Phytochelatin synthase activity assay

Phytochelatin synthase (PCS) activity was determined according to a modified protocol by Finkemeier et al.⁵². The enzyme was extracted from algal cells in buffer containing 20 mM HEPES-NaOH, pH 7.5, 10 mM β-mercaptoethanol, 100 µM Pb(NO₃)₂, 20% (w/v) glycerol and polyvinylpyrrolidone (100 mg mL⁻¹). The assay contained extract (400 µL), reaction buffer (25 mM GSH, 100 µM Pb(NO3)2, 10% (w/v) glycerol, 250 mM HEPES-NaOH, pH 8.0) and protease inhibitor. The samples were incubated for 90 min at 35 °C and terminated by the addition of 20% (w/v) trichloroacetic acid. Thiol groups were derivatized using mBBr and HPLC analysis was performed as described above. One unit of PCS activity was assumed as the amount of PC₂ (nmol) synthesized per mg of soluble protein per minute at 35 °C. The content of soluble proteins in algal extracts prepared for the determination of PCS activity was measured following the Bradford⁵³ method.