Acetylene reduction assay

Acetylene-reduction assay Estimates nitrogenase activity by measuring the rate of acetylene reduced to ethylene. 

The development of the acetylene reduction assay to measure nitrogenase activity. 

Seitzinger, S. P., and Garber, J. H. (1987). Nitrogen fixation and 15N2 calibration of the acetylene reduction assay in coastal marine sediments. Mar. Ecol. Prog. Ser. 37, 65—73. 

Patriquin, D. G., and McClung, C. R. (1978). In situ acetylene reduction assays of nitrogenase activity associated with the emergent halophyte Spartina alternijlora Loisel Methodological problems. Mar. Biol 47, 22-242. 

Nj-fixing activity by acetylene-reduction assay (micromoles of ethylene produced). Data averaged for seed treatment with cobalt (Co) at 15 mg per liter of seed and for control (-Mo) and treatment (+Mo) at 130mg Mo + 15mg Co per liter of seed. 

Nitrogen Fixation Rates (Estimated Acetylene Reduction Assay) in Select Wetland Ecosystems... 

N2 fixation was evaluated by the acetylene reduction assay of excised nodules (6). 

This new assay technique was developed independently by American and Australian workers who first established the connection between nitrogenase and hydrogenase, and showed the former could reduce a number of substrates other than nitrogen, for example nitrous oxide, azide, cyanide and, most usefully, acetylene. The latter is reduced to ethylene and this forms the basis of the acetylene reduction test. The material to be examined is briefly gassed with acetylene and the ethylene formed measured by gas chromatography. While this technique was being refined,... 

These earlier and more recent field measures all demonstrate the importance of these DDAs on the local conditions, however, in most experiments the collection of samples used towed nets, and thus are rather disruptive. Two experiments by Mague etal. (1974, 1977) found that preparing samples by concentration caused a significant (17—29%) reduction in acetylene reduction activity. It seems that more attention or creative sampling schemes need to be developed to accurately measure the N2 (and likely carbon) fixation by these DDAs. Studies similar to those presented by Zehr et al. (2007) and Needoba et al. (2007), which combine uptake rates with quantitative PCR approaches for the target diazotrophs are a plausible alternative since assays are run on bulk water. 

Light-induced nitrogenase activity measured as hydrogen evolution and/or acetylene reduction in the absence of DCMU was seen when CO2 evolution was evident. Apparently, nitrogenase activity ceased when substrates for respiration were exhausted. CO2 evolution was then replaced by uptake, and oxygen evolution showed up in the assay. 

TABLE 1. Photosynthetic oxygen evolution (A), respiratory oxygen consumption in the dark (B), chlorophyll a content (C)acetylene reduction (D) and ammonia excretion (E) of Nostoc 268 grown for 9 days (7 days for the assay of acetylene reduction) photoautotrophically on CO or mixotrophically on various carbon sources. The cultivation took place as described in the legend to Fig 1. 

A major difference between the V and Mo enzymes lies in substrate specificity and product formation.As is clearly shown in Table 7.9, the FeV nitrogenase has a much lower reactivity toward acetylene than does the Mo system. Furthermore, whereas the FeMo system exclusively produces ethylene from acetylene, the FeV system yields significant amounts of the four-electron reduction product, ethane. The detection of ethane in the acetylene assay may... 

Nitrogenase is a complex enzyme which catalyzes the reduction of a variety of substrates (Table IV) with the concomitant hydrolysis of ATP. It contains two, easily separable, iron-sulfur proteins one of which contains molybdenum. These require a reductant, a reducible substrate, an ATP-generating system, and an anaerobic environment to function. The ATP-generating system is necessary because ADP inhibits nitrogenase activity. Two important discoveries have helped to simplify the assay for nitrogenase. First, sodium dithionite proved to be an adequate reductant to replace the complex and unstable pyruvate phosphoroclastic system (Bulen et al., 1965) and, second, nitrogenase reduces acetylene to ethylene (Dilworth, 1966 Schollhorn and Burris, 1967) which can be measured at nanomolar concen-... 

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