Study 1
A preliminary investigation
Glutathione
S-Transferase as a Biological Marker of Aquatic Contamination Return to the Index
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A speculative investigation of the assay currently in use was undertaken, with a view to providing a basis for further work. The absorbance spectrum of each of the assay components was obtained, as well as the change in absorbance which occurs as the assay reaction progresses.
The specimen used was a randomly selected gill GST homogenate from a single swan mussel (Anodonta cygnae), which had been exposed to 3200 ng/l of lindane for 7 days in a previous study (Polak, M., 1992). The gill tissue was prepared as described in the introduction, and then stored at -25
°C for 22 months (from July 1991). Assays were conducted as described in the introduction.Firstly, a spectrum was obtained of the absorbance of a blank mixture (no cytosolic extract), with a distilled water blank (Figure 1.1). Then each component of the assay mixture was scanned individually, with the other components replaced by buffer (Figure 1.2 to 1.4).
Scans were then made of the assay after 5 minutes reaction time, zeroed against distilled water (Figure 1.5) or a cytosol free blank (Figure 1.6). The scans seem to show clearly an absorbance peak at around 340 nm, but also apparently show another strong peak at around 390 nm.
To investigate the kinetics of these two absorbances, readings were made at 340 nm over a period of three minutes (Figure 1.7). The activity at 390 nm was found to be very low, so the reading at 390 nm was made over 36 minutes (fig 1.8).
The increase at both wavelengths was measured over 36 minutes in the absence of tissue extract. Both showed a reduced, though still measurable activity (Figure 1.9 and 1.10).
The full reaction was followed over time at all wavelengths by scanning at 90 seconds, 180 seconds, 40 minutes and 19 hours (Figures 1.11-1.14).
A scan was made of the absorbance of an assay mixture sans tissue extract at 23 hours. The result (not shown), closely matched the absorbance spectra of the assay plus tissue extract.
Two absorbance peaks were found for the assay mixture. The double peak has since been identified as an aberration due to a mechanical fault in the spectrophotometer which causes the absorbance recorded to change markedly at 355 nm, and again at 325 nm. It therefore appears that there is a single absorbance maximum at 390 nm. This is unexpected given that Habig et al. (1974) report the dABS maximum as 340 nm, and a previously published absorbance spectrum of the progression of the reaction gives a smooth curve peaking at 338 nm (Dierickx, P.J., 1984). The increase in absorbance found in this study is initially maximal at 340 nm, with a time dependent progression to a peak at 390 nm. The anomolous peak and atypical appearance of the absorbance spectra suggests an additional unknown factor is responsible.