Nitrite Reduction Assay for Whole Pseudomonas Cells
全假单胞菌细胞的亚硝酸盐还原试验
发布: 2016年05月20日第6卷第10期 DOI: 10.21769/BioProtoc.1818 浏览次数: 11893
评审: Valentine V TrotterGeneviève BallAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Aug 2015
Abstract
The second step of the dissimilatory denitrification pathway in which nitrite (NO2-) is converted to nitric oxide (NO) is catalyzed by the enzyme nitrite reductase. Two distinct enzymes are found in nature that catalyze this reaction, and they contain different metal sites, either iron (Fe), in the form of heme, or copper (Cu) (Zumft, 1997). The Pseudomonas stutzeri (P. stutzeri) RCH2 strain used in this assay contains both an Fe and a Cu form of nitrite reductase. In this assay, total nitrite reductase activity can be measured in whole cells using fumarate or some other carbon source as an electron source by measuring the disappearance of nitrite over time (Thorgersen et al., 2015).
Keywords: Nitrite reductase (亚硝酸还原酶)
Pseudomonas (铜绿假单胞菌)
Griess (格里斯)
Materials and Reagents
- Borosilicate glass culture tubes, 16 x 125 mm (VWR International, catalog number: 47729-578 )
- Hungate tubes (16 x 125 mm) with butyl rubber stopper (Bellco Glass, catalog number: 2047-16125 )
- Pseudomonas cells
- M9, minimal salts, 5x (Sigma-Aldrich, catalog number: M6030 )
- Bacto yeast extract technical (BD Biosciences, catalog number: 288610 )
- Potassium phosphate (dibasic, powder) (VWR International, J.T. Baker®, catalog number: 3252-05 )
- Potassium phosphate (monobasic, crystal) (VWR International, J.T. Baker®, catalog number: 3246-05 )
- Sodium fumarate dibasic (Sigma-Aldrich, catalog number: F1506 )
- Sodium nitrate (VWR International, J.T. Baker®, catalog number: 3770-01 )
- Sodium nitrite (VWR International, J.T. Baker®, catalog number: 3780-01 )
- Griess reagent (Sigma-Aldrich, catalog number: G4410 )
- High purity 100% argon gas (Airgas, catalog number: ARHP300 )
- 50 mM potassium phosphate buffer (pH 7.0) (see Recipes)
- Assay buffer (see Recipes)
Equipment
- Allegra 25R centrifuge (Beckman Coulter)
- Spectrophotometer (measuring absorption in the visible range)
- Innova 4230 refrigerated incubator shaker (New Brunswick Scientific)
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Thorgersen, M. P. and Adams, M. W. W. (2016). Nitrite Reduction Assay for Whole Pseudomonas Cells. Bio-protocol 6(10): e1818. DOI: 10.21769/BioProtoc.1818.
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分类
微生物学 > 微生物生物化学 > 蛋白质 > 活性
生物化学 > 蛋白质 > 活性
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