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. 2013 Apr 5;288(20):14341–14361. doi: 10.1074/jbc.M113.466995

FIGURE 4.

FIGURE 4.

DMAV is an antioxidant that scavenges O2⨪. A, neutrophils were activated with PMA (5 nmol/liter) in the presence of increasing concentrations of DMAV. Superoxide was detected using Diogenes luminescent reagent for O2⨪. B, neutrophils were activated as in A, but O2⨪ was detected with Lucigenin (20 μmol/liter). C, O2⨪ was generated by an X/XO system; luminescence was detected using luminol (500 μmol/liter). D, several proteins (DM146, ixolaris, triplatin, 9.3 kDa, SVEP, and yellow protein; 2 μmol/liter) were expressed and purified as DMAV and tested side by side. Detection of O2⨪ was performed with Luminol (500 μmol/liter). E, O2⨪ was produced by the X (500 μmol/liter)/XO (1.2 milliunits/ml) system and detected by the reduction of cytochrome c (12 μmol/liter) at 550 nm. Other available salivary recombinant proteins (dipetalodipin (DPTL), ixolaris, and BSA; 2 μmol/liter) were tested side by side with DMAV (5 μmol/liter). F, DMAV (5 μmol/liter) does not affect the enzymatic function of X/XO, determined by uric acid production at 293 nm. G, effects of DMAV on generation of H2O2. X (500 μmol/liter)/XO (6.3 milliunits/ml) was used to generate H2O2, which was detected with the Amplex Red reagent. In control, spontaneous dismutation of O2⨪ is observed, which is increased in the presence of Cu,Zn-SOD (4 milliunits/ml). DMAV (5 μmol/liter) did not increase H2O2 production. H, DMAV does not display catalase activity. Different concentrations of H2O2 were incubated for 15 min with PBS or DMAV (2 μmol/liter) and detected with Amplex Red reagent as described under “Experimental Procedures.” I, catalase activity. XO (0.7 milliunits/ml) was added to a solution containing hypoxanthine (500 μm), and O2 consumption was detected with a Clark electrode. After 15 min, catalase (333 units/m) was added to the chamber, and a peak of O2 is observed. No effects were observed after the addition of DMAV (0.5 μm). J, DMAV displays metal-dependent antioxidant activity. DMAV (5 μmol/liter) or Cu,Zn-SOD (0.42 unit/ml) was incubated with EDTA (5 mmol/liter) for 15 min and tested in the cytochrome c reduction assay. Antioxidant activity of DMAV was abolished by EDTA (n = 3). Significance (*) was set at p < 0.05 (analysis of variance). A.U., arbitrary units. Error bars, S.E.