Nadph

The recombinant enzymes Fre (derived from E. coli) and XiaP (derived from Streptomyces sp. HKI0576) were tested for NAD(P)H:flavin oxidoreductase activity. Reactions were set up in XiaF assay buffer (20 μM Tris-HCl pH 8.0, 10% glycerol) containing 500 μM NAD(P)H (Sigma-Aldrich), 50 μM FAD/FMN/riboflavin (Sigma-Aldrich) and 1 μM flavin reductase at room temperature, unless stated otherwise. Reaction rates were monitored by following the decrease of NAD(P)H absorbance at 340 nm caused by the oxidation to NAD(P) on the Tecan Safire² multi-detection microplate reader. Assays were carried out in a volume of 100 μl. Reactions without enzyme or flavin served as negative controls. Blanc measurements were performed with XiaF assay buffer und subtracted from each value.

The enzyme activity of bovine aldose reductase was performed according to the method of Fujita et al. [54 (link)], with some modifications. For this purpose, a reaction mixture containing 50 mM potassium phosphate buffer pH 7.0 was used with DL-glyceraldehyde (Sigma-Aldrich, St. Louis, MO, USA) as a substrate and the NADPH (Sigma-Aldrich, St. Louis, MO, USA) 150 µM cofactor and crude protein extract of the bovine lens homogenate in a total volume of 200 µL. The reaction mixture without the cofactor was incubated at 37 °C for 5 min, after which the reaction was initiated by adding the NADPH and immediately the decrease in absorbance at 340 nm every 30 s for 20 min was quantified using a reader of TECAN microplates. The decrease in absorbance was proportional to the cofactor’s oxidation.

Cells were collected in lysis buffer (100 mM Tris pH 7.6, 5 mM EDTA, 1 mM NaN₃ and 0.1% peroxide-free Triton-X100). Lysates were complemented with 0.6 U/mL glutathione reductase (Sigma, G3664), 0.2 mM nicotinamide adenine dinucleotide phosphate hydrogen (NADPH, Sigma, N7505), 3 mM reduced glutathione (GSH, Sigma, G4251) and 200 µM of the substrate cumene hydroperoxide (CHP, Sigma, 247502). NADPH turnover was measured on an Infinite 200PRO reader (Tecan) at 340 nm over 10 min at 37 °C. Enzymatic activity was calculated after subtracting absorbance decay obtained from buffer without cell lysates by using NADPH extinction coefficient of 6220/M/cm and by normalizing to total protein content^{63 (link)}.