Nebraska Redox Biology Center Educational Portal

Nitric Oxide

NO is a small uncharged free radical containing one unpaired electron [ 1, 2, 3 ].

NO biosynthesis is carried out from L-arginine and is catalyzed by NO-synthases (NOS). In the presence of cofactors ( NADPH, oxygen, iron, tetrahydrobiopterine, FAD and FMN), enzyme NO-synthases, convert arginine into hydroxyarginine and finally into citrulline and NO according to the following reactions [ 4, 5, 6, 7 ]:

Essential biochemical reactions involving nitric oxide are also S-nitrosation of thiols and nitrosylation of transition metal ions [ 6, 8, 9, 10 ]. Hemoglobin structure may be altered by NO direct attachment to heme in the nitrosylation reaction, or by S-nitrosation of the thiol moieties, yielding S-nitrosothiols. [ 11 ].

Nitric oxide plays an important role in the maintenance of healthy blood pressure and, in turn, cardiovascular health. When arteries become clogged, they produce less nitric oxide than normal. Huge quantities of nitric oxide are produced in whole blood cells to kill invading bacteria and parasites.Nitric oxide formed in nerve cells stimulates the brain and modulate many functions, from behavior to gastrointestinal activity. [ 6, 10, 12, 13, 14, 15, 16, 17, 18 ].

Nitric oxide activates the guanylate cyclase in the smooth muscle cells and platelets risings the level of intracellular messenger cGMP. This rise causes smooth muscle relaxation and platelet aggregation inhibition, thus resulting in increasing blood flow. Nitric oxide can bind to oxi-Hb and Fe-SH complexes of other proteins, thus modulating the activity of many hepatic enzymes. NO induces hyperpolarization through the opening of K+ channels in the smooth muscles. NO work as effector essential in the antitumoral, antimicrobial, and antiviral action of activated immune cells [ 6, 10, 13, 18, 19 ].

Nitric oxide is involved in S-nitrosation of reactive cysteine thiols in the cell, thus, regulating protein function and serving as important signaling molecule [ 6, 8, 9, 10 ].

Biological roles of nitric oxide. NO can directly damage DNA, inhibit DNA repair, block apoptosis, and enhance oncogene expression. NO modulates transcription factors, especially redox-sensitive and zinc finger motifs containing transcription fac- tors. NO has also been found to contribute to angiogenesis

The major and commonly used methods for NO detection

1) Chemiluminescence technique using the reaction of NO with ozone or luminol [ 20, 21, 22, 23 ].

2) Fluorometry using a fluorescent NO indicator. DAF and DAN - like probes are commonly used for bioimaging of and quantification of nitric oxide [ 20, 21, 22 ].

Diaminofluoresceins (DAFs) were therefore designed as novel probes for NO. Conversion of DAFs to the corresponding triazole forms (DAF-Ts) by reaction with NO [ 21 ].

[ 21 ].

3) Spectrophotometry using the formation of NO hemoglobin (NO-Hb) [ 20, 21, 22 ].

4) EPR spectrometry using spin (NO) traps such as hemoglobin, organic compounds, and iron -dithiocarbamate complexes. The EPR signal of NO in biological systems is hardly detectable with a straightforward EPR technique, probably because of the short lifetime and low concentration of NO. Several spin-trapping compounds (spin traps) that selectively react with and trap NO have been developed and applied to biological NO measurements [ 20, 21, 22, 24, 25 ].

5) Electrochemical method using microelectrodes specific to NO [ 20, 21, 22 ]. Other indirect assays include spectrophotometry, fluorometry, and radiometry of stable biological metabolites of NO or L-arginine such as nitrite, nitrate, and L-citrulline

Summary for methods of NO detection.

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