Cytochrome $C_3$의 존재하에 수소의 발생 및 흡수를 촉매하는 Hydrogenase를 Desulfovibrio desulfricans로부터 정제하였다. 세포벽에 붙어 있는 이 효소는 박테리아를 초음파로 분쇄한 후 Triton X와 sodium deoxycholate로 처리하여 $100,000{ imes}g$로 초원심분리하고 다시 Sephadex G-150 및 DEAE-Cellulose 콜럼을 통과시킨 후 isoelectric focusing에 의하여 정제하였다. 분자량은 120,000으로 추정되었고 pH 적정점은 7.0 근처이었다. 이 효소는 Cytochrome $C_3$의 인위적인 전자전달 매개체인 환원된 methyl viologen으로부터 수소발생을 촉매하며, 이에 대한 Km 값은 0.2 M NaCl에서 $5.1{ imes}10^{-5}M$이었고 NaCl의 농도가 증가함에 따라 그 값이 감소하였다. Isoelctric focusing에 의하여 측청된 pI 값은 6.1 근처이었으며 열(heat)에 대하여 상당히 안정한 것으로 나타났다. 그리고 이 효소는 중금속과 리간드를 형성하는 $NaN_3$, EDTA 및 KCN에 의하여 그 활동도가 억제되었으나 $Fe^{3+}$ 및 $Fe^{2+}$와 같은 금속이온에 의해서는 그 활동도가 현저히 촉진되었다.
Hydrogenase which catalyzes the production and consumation of gaseous $H_2$ in the presence of cytochrome $C_3$ was purified from Desulfovibrio desulfricans. This particulate enzyme was solubilized in Tris-HCl buffer containing Triton X and sodium deoxycholate after sonication. This was further purified by ultracentrifugation ($100,000{ imes}g$) of sonicate, gel filtration on Sephadex G-150, ion-exchange on DEAE-cellulose and isoelectric focusing. Its molecular wight was estimated to be 120,000 by gel filtration on Sephadex G-200 column chromatography and showed maximium activity around pH 7.0. This enzyme catalyzes the $H_2$ evolving reaction of methyl viologen which is an artificial electron carrier of ferricytochrome $C_3$. Its Km value was $5.1{ imes}10^{-5}M$ but it was expected to show decrease according to the increase of salt concentration. pI value estimated by isoelectric focusing was 6.1 and it showed rather stable property upon heat inactivation. The enzyme was inhibited by metal complexing agents such as $NaN_3$, EDTA and KCN, but showed significant activation effect by metal ions such as $Fe^{3+}$ and $Fe^{2+}$.
