Enzymatic Assays for Aquatic Biomonitoring

For acetylcholinesterase (AChE) measurement, samples from head and tail were used. The head was considered from mouth until operculum and tail were considered from anal pore until caudal fin tip. For the measurement of glutathione-S-transferase (GST) activity, gill and body samples were used. We took body samples, considering from operculum until anal pore. For LDH, only samples from the tail were used. The samples were stored in phosphate buffer 0.1 M, pH 7.2, at −20 °C. Samples were defrosted on ice, triturated using scissors, homogenized using a sonicator, and centrifuged while refrigerated (4 °C) for 20 min at 10,000 g. The resulting post-mitochondrial supernatant (PMS) was isolated and placed in 96-well microplates for enzymatic determinations performed spectrophotometrically (Thermo, Waltham, MA, USA) in quadruplicate.
The Bradford method was used to quantify proteins [30 (link)]. The reactions were performed spectrophotometrically in quadruplicate, with the protocol adapted for microplates [31 (link)].
AChE activity was carried out using acetylthiocholine (ASCh) and propionylthiocoline (PSCh) as substrates. We followed the protocol described by Ellman and coworkers [32 (link)], with modifications (absorbance at 414 nm, every 40 s, for 5 min). AChE was expressed as nanomol of substrate hydrolyzed per minute and per mg of protein (U) after 10 min of absorbance reaction for measurement of enzymatic activity. Lactate Dehydrogenase (LDH) activity was performed using pyruvate as substrate and measuring the reduction of pyruvate and the oxidation of NADH at 340 nm, every 40 s, for 5 min. The determinations of the LDH activity followed the protocol described by Vassault [33 ]. The variations in Glutathione-S-Transferase (GST) activity were carried out according to the method of Habig and coworkers [34 (link)].