Hanna

Chemical composition (moisture, fat, ash, and protein) was measured as described by standard methods (AOAC, 1995). After calibration of the pH meter (Hanna; Methrom), trout samples (10 g) were homogenized by distilled water (100 ml) at a ratio of 1:10, and pH was measured.

The pH determination was performed with a pH potentiometer HANNA^® (Sao Paulo, Brazil), that was previously calibrated with standard buffer pH 4.0 and 7.0 and the results corresponded to the average of three independent determinations.

The Mo^IV/V and Mo^V/VI redox potentials of SorT were determined by an EPR-monitored redox titration carried out in a Belle technology anaerobic box. The protein solution (1.5 mL, 40-90 μM in Tris-HCl, pH 8.0 and 10% glycerol) also contained the following redox mediators at concentrations of ~50 µM: diaminodurol (2,3,5,6-tetramethylphenylene-1,4-diamine, E_m,7 +276 mV), dichlorophenolindophenol (E_m,7 +217 mV), 2,6-dimethylbenzoquinone (E_m,7 +180 mV), phenazine methosulfate (E_m,7 +80 mV), 2,5-dihydroxybenzoquinone (E_m,7 –60 mV) indigo trisulfonate (E_m,7 -90 mV), 2-hydroxy-1,4-naphthoquinone (E_m,7 -152 mV) and anthraquinone 2-sulfonate (E_m,7 -230 mV). The reductant was Ti(III) citrate and the oxidant was NaS₂O₈ (both ~100 mM). After addition of titrant and equilibration (15–30 min), the equilibrium potential was measured with a combination Pt wire/AgAgCl redox electrode attached to a Hanna 8417 meter calibrated against the quinhydrone redox couple (E^o′ (pH 7) = +284 mV vs NHE). A 100 μL aliquot of protein was withdrawn and transferred to an EPR tube (in the anaerobic box) which was then sealed and then carefully frozen in liquid nitrogen (outside the box). Potentials for all experiments were measured with a combination Pt wire-Ag/AgCl electrode attached to a Hanna 8417 meter. The intensity of the Mo^V signal (I) was recorded as a function of measured potential (E), Figure 6.