5% (w/v polyacrylamide) non-denaturating PAGE and the gels were treated as follows: A. transferred to a nitrocellulose
membrane and analyzed with antibodies directed against Hyd-1; B. transferred to a nitrocellulose membrane and analyzed with monoclonal MAPK inhibitor His-tag antibody; C. the gel containing purified Hyd-1 and the molecular mass standard was stained with Coomassie Brilliant Blue. The masses of the standard proteins (Sigma) are given on the right hand of the panel. Alternatively, the extracts and purified enzyme were: D. stained for 10 minutes under a 100% hydrogen CH5183284 clinical trial atmosphere with PMS and NBT as electron acceptors; or E. stained under a hydrogen atmosphere with BV and TTC as electron acceptors. The bands assigned to Hyd-1 activity or the His tagged version of HyaA-Hyd-1 activity are indicated on the right hand of the gels. Discussion Tetrazolium-based redox dyes are useful tools in zymographic detection of oxidoreductase enzyme
activity in non-denaturing PAGE because upon irreversible reduction they generate coloured, insoluble formazan complexes, which are advantageous in cumulative staining procedures. Triphenyl tetrazolium has been used for a considerable time as a means of distinguishing the hydrogenase enzymes in E. coli cell extracts [18, 19]. Measuring Hyd-3 activity in the presence of the H2-oxidizing enzymes was problematic in the past and visualizing it had not been successfully
accomplished check details until the current study was conducted. However, optimization of the in-gel assay conditions, together with the judicious use of defined mutants has allowed us for the first time to visualize Hyd-3 activity unequivocally after native-PAGE. The complexes exhibiting Hyd-3 activity migrate in native-PAGE at high molecular masses, similar to the trimer of trimers of the Fdh-N and Fdh-O with a mass of 500-550 kDa . This suggests that the stoichiometry of the individual components in the FHL complex might be greater than unity. Nothing is currently known about the stoichiometry of the FHL complex components or the architecture of the HycE/HycG large and small subunit within the complex, and this will form the subject of future studies. The findings of the current study suggest that while the Fdh-H component of the FHL complex is required Phosphoribosylglycinamide formyltransferase for maximal activity of the complex, in its absence activity of the Hyd-3 can still be detected and its migration position in the gel system is very similar in extracts of the wild-type and the fdhF mutant. This suggests perhaps that the Fdh-H component is separated from the rest of the complex during electrophoresis. The lability of the Fdh-H activity has been noted previously [15, 43]. One possible reason why the Hyd-3 activity was previously overlooked after in-gel staining is the considerable overlap in the staining pattern of Fdh-N/O, Hyd-3 and Hyd-2.