green part) were extracted by homogenizing in an equal volume of 25 mM Tris-HCl buffer (pH 8.5), which contained 1 mM ethylenediaminetetraacetic acid (EDTA) and 1% (w/v) ascorbate, with mortar-pestle. The homogenate was squeezed through two layers of nylon cloth and centrifuged at 11,500×g for 10 minutes, and supernatant was used as soluble protein solution for enzyme purification.
2.3. DEAE-cellulose chromatography
Proteins were precipitated by ammonium sulfate at 65% saturation from the supernatant and centrifuged at 11,500×g for 10 minutes. The proteins were dialyzed against 10 mM Tris-HCl buffer (pH 8) containing 0.01% (w/v) β-mercaptoethanol and 1 mM EDTA (buffer A) overnight to completely remove low molecular inhibitors. The dialyzate (crude enzyme solution) was applied to a column (1.8 cm i.d. × 20 cm) of DEAE-cellulose (DE-52, Whatman, UK) that had been equilibrated with buffer A and eluted with a linear gradient of 0 to 0.2 M KCl in 600 ml of buffer A. The high active GSTa fractions eluted at around 91.67 mM KCl was collected for further purification.
2.4. Hydroxyapatite chromatography
The pooled sample of GSTa, separated by DEAE-cellulose column chromatography, was
applied on a hydroxylapatite column (1.4 cm i.d. × 6 cm) that had been equilibrated with buffer A. The column was eluted with a 300 ml linear gradient of potassium phosphate buffer (0-20 mM; pH 7.0) in buffer A. The high active fraction (10 ml) was collected and further purified by affinity chromatography.
2.5. Affinity chromatography
The collected sample was applied to a column (0.76 cm i.d. × 4.0 cm) of Shexylglutathione-agarose (Sigma, St. Louis, MO) that had been equilibrated with 10 mM Tris-HCl buffer (pH 8.0) containing 0.01% (v/v) β-mercaptoethanol (buffer B). The column was washed with buffer B containing 0.2 M KCl and eluted with buffer B containing 1.2 mM S-hexylglutathione. The high active protein fractions eluted with S-hexylglutathione were combined and dialyzed against buffer B and the dialyzate was preserved as the purified GSTa.
2.6. SDS-PAGE and Staining
SDS-PAGE was done in 12.5% (w/v) gel containing 0.1% (w/v) SDS by the method of Laemmli (1970). The gel was stained with silver and coomassie brilliant blue (CBB).
2.7. Enzyme assay and protein quantification
GST activity was determined spectrophotometrically (Shimadzu UV-1800) by the method of Rohman et al., 2009. The reaction mixture contained 100 mM potassium phosphate buffer (pH 6.5), 1.5 mM reduced glutathione (GSH), 1 mM 1-chloro-2,4-dinitrobenzene (CDNB), and enzyme solutions in a final volume of 0.7 ml. The enzyme reaction was initiated by the addition of CDNB, and absorbance (A340) was monitored at 25°C for 1 min. Protein was estimated following the method of Bradford (1976).
2.8. Measurement of molecular weight
The Molecular weight was measure by gel documentation system (Alpha-Inotech)
2.9. Data analysis
Data generated from this study were analyzed by MSTAT software where need. The graphs were prepared in MS Excel, 2007.