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My Master's research investigated the kinetics of Nitric Oxide (NO) production by Suberoylanilide Hydroxamic Acid (SAHA, vorinostat) and other Histone deacetylases (HDAC) inhibitors, using UV-Vis Spectrometry. SAHA is known to cause growth arrest and death of a broad variety of transformed cells both in vitro and in vivo at concentrations not toxic to normal cells.

HDACs are a family of enzymes involved in the regulation of gene expression, DNA repair, and stress response. These processes often are altered in tumors, and HDAC inhibitors have had pronounced antitumor activity with promising results in clinical trials.

Histone deacetylase HDAC7 with SAHA at the catalitic site

The role of Nitric Oxide in the immune system

Protective and toxic effects of NO are frequently seen in parallel. There is no simple, uniform picture of the function of NO in the immune system. It is involved in the pathogenesis and control of infectious diseases, tumors, autoimmune processes and chronic degenerative diseases.

With its role as a pleiotropic regulator, NO is critical to numerous biological processes of interest for cancer research, including macrophage-mediated immunity. The modulation of NO levels might be therapeutically desirable depending on the condition and this poses considerable challenges for drug development.

Nitrite detection using the Griess Assay

The Griess Reaction

NO has a half-life of less than 30 seconds, and reacts rapidly with free oxygen, oxygen radicals, redox metals, sulphydryls, disulfides and oxygenated hemoglobin. [Stamler JS, Singel DJ, Loscalzo J, Science, 1992]

The stable degradation products of NO, nitrite (NO2) and nitrate (NO3), accumulate in solution and can be measured using the Griess reaction. The detection limit of the technique is in the micromolar range and the concentrations of nitrite and nitrate can be assessed as an index of NO production. [Griess JP, Ber Deutsch Chem Ges. 1879]

The Griess reaction is based on the two-step diazotization reaction in which acidified NO2 produces a nitrosating agent which reacts with sulfanilic acid to produce the diazonium ion. This ion is then coupled to N-(1-naphthyl) ethylenediamine to form the chromophoric azo-derivative which absorbs light at 540 nm.

Me using the NanoDrop 2000 Spectrometer (Thermo Scientific)

Griess JP, Ber Deutsch Chem Ges. 1879, 12: 426

Stamler JS, Singel DJ, Loscalzo J, "Biochemistry of nitric oxide and its redox-activated forms" Science, 1992, 258: 1898-1902

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