LIQUID-CHROMATOGRAPHIC FLUORESCENCE DETERMINATION OF AMMONIA FROM NITROGENASE REACTIONS - A 2-MIN ASSAY
LIQUID-CHROMATOGRAPHIC FLUORESCENCE DETERMINATION OF AMMONIA FROM NITROGENASE REACTIONS - A 2-MIN ASSAY
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DOI：
10.1128/aem.47.5.1027-1030.1984
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发表时间：
1984-01-01
影响因子：
4.4
通讯作者：
CORBIN, JL
中科院分区：
文献类型：
--
作者：
CORBIN, JL
氨与邻苯二醛巯基乙醇试剂在pH值为7时（非典型荧光）反应的分析潜力已经被证明。这一点，加上这里报道的其他发现，已经导致氨测定非常适合于氮酶的研究。因此，在温和的条件下，无需事先微扩散，高压液相色谱法可以快速分析大量样品。二亚硝酸钠（或通常的氮酶测定的其他组分）、替代底物（氰化物、叠氮化物、甲基异腈）及其产物（甲胺、二甲胺、肼）均不干扰。高压液相色谱分析表明，邻苯二醛巯基乙醇试剂-氨反应的荧光产物实际上不仅仅是一个单一的化合物。尽管如此，一旦找到合适的溶剂组成，高压液相色谱与小型廉价的C18保护柱证明了该测量非常快速和可重复性。对氨的荧光响应在至少40 nmol/ml时呈线性。之前的一个问题，荧光的长期稳定性，通过在黑暗中进行反应解决了。利用叔丁基硫醇和溶剂萃取，可以通过类似的邻苯二醛巯基乙醇试剂反应大幅度降低缓冲液中的背景氨。
The analytical potential of the reaction of ammonia with o-phthalaldehyde mercaptoethanol reagent at pH 7 (an atypical fluorescence) has already been demonstrated. This, coupled with additional findings reported here, has led to an ammonia determination well suited to nitrogenase studies. As a result, large numbers of samples can be rapidly analyzed by high-pressure liquid chromatography methods under mild conditions and without prior microdiffusion. Neither sodium dithionite (or other components of the usual nitrogenase assay), nor alternative substrates (cyanide, azide, methyl isonitrile), nor their products (methylamine, dimethylamine, hydrazine) interfere. High-pressure liquid chromatography showed that the fluorescent product of the o-phthalaldehyde mercaptoethanol reagent-ammonia reaction was, in fact, more than just a single compound. Despite this, once the proper solvent composition was found, high-pressure liquid chromatography with a small inexpensive C18 guard column proved quite fast and reproducible for this measurement. Fluorescence response to ammonia was linear to at least 40 nmol/ml. A previous problem, long-term stability of the fluorescence, was solved by running the reactions in the dark. Background ammonia in the buffer could be substantially reduced by an analogous o-phthalaldehyde mercaptoethanol reagent reaction, using tert-butyl mercaptan and solvent extraction.