拟南芥粗提物中硝酸还原酶体外活性测定

Joo Yong  Kim; Hak Soo Seo

doi:10.21769/BioProtoc.2785

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Peer-reviewed

In vitro Nitrate Reductase Activity Assay from Arabidopsis Crude Extracts

拟南芥粗提物中硝酸还原酶体外活性测定

JK Joo Yong Kim

HS Hak Soo Seo email

发布: 2018年04月05日第8卷第7期 DOI: 10.21769/BioProtoc.2785 浏览次数: 9426

评审: Renate WeizbauerVenkatasalam ShanmugabalajiAswad Khadilkar

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Abstract

Nitrate reductase (NR) reduces the major plant nitrogen source, NO₃^-, into NO₂^-. NR activity can be measured by its final product, nitrite through its absorbance under optimized condition. Here, we present a detailed protocol for measuring relative enzyme activity of NR from Arabidopsis crude extracts. This protocol offers simple procedure and data analysis to compare NR activity of multiple samples.

Keywords: Nitrate reductase (硝酸盐还原酶)

in vitro NR activity assay (体外硝酸盐还原酶活性测定)

Nitrite concentration (亚硝酸盐浓度)

Background

Nitrogen is crucial macronutrient required by plants and is mainly absorbed in the form of nitrate. Nitrate reductase is the first enzyme of the nitrogen assimilation in higher plants. Homodimers of plant nitrate reductase catalyze the NAD(P)H-dependent reduction of nitrate to nitrite as follows:

NO₃^- + NADH + H⁺ → NO₂^- + NAD⁺ + H₂O

Methods to measure NR activity may be a powerful tool to investigate biological factors influencing NR activity (Park et al., 2011). Nitrogen assimilation affects the contents of amino acid in plant, thus regulating NR activity could be used for increasing quality of some crop (Croy and Hageman, 1970; Dalling and Loyn, 1977; Ruan et al., 1998).

In this protocol, increased nitrite concentrations during limited time in optimized buffer condition are acquired as comparable values. Nitrite concentration is measured by its absorbance at 540 nm through Griess assay. Briefly, nitrite forms a diazonium salt with sulfanilic acid, then N-(1-naphthyl) ethylenediamine dihydrochloride is formed colored azo compound. It is possible to compare the values to determine how samples have different NR activity. Furthermore, the values could be converted to exact increased nitrite concentration through a simple process.

Materials and Reagents

3M^TM Micropore^TM surgical tape (3M, Micropore^TM, catalog number: 1530-1 )
Reaction tube, 1.5 ml (Greiner Bio One International, catalog number: 616201 )
150 x 25 mm (d x h) plastic Petri dish (SPL Life Sciences, catalog number: 10151 )
Cuvette (Ratiolab, catalog number: 2712120 )
Arabidopsis seeds
Ethanol (Merck, EMSURE^®, catalog number: 1009831011 )
Liquid nitrogen
Potassium nitrite (Sigma-Aldrich, catalog number: P8394 )
Murashige & Skoog medium including vitamins (Duchefa Biochemie, catalog number: M0222 )
MES monohydrate (Duchefa Biochemie, catalog number: M1503 )
Potassium hydroxide (Merck, catalog number: 814353 )
Sucrose (Duchefa Biochemie, catalog number: S0809 )
Plant agar (Duchefa Biochemie, catalog number: P1001 )
Tris-HCl (Duchefa Biochemie, catalog number: T1513 )
Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: EDS )
Sodium molybdate dihydrate (Na₂MoO₄·2H₂O) (Sigma-Aldrich, catalog number: M1003 )
Flavin adenine dinucleotide disodium salt hydrate (FAD-Na₂) (Sigma-Aldrich, catalog number: F8384 )
Dithiothreitol (DTT) (Duchefa Biochemie, catalog number: D1309 )
Bovine serum albumin (BSA) (Merck, Probumin^®, catalog number: 821006 )
2-Mercaptoethanol (Sigma-Aldrich, catalog number: M6250 )
Phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: 78830 )
Sodium nitrate (NaNO₃) (Sigma-Aldrich, catalog number: S5506 )
Sodium phosphate dibasic (Na₂HPO₄) (Bio Basic, catalog number: S0404 )
Sodium phosphate monobasic, anhydrous (NaH₂PO₄) (Bio Basic, catalog number: SB0878 )
Nicotinamide adenine dinucleotide (NADH) (Sigma-Aldrich, catalog number: 43420 )
Hydrochloric acid (HCl) (DAEJUNG CHEMICAL & METALS, catalog number: 4090-4405 )
Sulfanilamide (Sigma-Aldrich, catalog number: S9251 )
N-(1-naphthyl) ethylenediamine dihydrochloride (Sigma-Aldrich, catalog number: 222488 )
MS agar media (see Recipes)
Extraction buffer (see Recipes)
Reaction buffer (see Recipes)
1% sulfanilamide solution (see Recipes)
0.05% N-(1-naptyl) ethylenediamine hydrochloride (see Recipes)

Equipment

Pipette kit (Gilson, model: PIPETMAN^® Classic, catalog number: F167300 )
2 L flask (DWK Life Sciences, Duran^®, catalog number: 21 216 63 )
Arabidopsis growth chamber (Hanbaek, model: HB-301L-3 )
Stainless steel tweezers
Mortar & pestle (Silico & Chemico Porcelain Works, catalog number: J-753 )
Centrifuge (Thermo Fisher scientific, model: Sorvall^TM Legend^TM 17 , catalog number: 75002431)
Spectrophotometer (Biochrom, model: Libra S22 )
Magnetic stirrer (Vision Scientific, model: VS-130SH )
Stirring bar
pH meter (Fisher Scientific, model: accumet^TM AB15 )
Autoclave (Hanbaek, model: HB-506-6 )

Procedure

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Kim, J. Y. and Seo, H. S. (2018). In vitro Nitrate Reductase Activity Assay from Arabidopsis Crude Extracts. Bio-protocol 8(7): e2785. DOI: 10.21769/BioProtoc.2785.