TABLE 3

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.