Nitrate Reductase

We have recently described the modifications made to the DCFDA dependent fluorescence method for assaying the level of ROS in different sub-cellular fractions including mitochondria [23 (link),24 (link)]. Since mitochondrial superoxides are easily converted to hydrogen peroxide by superoxide dismutase in the mitochondrial inner membrane space and in the matrix, we used the DCFDA method (5 μM DCFDA in 1.0 mL assay containing 50 μg of proteins from mitochondria, microsomes and cytosol) as described before [24 (link)] for determination of total peroxides produced in the tissues of control and diabetic rats. The validity of the method for detection of superoxides and peroxides were tested by adding superoxide dismutase (SOD) and catalase to the reaction mixture as negative and positive controls respectively.
Nitric oxide production, based on nitric oxide synthase (NOS) activity, was measured in the control and diabetic rat tissues by using a kit 482702 from Calbiochem (CN Biosciences Inc, La Jolla, CA, USA) according to the vendor’s protocol. This assay is based on the measurement of total NO produced as the sum of both nitrate and nitrite by Griess reagent in the presence of nitrate reductase, NADPH and lactate dehydrogenase. The assay was developed in 96-well ELISA plates using 500 μg proteins from control and diabetic rat tissues and the total NO concentration was measured at 540 nm using a plate reader spectrophotometer.

The level of NO was estimated as nitrite, a NO metabolite, in control and case group samples, because NO is a highly reactive free radical gas that is a ready oxidizer and remains stored in tissues as Nitrates (NO₃^-) or Nitrite (NO₂^-). Thus, NO concentration can be estimated by measuring concentrations of NO₃^- and NO₂^- in combination. The simplest technique is the monitoring of reduction of NO₃^- to NO₂^- by nitrate reductase or metallic catalyst, followed by the calorimetric Griess Reaction to measure NO₂^- levels (nitrite levels).[8 (link)]
Sample solutions were taken in test tubes and treated with Griess reagent (1% sulphanilamide, 0.1% naphthylethylenediamine dihydrochloride and 2.5% hydrochloric acid). The colorimetric reaction was allowed to proceed for 10 min at room temperature, and optical density was measured at 550 nm using a spectrophotometer. The concentrations of nitrite were calculated from a standard curve established with serial dilutions of sodium nitrite.

The target segments of five nitrogen cycle functional genes, i.e., nitrogen-fixing enzyme (nifH), bacterial ammonia monooxygenase (AOB-amoA), archaeal ammonia monooxygenase (AOA-amoA), nitrate reductase (narG), and nitrite reductase (nirK), were quantified by qPCR. In Table 1, the specifics of the primer sequences for the nitrogen cycle functional genes are displayed.
An ABI Model 7,300 Fluorescent Quantitative PCR instrument (Applied Biosystems, United States) and ChamQ SYBR Color qPCR Master Mix (2X) reagent (Nanjing Novozymes Biotechnology Co., Ltd.) were used to perform real-time fluorescence quantitative PCR (q-PCR). The final reaction mixture volume for the quantification technique was 20 μl and contained 10 μl 2X Taq Plus Master Mix, 1 μl template DNA, 0.8 μ primer F (5 μM), 8 μl primer R (5 μM), and 7.4 μl ddH₂O. Each qPCR reaction was run in triplicate for each set of primers, and the non-template control served as a negative control. Furthermore, q-PCR was carried out using a three-step thermal cycling method that involved 35 cycles of 95°C pre-denaturation for 5 min, 95°C denaturation for 30 s, 58°C annealing for 30 s, and 72°C extension for 1 min (Li et al., 2022 (link)). Standard curves were created by diluting pMD18-T, a plasmid for five functional genes, in a 10-fold gradient. Additionally, the R² value of each standard curve exceeded 0.95, demonstrating a linear relationship throughout the concentration range used in this work, with amplification effectiveness ranging from 89.28 to 103.81% (mean 96.31%).

The remaining DNA samples used for sequencing in Section 2.3 were used for quantitative real-time PCR (q-PCR) analysis. The key genes, including narG (encoding the membrane-bound nitrate reductase), napA (encoding the periplasmic nitrate reductase), nirK (encoding the copper-containing nitrite reductase), nirS (encoding the haem-containing nitrite reductases), norB (encoding nitric oxide reductase), and nosZ (encoding nitrous oxide reductase), were further quantified by q-PCR using a ChamQ SYBR Color qPCR Master Mix (2X) with an ABI PRISM 7300 Sequence Detection System (Applied Biosystems, USA), and were conducted in triplicate in different experimental groups. Each PCR tube (20 μl) contained 10 μl 2X ChamQ SYBR Color qPCR Master Mix (Nanjing Novizan Biotechnology Co., LTD, China), 2 μl DNA, 0.8 μl each of forward and reverse primer, 0.4 μl ROX Reference Dye II (50×), and sterile ddH₂O to a total volume of 20 μl. The primers used for the PCR amplification of each gene are listed in Table 2.