## 3 Results  
### In-Gel Enzyme Protocol Development  
In this work, we adapted in-gel electrophoresis assay protocols to test the activities of antioxidant enzymes (CAT, SOD, and GPX) in tissues of the ectothermic marine bivalve _Mytilus galloprovincialis_ (Lam. ), an ecologically relevant bioindicator of anthropogenic marine pollution and or disturbance (Figure 1). In-gel CAT and GPX detection is based on negative staining with ferric chloride/ferrocyanide. Ferrocyanide (III) competes with these enzymes for a peroxide substrate and forms a stable blue-green precipitate, ferrocyanide (II), and achromatic bands at the migration position of the enzyme [41]. Figure 1: In-gel anti-oxidant assay in _Mytilus_ tissue protein extracts. (**A,B**) Representative native PAGE gels stained for the in-gel activity of CAT in the 20,000 g supernatant (**A**) and pellet (**B**), obtained from digestive gland. Mussel CAT is visible much above the bovine liver commercial standard. (**C**) Representative in-gel assay for SOD. Two bands are always visible. (**D**) Representative in-gel assay for GPX. Mussel GPX is visible just above the bovine erythrocyte GPX commercial standard. Legend: M, commercial marker; B, blank; S, mussel protein extract (digestive gland) showing some interindividual variability. The two enzyme activities can be distinguished by the different reaction mixtures. The improvements over previous protocols [41; 43; 44] mainly concern the run of native polyacrylamide gel electrophoresis, which was standardized to only 90 min at 40 mA, using commercial precast gels and a standard native Tris-glycine buffer. Pre-electrophoresis and/or a discontinuous buffer system are no longer required, resulting in significant optimization of overall process times. Other adjustments include the standardization of substrates. For CAT, the concentration of hydrogen peroxide in the reaction mixture was increased to 0.01%, resulting in satisfactory contrast enhancement and a satisfactory dynamic determination range of at least 1 order of magnitude (Figure 2). This is not trivial because the intensity of the achromatic bands may depend on the amount of ferrycyanide available and not on the activity of the enzyme and/or its concentration in the gel. It was found that the specific activity of CAT was similar in the SN20 fraction and the pellet, but the absolute activity was larger in the supernatant, where more proteins are present. This result is in agreement with [PERSON] et al. [45], who explain the presence of CAT in SN20 by the degradation of peroxisomes during centrifugation. However, it seems to be very convenient to measure the enzyme activity in this fraction, so it was finally decided to use it for measuring the CAT reactions upon silver exposure (see Section 3.2). For the in-gel GPX assay, [PERSON] et al. [43] was used as a reference to establish a mussel protocol. After 12% native PAGE and washing in Tris-HCl pH 7.9, a single reaction solution containing 1 mM GSH and peroxide was used to develop the achromatic bands due to GPX activity. Purified bovine GPX and protein extracts (SN20) from the digestive glands and gills of _Mytilus galloprovinticalis_ (Lam.) were used to standardize our method. Specifically, we tested the reaction in the presence and absence of 1 mM GSH and 10 mM sodium azide--a catalase inhibitor--in conjunction with two different substrates: hydrogen peroxide or cumene hydroperoxide. Cumene hydroperoxide at a concentration of 0.008% was confirmed as the optimal substrate to detect the total and genuine GSH-dependent GPX activity (Figure 3). Indeed, the ferricyanide reaction reveals several achromatic bands (Figure 1, Panel D), most likely representing selenium-dependent GPX, selenium-independent GPX, and an intrinsic GSH-dependent peroxidase activity of GST as suggested by [PERSON] et al. [45]. Once the protocol was established, the linearity of the in-gel reaction was tested using a purified standard (Figure 4). Figure 2: In-gel CAT assay.