8. REACTIONS OF INORGANIC SUBSTANCES
389
This reaction permits the quantitative estimation of hydrogenase activity from the rate of decoloration of methylene blue by bacterial suspensions or extracts in Thunberg tubes under H 2 . The reaction can also
be followed colorimetrically or volumetrically by measuring H 2 consumption by the Warburg technique. Other acceptors may be employed, depending on the organism or enzyme preparation under study,
e.g., ferricyanide, oxygen, nitrate, sulfate; other colored indicators such
as safranine O, dichlorophenolindophenol, indigo disulfonate, or triphenyltetrazolium may be used.
The reaction catalyzed by hydrogenase
H 2 ^ 2H+ + 2e
is that of the hydrogen electrode and has a very low oxidation-reduction
potential (E' 0 = —0.420 volt at pH 7; ΔΕΌ/ΔρΗ = —0.060 volt). Artificial acceptors are therefore useful whose potential lies closer to that
of hydrogenase than that of methylene blue (E' 0 = +0.011 volt). For
this reason, several authors have recommended the viologens, whose
chemical and physicochemical properties were studied by Michaelis
and Hill (160). In contrast to other oxidation-reduction indicators, the
viologens are colorless in the oxidized state and colored in the reduced
state; they fix only a single electron per molecule and have an unchanging potential with pH. The most commonly used H 2 acceptor for
hydrogenase is benzyl viologen (E' 0 = —0.359 volt).
In contrast with dye-reduction procedures, Peck and Gest (161)
described a new technique for the determination of hydrogenase activity based on the liberation of H 2 from a single-electron dye, methyl
viologen (E' 0 = —0.446 volt) reduced by an excess of hydrosulfite. At
pH 7.0, this indicator has an E' 0 below that of the hydrogenase-catalyzed reaction and consequently tends to be reoxidized by the enzyme
with the removal of H 2 . By this method it was shown that the cells of
C. butylicum have high hydrogenase activity although they appear inactive by the conventional methylene blue reduction method. Similarly,
the technique based on H 2 evolution gives higher values for hydrogenase
activity than methylene blue assays for suspensions of E. colt, C.
pasteurianum, and M. lactilyticus. However, A. vinehndii and R. rubrum, which reduce methylene blue in an H 2 atmosphere, do not release
hydrogen from reduced methyl viologen.
It has been stressed that the number and nature of dyes reduced
vary in an apparently erratic manner from one organism to the next.
One of the reasons for this is the absence of a cofactor or intermediary
carrier required for the reduction of a given acceptor. Curtis and Ordal
(162) showed, moreover, that methylene blue and other dyes reversibly
389
This reaction permits the quantitative estimation of hydrogenase activity from the rate of decoloration of methylene blue by bacterial suspensions or extracts in Thunberg tubes under H 2 . The reaction can also
be followed colorimetrically or volumetrically by measuring H 2 consumption by the Warburg technique. Other acceptors may be employed, depending on the organism or enzyme preparation under study,
e.g., ferricyanide, oxygen, nitrate, sulfate; other colored indicators such
as safranine O, dichlorophenolindophenol, indigo disulfonate, or triphenyltetrazolium may be used.
The reaction catalyzed by hydrogenase
H 2 ^ 2H+ + 2e
is that of the hydrogen electrode and has a very low oxidation-reduction
potential (E' 0 = —0.420 volt at pH 7; ΔΕΌ/ΔρΗ = —0.060 volt). Artificial acceptors are therefore useful whose potential lies closer to that
of hydrogenase than that of methylene blue (E' 0 = +0.011 volt). For
this reason, several authors have recommended the viologens, whose
chemical and physicochemical properties were studied by Michaelis
and Hill (160). In contrast to other oxidation-reduction indicators, the
viologens are colorless in the oxidized state and colored in the reduced
state; they fix only a single electron per molecule and have an unchanging potential with pH. The most commonly used H 2 acceptor for
hydrogenase is benzyl viologen (E' 0 = —0.359 volt).
In contrast with dye-reduction procedures, Peck and Gest (161)
described a new technique for the determination of hydrogenase activity based on the liberation of H 2 from a single-electron dye, methyl
viologen (E' 0 = —0.446 volt) reduced by an excess of hydrosulfite. At
pH 7.0, this indicator has an E' 0 below that of the hydrogenase-catalyzed reaction and consequently tends to be reoxidized by the enzyme
with the removal of H 2 . By this method it was shown that the cells of
C. butylicum have high hydrogenase activity although they appear inactive by the conventional methylene blue reduction method. Similarly,
the technique based on H 2 evolution gives higher values for hydrogenase
activity than methylene blue assays for suspensions of E. colt, C.
pasteurianum, and M. lactilyticus. However, A. vinehndii and R. rubrum, which reduce methylene blue in an H 2 atmosphere, do not release
hydrogen from reduced methyl viologen.
It has been stressed that the number and nature of dyes reduced
vary in an apparently erratic manner from one organism to the next.
One of the reasons for this is the absence of a cofactor or intermediary
carrier required for the reduction of a given acceptor. Curtis and Ordal
(162) showed, moreover, that methylene blue and other dyes reversibly