Daday A, Lambert G R, Smith G D
Biochem J. 1979 Jan 1;177(1):139-44. doi: 10.1042/bj1770139.
A method was devised that allows measurement in vivo of hydrogenase-catalysed H2 evolution from the cyanobacterium Anabaena cylindrica, independent of nitrogenase activity, which is also present. Addition of low concentrations of reduced Methyl Viologen (1-10mM) to intact heterocystous filaments of the organism resulted in H2 evolution, but produced conditions giving total inhibition of nitrogenase (acetylene-reducing and H2-evolving) activity. That the H2 formed under these conditions was not contributed to by nitrogenase was also supported by the observation that its rate of formation was similar in the dark or with Ar replaced by N2 in the gas phase, and also in view of the pattern of H2 evolution at very low Methyl Viologen concentrations. Conclusive evidence that the H2 formed in the presence of Methyl Viologen was solely hydrogenase-mediated was its evolution even from nitrogenase-free (non-heterocystous) cultures; by contrast 'uptake' hydrogenase activity in such cultures was greatly decreased. The hydrogenase activity was inhibited by CO and little affected by acetylene. Finally the hydrogenase activity was shown to be relatively constant at different stages during the batch growth of the organism, as opposed to nitrogenase activity, which varied.
设计了一种方法，可在体内测量圆柱鱼腥藻中氢化酶催化产生氢气的过程，该过程独立于同样存在的固氮酶活性。向该生物体完整的异形胞丝状体中添加低浓度的还原甲基紫精（1-10 mM）会导致氢气产生，但会产生完全抑制固氮酶（乙炔还原和氢气产生）活性的条件。在这些条件下形成的氢气并非由固氮酶产生，这一观点得到了以下观察结果的支持：在黑暗中或气相中用N2替代Ar时，氢气的形成速率相似，而且考虑到极低甲基紫精浓度下氢气产生的模式。在甲基紫精存在下形成的氢气完全是由氢化酶介导的，确凿证据是即使从无固氮酶（无异形胞）的培养物中也会产生氢气；相比之下，此类培养物中的“吸收”氢化酶活性则大大降低。氢化酶活性受到CO的抑制，而受乙炔的影响很小。最后，结果表明，在该生物体分批生长的不同阶段，氢化酶活性相对恒定，而固氮酶活性则有所不同。
