Hydrogenase assays

Enzyme activity Hydrogenase assay or sulfate reductase assay Measures enzyme activity in a field sample as a rate of reaction. Commercial kits are available. Hydrogenase activity may be related to MIC, while sulfate reductase assesses the presence of SRB. 

As only the soluble hydrogenase utilized NADH, in vivo assays could be applied to investigate this activity further. Hydrogen-driven MMO activities were measured to obtain information on the in vivo function of this hydrogenase. The apparent Ks for hydrogen was again 0.8 mM in both assays. Maximal rates of MMO activities were 140 nmol min 1... 

Activation of an [NiFe] hydrogenase such as that from D. gigas, by incubation with H2, as measured by different assay methods... 

Hydrogenase was assayed directly in aliquots from the cultures at an OD q °f I -5-... 

Four general methods may be used for the assay of hydrogenases based on their ability to catalyse the following reactions. 

Figure 5.5 Activition of an [NiFe] hydrogenase such as that from D.gigas, by incubation with H2, as measured by different assay methods.These are production of H2 with the low-potential donor methyl viologen consumption of H2 with the high-potential acceptor dlchlorolndophenol and Isotope exchange of H2 with H2O. The proportions of the enzyme in three different states, unready, ready and active, are indicated by the shading.

This review describes our unique assay system to investigate the role of cell-free hydrogenases in anaerobic biocorrosion, which resulted in proposing a new biocorrosion model. 

For reactions in which one or more reactants or products is a gas, manometry (the measurement of pressure differences) can provide a convenient means for monitoring the course and kinetics of the reaction Thus, enzymes that can be assayed with this method include oxidases, urease, carbonic anhydrase, hydrogenase, and decarboxylases. For example, bacterial glutamate decarboxylase is readily assayed by utilizing a Warburg flask and measuring the volume of gas evolved at different times using a constant-pressure respirometer. ... 

This new assay technique was developed independently by American and Australian workers who first established the connection between nitrogenase and hydrogenase, and showed the former could reduce a number of substrates other than nitrogen, for example nitrous oxide, azide, cyanide and, most usefully, acetylene. The latter is reduced to ethylene and this forms the basis of the acetylene reduction test. The material to be examined is briefly gassed with acetylene and the ethylene formed measured by gas chromatography. While this technique was being refined,... 

Fig. 4. H2 photoevolution from ascorbate via P. laminosum PSI particles under different conditions of immobilization. The complete assay system contained 100 mM Mes-NaOH buffer, pH 7.0 75 mM sodium ascorbate 15 mM DTT 2 mM TMPD 1% (w/v) BSA PSI particles (30 pg Chi) 50 pi (saturating amount) of Clostridium pasteurianum hydrogenase and 12.5 pM Spirulina maxima Fd as electron mediator, (a) Conditions all components free (b) hydrogenase immobilized in Ca alginate according to Gisby and Hall (1980) (c) PSI particles immobilized in Ca alginate (d) hydrogenase and PSI coimmobilized in Ca alginate.

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