8. REACTIONS OF INORGANIC SUBSTANCES
391
for several bacteria. They obtained the values shown in Table X, to
which their own data on the rate of conversion of para- to ortho-hydrogen should be added. The latter, expressed as in the other assays in
terms of —ζ> Η2 ,* is 10,000 for Proteus vulgaris and only 25 for R.
rubrum.
These observations and the entirely concordant findings of Peck et
al. (167) show that hydrogenase activity varies considerably from one
bacterial species to the next. In D. desulfuricans, the most active organism, the activity is indeed about two thousand times greater than in R.
rubrum. Another finding is that none of the methods available at present
invariably yield the highest or most accurate results. After all, it appears
that despite the hopes placed on the exchange and conversion reactions
none of the available methods really measures the true activity of the
enzymes, i.e., the activation of the hydrogen molecule, under conditions
which are independent of any accessory factor or system.
2. Purification of the Enzyme
The first cell-free extract with hydrogenase activity was obtained by
Bovarnik (168) from E. coll. Later, crude active extracts were prepared
from cells of other microorganisms, notably R. rubrum (138), A. vinelandii (169), H. facilis (170), and Micrococcus aero genes (162). The
attempts to purify these hydrogenases have been stymied by the fact
that the enzyme was in particulate form (171).
Shug et al. (172) extracted a soluble hydrogenase from the aerobic
nitrogen-fixing bacterium Clostridium pasteurianum and achieved considerable purification by standard protein separation techniques. Peck
and Gest (148) extracted another soluble hydrogenase from C. butylicum and obtained purified preparations whose specific activity, measured by H 2 evolution from reduced methyl viologen, reached 5-9 X 10
6
ju.1. hydrogen liberated per hour per milligram nitrogen. Sadana and
Jagannathan (173) purified the hydrogenase of D. desulfuricans and
obtained a specific activity of 2.6 X 10
6 , which was determined by
methylene blue reduction.
According to Kondo et al. (174), the hydrogenase contained in
crude extracts of D. desulfuricans is a particulate enzyme which sediments to the extent of 90% in about 2 hours of centrifugation at 100,000 g.
These investigators solubilized this hydrogenase and those of E. colt and
P. vulgaris by treatment with sodium deoxycholate, partial trypsin digestion, and ammonium sulfate precipitation.
The hydrogenase of C. pasteurianum (172) and the other purified
* —-(?H2
e( l
ua l
s nricroliters of H 2 consumed, evolved, exchanged, or converted
per hour and per milligram of bacterial nitrogen.
391
for several bacteria. They obtained the values shown in Table X, to
which their own data on the rate of conversion of para- to ortho-hydrogen should be added. The latter, expressed as in the other assays in
terms of —ζ> Η2 ,* is 10,000 for Proteus vulgaris and only 25 for R.
rubrum.
These observations and the entirely concordant findings of Peck et
al. (167) show that hydrogenase activity varies considerably from one
bacterial species to the next. In D. desulfuricans, the most active organism, the activity is indeed about two thousand times greater than in R.
rubrum. Another finding is that none of the methods available at present
invariably yield the highest or most accurate results. After all, it appears
that despite the hopes placed on the exchange and conversion reactions
none of the available methods really measures the true activity of the
enzymes, i.e., the activation of the hydrogen molecule, under conditions
which are independent of any accessory factor or system.
2. Purification of the Enzyme
The first cell-free extract with hydrogenase activity was obtained by
Bovarnik (168) from E. coll. Later, crude active extracts were prepared
from cells of other microorganisms, notably R. rubrum (138), A. vinelandii (169), H. facilis (170), and Micrococcus aero genes (162). The
attempts to purify these hydrogenases have been stymied by the fact
that the enzyme was in particulate form (171).
Shug et al. (172) extracted a soluble hydrogenase from the aerobic
nitrogen-fixing bacterium Clostridium pasteurianum and achieved considerable purification by standard protein separation techniques. Peck
and Gest (148) extracted another soluble hydrogenase from C. butylicum and obtained purified preparations whose specific activity, measured by H 2 evolution from reduced methyl viologen, reached 5-9 X 10
6
ju.1. hydrogen liberated per hour per milligram nitrogen. Sadana and
Jagannathan (173) purified the hydrogenase of D. desulfuricans and
obtained a specific activity of 2.6 X 10
6 , which was determined by
methylene blue reduction.
According to Kondo et al. (174), the hydrogenase contained in
crude extracts of D. desulfuricans is a particulate enzyme which sediments to the extent of 90% in about 2 hours of centrifugation at 100,000 g.
These investigators solubilized this hydrogenase and those of E. colt and
P. vulgaris by treatment with sodium deoxycholate, partial trypsin digestion, and ammonium sulfate precipitation.
The hydrogenase of C. pasteurianum (172) and the other purified
* —-(?H2
e( l
ua l
s nricroliters of H 2 consumed, evolved, exchanged, or converted
per hour and per milligram of bacterial nitrogen.