Study 5
pH dependence of the gill GST
Glutathione
S-Transferase as a Biological Marker of Aquatic Contamination Return to the Index
Page | Visitor number:
|
As a result of the previous study, it was decided to proceed with a CDNB based assay as the basis of further investigation. With this in mind, the pH dependence of the assay was investigated in order to ascertain the optimum pH. The pH used previously (pH 6.5) was derived from the work of Habig et al. (1974) with rat liver GST, but groups working with other animals have found different pH values to be optimal.
The samples used were Anodonta gill cytosolic extracts of the upstream group used in study 4. These samples were used because; (1) they had been found to have the highest activity and, (2) gill samples were to be used in subsequent studies since they showed the greatest inducibility. Five samples were chosen at random from this group, disregarding those which had been used in study 4.
Six 0.1 M phosphate buffers were made at a range of pHs; 6.0, 6.5, 7.0, 7.5, 8.0, 8.5. This range of pH was used because previous studies had found that optimal GST activity occurs at a pH of around 7.5 (for example Davies, P.E., 1985).
Assay conditions were as used in study 4. The uncatalysed rate at each pH was measured at regular intervals (a total of five times). The average uncatalysed rate at the appropriate pH was subtracted from each GST assessment. The results are summarised below and shown in Figure 5.1.
| pH | Uncatalysed Activity dABS/min |
Catalysed Activity dABS/min mean +/- S.E.M. |
| 6.0 | 0.008 | 0.195 +/- 0.038 |
| 6.5 | 0.031 | 0.259 +/- 0.048 |
| 7.0 | 0.073 | 0.299 +/- 0.065 |
| 7.5 | 0.207 | 0.275 +/- 0.065 |
| 8.0 | 0.371 | 0.215 +/- 0.053 |
| 8.5 | 0.542 | 0.066 +/- 0.023 |
Peak GST activity with CDNB occurs at pH 7.0. This is somewhat lower than that found in other species. However an assay pH of 6.5 is still recommended since the background activity at this pH is lower. It has previously been observed that a high relative background activity introduces uncertainty into the results. Some evidence for this is provided by the observation that the coefficient of variation is lower at pH 6.5 than any other pH.