Newton William E, Dilworth Michael J
Department of Biochemistry, Virginia Polytechnic Institute & State University, Blacksburg, VA 24061, USA.
Methods Mol Biol. 2011;766:105-27. doi: 10.1007/978-1-61779-194-9_8.
Steady-state assays of nitrogenases share at least five requirements: an anaerobic environment, a consistent source of magnesium adenosine triphosphate (MgATP), a suitable source of reductant, a buffer system compatible with the product-quantification protocol to be used, and the desired substrate. The assay is initiated by injection of the component protein(s) of the enzyme or MgATP and terminated by injection of either acid or a solution of Na(2)EDTA. The various nitrogenases catalyze the reduction of a wide variety of substrates. This chapter outlines the methods used to analyze the products of nitrogenase-catalyzed reactions involving nitrogen-nitrogen bonds, nitrogen-oxygen bonds, carbon-nitrogen bonds, carbon-carbon bonds, carbon-oxygen bonds, carbon-sulfur bonds, and hydrogen only. The usefulness of measurements of residual amounts of other components of nitrogenase assays is also discussed.
厌氧环境、三磷酸镁腺苷（MgATP）的稳定来源、合适的还原剂来源、与要使用的产物定量方案兼容的缓冲系统以及所需的底物。分析通过注入酶的组分蛋白或MgATP开始，并通过注入酸或Na₂EDTA溶液终止。各种固氮酶催化多种底物的还原反应。本章概述了用于分析固氮酶催化反应产物的方法，这些反应涉及氮-氮键、氮-氧键、碳-氮键、碳-碳键、碳-氧键、碳-硫键以及仅涉及氢的反应。还讨论了测定固氮酶分析中其他组分残留量的用途。
