Determination of nitric acid reductase vitality in plants (ionosome method)

Principle Nitrate reductase (NR) is a key enzyme for plant nitrogen assimilation, which catalyzes nitrates in plants to be reduced to nitrites: nitrites produced byand parasyl sulphate (or amino sulphonamide) and α-vinyl ethylene diamines produce quantitative red adrenaline
compounds
under acidic conditions. The reaction is as follows:a red ad-nitrogen compound produced by the united States has the maximum absorption peak at 540nm wavelength, which can be determined by phosphorescies. The activity of nitric acid reductase can be expressed by the amount of nitrosity nitrogen produced. It is generally in N'g-1-h-1. The determination of NR can be divided into living and ionosphere methods. The living method step is simple and suitable for fast, multi-group determination. The ionosphere method is complex, but the repetition is better.equipment centrifuges
; terra photon meters; refrigerators; research
;
test tubes
and other appliances are living with the same body method.
reagent0.1mol/L pH7.5 phosphoric acid buffer; 0.1mol/L KNO3 phosphoric acid buffer; 1% (W/V) to aminobenzene sulfonate solution; 0.2% (W/V) α-amine solution (the above solution is accompanied by a living method). 。 NADH2 solution: 2.0mg NADH2 is said to be dissolved in 1 ml of 0.1mol/L phosphoric acid buffer (refrigerator storage available for one week);extraction buffer (25mmol/L phosphate buffer, 5mmol/L cysteine, 5mmol/L
(EDTA
-Na2 mixture): Take 250 ml 0.1mol/L phosphate buffer, add cysteine 0.61g;1. Take2g of material, wash and shred, and place it in a research center to freeze 30min in a low-temperature refrigerator. Remove and add a small amount of quartz sand to the ice bath and extract the buffer (corn, wheat and rice are added twice in 1 ml, 2 ml and 4 ml per gh of fresh weight, respectively). Grinding for homogenous slurry, low temperature centrifugation 5min (4000r/min). The upper liquid is the crude enzyme extract. Try to do this in an ice bath.。 2. Take 0.4 ml crude enzyme extract, 1.2 ml 0.1mol/L KNO3 phosphoric acid buffer, 0.4 ml NADH2 solution mixed in a prepared scale test tube, insulation at 30 degrees C 30min, control without NADH2, 0.4 ml distilled water instead. 。 3. Immediately after insulation, add 1 ml of a solution of amino benzene sulfonate to terminate the reaction, add 1 ml of alpha-amine solution, color 20min, centrifuge 10min on a desktop centrifuge, and measure the light density at a wavelength of 540nm on a hydrometer.。 4. Standard curve production and result calculation of the same living method.Note:1. The nitrate reduction process should be carried out in the dark to prevent nitrites from being reduced to ammonia. Addition of isopropyl alcohol increases the
tissue
's permeability to NO3-and NO2-, and anaerobic conditions prevent oxygen competition to reduce the nucleotide
of
. 。 2. The pre-sampling material should be lighted above 3h, and the Oda sampling is appropriate after 9 a.m. and not suitable for sampling on rainy days. The sampling site should be as consistent as possible.。 3. The KNO3 phosphoric acid buffer should be kept at low temperature, otherwise it is easy to breed
microorganisms
will NO3-restore, so that the control absorbent value is high. 。 4. From color to color ratio time is consistent, too short too long has an effect on the color