The success of the anaerobic induction of hydrogenase activity can be monitored by an in vitro hydrogenase activity assay. The reaction mixture of this assay contains Triton-X 100, a mild detergent which lyses the algal cells. It should be noted that some algal species have different types of cell walls which might be too resistant to Triton. Selleck SB-715992 The assay described here performs well in C. reinhardtii, C. moewusii, Scenedesmus obliquus, S. vacuolatus, and some other species tested to date (Winkler et al. 2002b; Kamp et al. 2008). The assay furthermore contains methyl viologen as a potent artificial electron donor to FeFe-hydrogenases and sodium dithionite
(Na2S2O4) as an efficient reductant for methyl viologen. The details: First, 1.6 ml of 60 mM potassium phosphate buffer pH 6.8, 1% Triton X-100 (0.2 ml of a 10% (v/v) stock solution in the above mentioned phosphate buffer) and 10 mM methyl viologen (of a 1 M stock solution in phosphate buffer, which can be stored in the fridge for several weeks) are mixed in a 8–10-ml edge rolls bottle (e.g., 10-ml headspace bottles ND20/ND18, cat. no. 3205550 at www.de.fishersci.com/) (Fig. 2b). The flask is then sealed by a Red Rubber Suba Seal (e.g., No. 25, cat. no. Z12,459-1 at www.sigmaaldrich.com/germany.html) and gassed with Ar (N2) for 5 min. For this purpose, a needle connected to a gas cylinder via an adequate tube is pierced through
the septum, and another needle serves as gas exhaust. In parallel, a 1-M freshly prepared sodium selleck products dithionite solution is prepared in a sealed headspace bottle by injecting the required amount of phosphate buffer through the septum of the vessel, in which the required amount of sodium dithionite is already present. This solution is also flushed with Ar (N2) for 5 min. Finally, 200 μl of the anaerobic sodium dithionite stock PAK5 solution is added to the pre-mix containing buffer, Triton, and methyl viologen by a syringe piercing through the rubber septum. The reaction mixture should turn deep blue to
purple, an indication of methyl viologen being reduced (Fig. 2b). As an alternative to applying Ar gassing, all the reaction mixtures can be prepared in an anaerobic glove box (e.g., of Coy Laboratories, Detroit, USA). Fig. 2 a Development of in vitro hydrogenase activity in a concentrated C. reinhardtii culture sparged with Ar starting at 0 min. Samples of 200 μl containing the algal suspension were removed from the shaded incubation flask at the depicted time points and injected into an in vitro assay reaction mixture containing Triton X-100 used for cell lysis, and sodium dithionite reduced methyl viologen as an efficient, in vitro electron donor to FeFe-hydrogenases. After 15 min of incubation in a shaking water bath at 37°C, the headspace within the reaction vessel was analyzed by gas chromatography (GC).