Figure 4.

Assessing stability of SAH and Hcy in air-exposed reaction buffer. The fluorescent readouts of 1 nmol SAH (20 μM in 50 μl reaction buffer) were measured after pre-incubating for 0–12 h in the absence or presence of 10 μM SsSAHH and 0.35 μM PfADA. Since the amount of the coupling enzymes is sufficient to process 1 nmol SAH to Hcy within 1 h (data not shown), the fluorescence readouts in the absence and presence of the couple enzymes are proportional to the amount of residual SAH and Hcy after the pre-incubation step, respectively. The constant fluorescence readout in the former indicates that SAH is stable under the assay condition. In contrast, the gradual loss of fluorescence readout in the latter scenario suggests that Hcy is subject to air-mediated oxidation under the assay condition