Activity Assay and Methods for Purification of an Uptake Hydrogenase from Frankia strain ArI-5
The nitrogen-fixing actinomycete Frankia contains an uptake hydrogenase which greatly improves the overall efficiency of the nitrogenase complex. The function of the enzyme is to catalyze the uptake and oxidation of molecular hydrogen. The electrons liberated by this process proceed along an electron-transport chain which ultimately catalyzes the formation of ATP. The protons created by the hydrogenase participate in the reduction of molecular oxygen, thus protecting the delicate nitrogenase complex from oxidation. A proceedure was developed which attempted to purify hydrogenase from free-living Frankia. Methodology included: optimization of growth conditions to stimulate growth of the microorganism and expression of functional hydrogenase; solubilization of hydrogenase from membrane and purification using a Reactive Red affinity column; and detection of hydrogenase activity using an amperometric assay. The assembly used for the activity assay consisted of a water-jacketed cuvette and modified Clark electrode operating at a potential of 0.6 V. Data was recorded through an autoranging picoammeter-Macintosh interface, and manipulated with a script designed for Matlab.