Estimation of Nitrogenase Using a Colorimetric Determination for Ethylene

Abstract

Ethylene is measured by oxidizing it to formaldehyde and determining the formaldehyde colorimetrically. The assay is applied to estimation of nitrogenase in nodulated legume roots by measuring the ethylene produced from acetylene.flask is agitated vigorously on a rotary shaker at 300 rpm for 90 min at room temperature (22 C). One-fourth ml of 4 M NaAsO2 and one-fourth ml of 4 N H2SO are added, mixing to destroy excess oxidant. One ml of Nash reagent is added, and the absorbance at 412 nm is determined after 60 min. Standards containing known amounts of C2H4 are carried through the analysis at the same time as the samples. RESULTSIn 1966, Dilworth (2) reported that nitrogen-fixing extracts of Clostridium pasteurianum reduced acetylene to ethylene. It was soon apparent that this provided a convenient and sensitive assay of nitrogenase, for ethylene and acetylene are readily separated and determined by gas chromatography. The acetylene reduction assay is now well established in the study of biological nitrogen fixation. Its utility is attested to by its reported use in over 200 publications (3).Until now gas chromatography has been the only means of measuring ethylene in the presence of acetylene. This publication reports a colorimetric assay for ethylene sufficiently sensitive to estimate nitrogenase in nodulated legume roots. In designing the procedure, stress was placed on economy, safety, and simplicity. All reagents are inexpensive, none are particularly toxic or dangerous to the operator, and no heating steps are necessary.MATERIALS AND METHODS Distilled water should be used in making up all reagents. Sodium metaperiodate: 0.05 M NaIO4, 16.5 g/l; store in a dark bottle for no more than 4 weeks. Potassium permanganate: 0.005 M KMnO4, 0.79 g/l; store in a dark, well stoppered bottle. A slight precipitate usually forms after 1 to 2 days; if there is much precipitate, use another lot of KMnO4. Sulphuric acid: 4 N. Sodium arsenite: 4 M, NaAsO2, 52 g/ 100 ml. Oxidant solution: 80 ml of 0.05 M NaIO4, 10 ml of 0.005 M KMnO4, adjust pH to 7.5 with KOH, dilute to 100 ml. Acetyl acetone (2, 5-pentanedione): purify by distillation. Nash reagent (1, 6): 150 g of ammonium acetate, 3 ml of acetic acid, 2 ml of acetyl acetone diluted to 1 liter.Procedure. Oxidant solution (1.5 ml) is placed in a 10-ml conical flask which is then sealed with a rubber serum bottle cap. From 1 to 5 ml of gas containing up to 1 ,umole of C,H4 are transferred by syringe from a chamber in which plant roots are incubated with C2H2. If necessary, some of the atmosphere in the conical flask is removed first with a syringe. The conical 'NRCC No. 13011.

Response of nitrogen fixation, nodule activities, and growth to potassium supply in water‐stressed broad bean

“…Viciafaba plants were sampled from each treatment five weeks after starting the water stress. Analyses Nitrogenase activity (C2H2 reduction) was determined on a detached roots in a closed system as described previously (Abd-Alla, 1992) using the method of LaRue and Kurz (1973). Subsequently, nodule number per plant was recorded.…”

Seasonal Variation in Nitrogen Fixation by Different Ages of Root Nodules of Alnus nepalensis Plantations, in the Eastern Himalayas

“…Nitrogenase activity was estimated by acetylene reduction (Stewart, Fitzgerald & Burris 1967) and calorimetric determination of ethylene (La Rue & Kurz 1973). Standardization of the above method was tested repeatedly and experimental standard error was in the range 0.5-3%.…”

Differential Response of Faba Bean (Vicia Faba L.) Plants to Water Deficit and Waterlogging Stresses

“…Nodule number, nodule fresh and dry weights were recorded. The activity nitrogenase enzyme was measured as described previously by Abd-Alla (1992) using the method recorded by Larue and Kurz (1973). Save each bottle at room temperature with 5% (T/T) acetylene for 1 hour and the emitted ethylene in the reaction bottle was measured by a gas chromatograph (Thermo Scientific TRACE GC Ultra equipped with FID detector).…”