Superoxide production of mAOX1, mAOX3, mAOX4, and mAOX2
Superoxide production of enzymes was calculated by following the reduction of 100 μM cytochrome c at 550 nm in the presence of 50 μM substrate (p-DMAC or vanillin) in 50 mM Tris-HCl, 200 mM NaCl, and 1 mM EDTA (pH 8.0) buffer. Substrate consumption was monitored directly by the decrease in UV absorbance at wavelengths of 398 and 347 nm for p-DMAC and vanillin, respectively, in the presence of oxygen as electron acceptor. Extinction coefficients used are 21,000 M–1cm−1 for cytochrome c, 30,500 M–1cm−1 for vanillin, and 25,100 M–1cm−1 for p-DMAC.
Substrate | mAOX1 | mAOX3 | mAOX4 | mAOX2 | |
---|---|---|---|---|---|
Vanillin | Substrate consumption (U μmol−1 of enzyme) | 51.85 | 29.72 | 9.17 | 5.78 |
Superoxide formation (U μmol−1 of enzyme) | 16.45 | 6.92 | 1.88 | 2.42 | |
Ratio (%) | 31.7 | 23.3 | 20.4 | 41.8 | |
p-DMAC | Substrate consumption (U μmol−1 of enzyme) | 142.28 | 27.44 | 19.37 | 15.83 |
Superoxide formation (U μmol−1 of enzyme) | 43.47 | 5.25 | 3.84 | 6.21 | |
Ratio (%) | 30.6 | 19.1 | 19.9 | 39.3 |
One unit (U) is defined as the oxidation of 1 μmol substrate per minute under assay conditions.