FIGURE 3.

Assays for sepiapterin reduction and redox cycling activity of sepiapterin reductase. Panel A, changes in the absorbance spectra of sepiapterin in the sepiapterin reductase assay. Note the decrease in absorption of sepiapterin over time. Standard reaction mixes in a total volume of 0.2 ml contained 50 mm phosphate buffer, pH 7.8, 200 μm NADPH, 50 μm sepiapterin, and 1 μg/ml sepiapterin reductase and were analyzed after 0 min (curve a), 1 min (curve b), 2 min (curve c), and 3 min (curve d). Controls contained the standard reaction mix without the enzyme and sepiapterin (curve e), with the standard reaction mix buffer plus 250 μm menadione (curve f), or with the standard reaction mix buffer plus 50 μm BH2 (curve g). Panel B, comparison of sepiapterin consumption and BH2 production in sepiapterin reductase enzyme assays. Both sepiapterin reduction reactions were run under the conditions indicated above. Sepiapterin reduction was measured by decreases in absorbance at 420 nm and was presented as micromolar concentrations of the substrate remaining in the assay over time. BH2 was measured using HPLC as described under “Experimental Procedures” and was presented as micromolar concentrations of the product formed in the assay over time. Panel C, effects of DTPA on H₂O₂ formation by sepiapterin reductase. Redox cycling was run in standard reaction mixes without sepiapterin and supplemented with 5 μm 9,10-phenanthrenequinone without (open triangles) and with 250 μm DTPA (closed triangles). In control experiments, sepiapterin reductase was left out of reaction mixes without (open circles) and with DTPA (closed circles). Panel D, comparison of H₂O₂ production by sepiapterin reductase redox cycling and autooxidation of BH2 and/or BH4. H₂O₂ production in the redox cycling reaction was compared with autooxidation of 50 μm BH2, 50 μm BH4, or the combination of BH2 and BH4. Autooxidation reactions were run in the buffer of the standard reaction mix. Inset, enlarged scale for autooxidation reactions run over 10 min. Note that the redox cycling reaction generated much greater amounts of H₂O₂ when compared with the autooxidation reactions.