M1882

The measurement of antioxidant defenses was determined by total antioxidant capacity and superoxide dismutase (SOD), antioxidant activities of catalase (CAT), and glutathione S-transferase (GST) using previously described protocols [4 (link)]. In brief, total antioxidant capacity was measured as the inverse of the amount of ABTS radical (ABTS^•+) formed when 2,2′-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid (ABTS; A1888, Sigma-Aldrich, Madrid, Spain) is oxidized by hydrogen peroxide (H1009; Sigma Aldrich, Madrid, Spain) and metmyoglobin (M1882, Sigma-Aldrich, Madrid, Spain) [35 (link)]. CAT (CAT, EC 1.11.1.6) activity was measured following the decrease in H₂O₂ for 30 s at 240 nm. SOD (SOD, EC 1.15.1.1) activity was based on the inhibition of the rate of reduction in cytochrome c (C2506, Sigma-Aldrich, Madrid, Spain) by O₂^•−, with a xanthine (X7375, Sigma-Aldrich, Madrid, Spain)/xanthine oxidase (X4500, Sigma-Aldrich, Madrid, Spain) system as a source of O₂^•−. GST (GST, EC 2.5.1.18) catalyzes the reaction of the sulfhydryl groups of the GSH with 1-chloro-2,4-dinitrobenzene (CDNB) (237329, Sigma Aldrich, Madrid, Spain). The result is a conjugate GSH-CDNB, which can be detected by spectrophotometry at 340 nm.

Urine samples were diluted in 0.1% (v/v) formic acid to reach 0.1 μg of protein column (5 pmol) and centrifuged at 13,000 g for 10 min. All analyses were performed on a Synapt G2 mass spectrometer (Waters Corporation), fitted with an API source. Samples were desalted and concentrated on a C4 reverse phase trap (Thermo Scientific) and protein was eluted at a flow rate of 10 μL/min using three repeated 0–100% (v/v) acetronitile (ACN) gradients. Data was collected between 800 and 3500 Th (m/z), processed and transformed to a neutral average mass using MaxEnt 1 (Maximum Entropy Software, Waters Corporation). The instrument was calibrated using a 2 pmol injection of myoglobin from equine heart (Sigma-Aldrich; M1882).

To a PBS solution of myoglobin (Sigma-Aldrich, M1882, 0.5 mg/mL, 6 mL, pH 8.0) was added a DMSO solution of Alexa fluor 647-NHS ester (Thermo Fisher Scientific, A-20006, 50 mM, 6.72 µL). The reaction mixture was incubated at 4 °C for 24 h. The resulting mixture was diluted with PBS (18 mL) and dialyzed by Spectra/Por dialysis membrane (MWCO 8000) against PBS (500 mL, 3 times) and 100 mM HEPES (1 L, pH 8.0). The solution was concentrated by an Amicon-Ultra Centrifugal filter unit (NMWL 3500) to obtain Ax647-Mb as a blue transparent solution (Mb: 718 µM, Alexa fluor 647: 775 µM determined by UV-vis absorption spectroscopy in supplementary Fig. 41). The molar absorption coefficients of Mb (18,800⁷⁰ ) and Alexa Flour 647 (290,000⁶⁹ ) were used.

This method studies the ability of antioxidant molecules to metabolize free radicals present in cells, including small proteins and different enzymes. To form an ABTS radical (ABTS^•+), 2,2’-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid (ABTS; A1888, Sigma-Aldrich, Madrid, Spain) can be oxidized by metmyoglobin (M1882, Sigma-Aldrich, Madrid, Spain) and hydrogen peroxide (H1009; Sigma Aldrich, Madrid, Spain). The blue-green product can be measured at 405 nm and its level is inversely proportional to the concentration of antioxidant molecules in biological samples [52 (link)]. Results are expressed in mM Trolox equivalents per mg of protein, with Trolox (238813, Sigma-Aldrich, Madrid, Spain) being a water-soluble tocopherol analogue used to standardize antioxidants.