Reagents 
 

• Phosphate buffer solution (PBS), 0.03M pH 6.7 (Sigma Chemical Co., U.S)

• 1.2 mM 1-chloro-2, 4-dinitrobenzene (CDNB) in 2% v/v aqueous Ethanol (Sigma Chemical Co., U.S)

• GST form rat liver and equine liver (Sigma Chemical Co., U.S)

• TBT (Aldrich Chemical Co. U.S) 

GSH Standard Assay

• The reaction mixture contained 2 ml PBS, 0.5 ml GSH (0-50µM final concentration), and 0.4 ml, 2.5 U/ml GST in a 3.5 ml cuvette. The blank contained the same reagents except that GSH was replaced with PBS.  The addition of 0.5 ml CDNB to the cuvettes initiated the reaction.

• The kinetics of the reaction was monitored at 340 nm, the cuvettes were incubated for 3 hours at 37⁰C, and the absorption value recorded upon equilibrium. 

Molluscan GST Assay

• Helix aspersa were dissected to obtain the visceral complex. The homogenized visceral mass was centrifuged at 4^oC for 1 hour at 100,00 x g to obtain the supernatant composed primarily of cytosol. The presence of GST activity in the cytosol was monitored as above using 50 µM GSH assay with 20 µl cytosol in place of the mammalian GST. 

• The intrinsic GST activity in the cytosol was quantified by the method of standard additions. Reaction mixtures containing 100 µl GSH (1 mM), 20 µl cytosol, 150 µl of mammalian GST (0, 0.5,1,2…5 U/ml), 150?l CDNB were made up to 1ml total volume by adding PBS. The blank contained only cytosol, CDNB and PBS.

• The change in absorbance was monitored at 340 nm and 25⁰C. The rate of change in absorbance during the linear phase of the reaction was monitored over 5 minutes.

Enzyme Kinetics for Mammalian and Molluscan GST
 

• The reaction mixture contained 250 µl PBS, 150 µl CDNB, 500 µl GSH (0-500 µM serial dilutions) and 150 µl mammalian GST. The reaction was monitored for 5 minutes at a wavelength of 340 nm at room temperature.

• The procedure outlined above was repeated using 10 µl molluscan cytosol in place of the mammalian GST and additional PBS was added to bring up to a total reaction volume of 1 ml. 

TBT Inhibition of Molluscan GST

The procedure for molluscan enzyme kinetics was repeated as above except the cytosol was initially mixed with 10 µM and 100 µM TBT for approximately 1 hour on ice.