Bravo M, Eran H, Zhang F X, McKenna C E
Department of Chemistry, University of Southern California, Los Angeles 90089-0744.
Anal Biochem. 1988 Dec;175(2):482-91. doi: 10.1016/0003-2697(88)90572-6.
A dansyl chloride precolumn derivatization method has been developed for high-performance liquid chromatography analysis of NH3 and/or CH3NH2 produced by nitrogenase-catalyzed reduction of substrates such as N2 (NH3) or diazirine (NH3, CH3NH2). The dansyl chloride reagent can be used immediately after preparation and is stable at 4 degrees C for 1 month. The derivatization products from NH3 and CH3NH2 are prepared by direct treatment of the assay mixture (30 min of incubation) and are then stable in air and ambient light at room temperature for at least 1 day. They are separated isocratically within several minutes on a mu Bondapak C-18 reversed-phase column (Altex) at 2000-2500 psi using an eluent of 7:7:3 H2O:methanol:acetonitrile and are detected fluorometrically (368 nm excitation; 500 nm emission). The NH3 sensitivity is limited by the background NH3 in the reagents and under practical conditions is ca. 0.02 nmol (20-microliters injection volume). Methylamine sensitivity is an order of magnitude greater. The detector response for either product is linear to at least 2.4 nmol. The method is compared to alternative analytical procedures with respect to sensitivity, convenience, and absence of interferences.
已开发出一种丹磺酰氯柱前衍生化方法，用于高效液相色谱分析由固氮酶催化还原诸如N2（生成NH3）或重氮甲烷（生成NH3、CH3NH2）等底物所产生的NH3和/或CH3NH2。丹磺酰氯试剂制备后可立即使用，在4℃下稳定1个月。NH3和CH3NH2的衍生化产物通过直接处理分析混合物（孵育30分钟）制备，然后在室温下的空气和环境光中至少稳定1天。它们在μ Bondapak C - 18反相柱（Altex）上，于2000 - 2500 psi压力下，使用7:7:3的H2O:甲醇:乙腈洗脱液，在几分钟内等度洗脱，并通过荧光检测（激发波长368 nm；发射波长500 nm）。NH3的灵敏度受试剂中背景NH3的限制，在实际条件下约为0.02 nmol（进样体积20微升）。甲胺的灵敏度高一个数量级。两种产物的检测器响应至少在2.4 nmol范围内呈线性。该方法在灵敏度、便利性和无干扰方面与其他分析方法进行了比较。
