A Hidden Transhydrogen Activity of a FMN-Bound Diaphorase under Anaerobic Conditions - Fig 5

<p>(A) Real-time monitoring of the hydride exchange from NADPH to thio-NAD⁺ at 400 nm, and the consumption of thio-NADH with the addition of LDH. Condition: 1 mM NADPH and 1 mM thio-NAD⁺ were first incubated with 500 nM FMN-DI in 1 × TBS buffer (pH 7.4) at room temperature. Then 10 nM LDH and 1 mM pyruvate were added to oxidize thio-NADH. (B) The LDH-catalyzed oxidation was activated by the addition of a FMN-DI to convert NADPH to NADH (black) and controls of no addition of DI (red), no addition of LDH (red) and no addition of enzymes (blue). Condition: 1 mM NADPH and 1 mM NAD⁺ were first incubated with or without 1 μM FMN-DI for one hour in 1 × TBS buffer (pH 7.4). Then 1 nM LDH and 1 mM pyruvate were added to evaluate the oxidation of the mixture of NADPH and NAD⁺. Anaerobic solution was used for the assay. Error bars were generated as the range of at least three replicates.