Fig. 3

Single-product OAS assay using dNTPs and NAD⁺ as substrates. The purified human recombinant His-OAS1 enzyme was incubated with NAD⁺ and dNTP (dATP, dCTP, dGTP or TTP) for 30 min at 37 °C, and the reactants and product resolved using a HiTrap Q column. Each figure (a-d) includes three chromatograms that have been superimposed. The position of the reactants and product are indicated in the chromatograms. The substrates used were: (a) NAD⁺ and dATP, (b) NAD⁺ and dCTP, (c) NAD⁺ and dGTP and (d) NAD⁺ and TTP. Blue curves: Profiles from reactions using His-OAS1 together with NAD⁺ and dNTP. Red and green curves: Control reactions after incubation of His-OAS1 with either NAD⁺ or dNTP alone, respectively. The insert in (a) shows the structural formula of NAD⁺. The 2′ hydroxyl (OH) to be linked with dNMP during the reaction has been highlighted in red. Brown curves: experimental salt gradients. Chromatograms were obtained by using 254 nm as the absorbance wavelength. mAU, milli-absorbance unit and mS/cm, milliSiemens/centimeter