Central Research Department, Experimental Station, E. I. du Pont de Nemours and Company, Wilmington, Delaware 19898.
Plant Physiol. 1968 Aug;43(8):1185-207. doi: 10.1104/pp.43.8.1185.
The methodology, characteristics and application of the sensitive C(2)H(2)-C(2)H(4) assay for N(2) fixation by nitrogenase preparations and bacterial cultures in the laboratory and by legumes and free-living bacteria in situ is presented in this comprehensive report. This assay is based on the N(2)ase-catalyzed reduction of C(2)H(2) to C(2)H(4), gas chromatographic isolation of C(2)H(2) and C(2)H(4), and quantitative measurement with a H(2)-flame analyzer. As little as 1 mumumole C(2)H(4) can be detected, providing a sensitivity 10(3)-fold greater than is possible with (15)N analysis.A simple, rapid and effective procedure utilizing syringe-type assay chambers is described for the analysis of C(2)H(2)-reducing activity in the field. Applications to field samples included an evaluation of N(2) fixation by commercially grown soybeans based on over 2000 analyses made during the course of the growing season. Assay values reflected the degree of nodulation of soybean plants and indicated a calculated seasonal N(2) fixation rate of 30 to 33 kg N(2) fixed per acre, in good agreement with literature estimates based on Kjeldahl analyses. The assay was successfully applied to measurements of N(2) fixation by other symbionts and by free living soil microorganisms, and was also used to assess the effects of light and temperature on the N(2) fixing activity of soybeans. The validity of measuring N(2) fixation in terms of C(2)H(2) reduction was established through extensive comparisons of these activities using defined systems, including purified N(2)ase preparations and pure cultures of N(2)-fixing bacteria.With this assay it now becomes possible and practicable to conduct comprehensive surveys of N(2) fixation, to make detailed comparisons among different N(2)-fixing symbionts, and to rapidly evaluate the effects of cultural practices and environmental factors on N(2) fixation. The knowledge obtained through extensive application of this assay should provide the basis for efforts leading to the maximum agricultural exploitation of the N(2) fixation reaction.
本文全面介绍了用于实验室中氮酶制剂和细菌培养物以及豆类和自由生活细菌原位固氮的敏感 C(2)H(2)-C(2)H(4)测定法的方法学、特点和应用。该测定法基于 N(2)ase 催化 C(2)H(2)还原为 C(2)H(4)、C(2)H(2)和 C(2)H(4)的气相色谱分离以及用 H(2)-火焰分析仪进行定量测量。该测定法的检测灵敏度非常高，即使痕量（1 mumumole）C(2)H(4)也能被检测到，比 (15)N 分析的灵敏度高 10(3)倍。本文还描述了一种利用注射器型测定室分析田间 C(2)H(2)-还原活性的简单、快速和有效的程序。该测定法在现场样本中的应用包括对商业种植大豆的固氮能力进行评估，该评估是在生长季节进行了 2000 多次分析的基础上进行的。测定值反映了大豆植株的结瘤程度，并表明计算出的季节性 N(2)固定率为每亩 30 至 33 公斤 N(2)，与基于凯氏定氮分析的文献估计值非常吻合。该测定法成功地应用于其他共生体和自由生活土壤微生物的固氮测量，也用于评估光照和温度对大豆固氮活性的影响。通过使用定义明确的系统（包括纯化的 N(2)ase 制剂和纯培养的 N(2)固定细菌）对这些活性进行广泛比较，证明了用 C(2)H(2)还原来测量 N(2)固定的有效性。有了这个测定法，现在可以对 N(2)固定进行全面调查，可以对不同的 N(2)固定共生体进行详细比较，并可以快速评估文化实践和环境因素对 N(2)固定的影响。通过广泛应用这种测定法获得的知识将为最大限度地利用 N(2)固定反应的农业应用提供基础。
