GST exists as a family of isoenzymes. Six have been identified in the rat, and three in humans (Habig, W.H., Jakoby, W.B., 1981). Although the isoenzymic makeup of the mollusc Anodonta is unknown, at least two (and possibly three) isoenzymes have been identified in Sphaerium (Johnson, I., Fleming, R., Garrood, A. C., Beverley, M., 1992). A generalised pattern is hard to establish, however it is readily apparent that the isoenzymes of GST have differing activities towards a range of substrates. These can provide the basis of a mechanism to differentiate between them (Habig, W.H., Jakoby, W.B., 1981).

George et al. (1989) found that activities towards other substrates in a range of fish were very low (typically less than 10% of the CDNB conjugating activity) (George, S., Buchanan, G., Nimmo, I., Hayes, J., 1989). However Jewell et al. (1989) also found that, in the red swamp crayfish, GST activity towards CDNB is associated with two isoenzymes which can be physically separated from GST activity towards p-nitrophenylacetate and p-nitrophenyltrimethylacetate (Jewell, C.S., Lee, R.F., Winston, G.W., 1989). Since there is good evidence that isoenzymes may be differentially regulated (see earlier literature review), an assay specific toward a different GST isoenzyme may provide a test which overcomes some of the problems encountered with a CDNB based assay. Specifically, interference encountered at 340 nm, lack of inducibility, and fluctuations in control levels.

To this end, a range finding study of 6 potential GST substrates was undertaken, with a view to eliminating those with no discernable activity, and identifying optimum assay conditions for those with demonstrable activity.

The assay was a modification of that given in the introduction. The cytosolic extracts used were those of swan mussels which had been exposed to 3200 ng/l lindane for 7 days and then frozen (Polak, M., 1992). 2 samples of gill tissue, and 2 samples of digestive gland tissue, were chosen at random from those available (G 19, G6, DG 134, DG 114).

The test substances used were;

1-chloro-2,4-dinitrobenzene (CDNB),
p-nitrobenzyl chloride (PNBC),
p-nitrophenyl acetate (PNPA),
ethacrynic acid (EA),
nitropyridine oxide (NPD),
1,2-epoxy-3-(p-nitrophenoxy)propane (ENP).

These substrates were chosen on the basis of their identification in previous work (Johnson et al., 1992) as substrates of GST, and on their availability from commercial suppliers. The concentrations used were based on those used in the previous in work or derived from the literature (Habig et al., 1974). The pH of the buffer and the wavelength at which absorbance was initially measured were also derived from Johnson, et al. 1992 and Habig & Jakoby (1974). Where sources were in disagreement both permutations were assessed. The basic conditions are outlined below:

Intially, an absorption spectrum (blanked against water) was obtained for the test substance in an assay mixture of 1.6 ml buffer, 0.2 ml GSH, 0.1 ml test substance (total volume 1.9 ml). A 0.1 ml aliqout of cytosolic extract (DG 134) was added and the absorbance change (dABS) over 3 minutes at a single wavelength was measured. This mixture was then allowed to stand. If no significant change in absorbance at the selected wavelength was detected, the test was repeated with extract G 19.

An absorbance spectrum was then obtained of the assay mixture which had been allowed to stand. This gave the absorbance of the reaction after around 10 minutes, and was analysed to identify any potentially more suitable wavelengths for further study. If no activity could be found, the remaining 2 extracts were studied with 0.1 ml of the buffer replaced by an additional 0.1 ml of extract. If activity was detected, further permutations were investigated in order to find the optimum assay conditions.

Absorbances of the assays sans extract are given in Figures 3.1-3.6. For three of the test substrates (PNBC, NPD, ENP), no activity at any wavelength was discernable over the time course of the experiment (PNBC, NPD, ENP). The absorbance spectra of the other three substrates (CDNB, PNPA, EA) after around 10 minutes reaction are given in Figures 3.7-3.9. The optimum assay absorbance for PNPA was identified as 330 nm, rather than the 400 nm previously suggested (Johnson et al., 1991). Typical changes in absorbance at the test wavelength over 3 minutes are given in Figures 3.10-3.12.