Nitric oxide (NO) is a biological signaling and effector molecule involved in many biological functions, including maintaining blood pressure, modulating neural transmissions and stimulating immune responses. Nitric oxide is also implicated in the regulation of hypoxia-inducible factor 1?, a transcription factor that is a key regulator of oxygen homeostasis1, 2 (see TransAM® HIF-1). Consequently, Nitric oxide has become the subject of extensive research.
Figure 1: Dynamic range of nitric oxide standard curves.
Nitrate standard curves prepared using the Nitric Oxide Quantitation Kit and a conventional LDH-method kit. The Active Motif method provides linear results over a wider range.
- Huang, LE et al. (1999) J. Biol. Chem. 274: 9038-9044.
- Liu, Y et al. (1998) J. Biol. Chem. 273: 15257-15262.
Principle behind traditional nitric oxide assays
Nitric oxide has an extremely short half-life (< 10 seconds), which makes it difficult to detect and study directly. However, because nitric oxide is metabolized to nitrite and nitrate, quantitation of these stable anions can be used to indirectly determine the amount of nitric oxide originally present.
Typically, nitric oxide assays are performed using either a two-step assay or a three-step lactate dehydrogenase (LDH) assay. In both methods, the first step is the reduction of nitrate into nitrite by nitrate reductase. In the final step, Griess Reagent converts the nitrite into a purple-colored azo compound, which is quantitated by spectrophotometer at A540 (Figure 1). However, excess NADPH, an essential cofactor in nitrate reduction, interferes with the Griess reaction, limiting the sensitivity of the two-step assay. To improve sensitivity, an LDH step that degrades NADPH can be added before the Griess Reagent step, resulting in the more time consuming, more complicated three-step LDH assay.
Figure 1: The principle of nitric oxide assays.
Nitric oxide is converted to nitrite, then assayed using Griess Reagent and a spectrophotometer.
Unique features of the Nitric Oxide Quantitation Kit
Active Motif’s Nitric Oxide Quantitation Kit offers advantages in time, ease-of-use and accuracy over the methods described above. It employs an innovative cofactor technology that accelerates the conversion of nitrate to nitrite while simultaneously degrading NADPH. Thus, the reductase reaction is reduced to only 30 minutes and sensitive colorimetric quantification can be performed without the need for LDH treatment. Moreover, the Active Motif method is linear over a broader dynamic range, which increases the range of sample concentrations that can be measured accurately (Figure 1). This means that you’ll need to dilute and re-assay fewer samples. For additional information, please download the Nitric Oxide Quantitation Kit manual.
Advantages of the Nitric Oxide Quantitation Kit