Iodonitrotetrazolium chloride,INT

2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride; p-Iodonitrotetrazolium violet

Tetrazolium dye precursor that forms a purple formazan dye on reduction. Used as an electron acceptor for the colorimetric assay of dehydrogenase activity. Also used for western blot staining and as a staining agent for soft agar colony formation assays.

Iodonitrotetrazolium (INT) (chloride) is a monotetrazolium salt used as an indicator dye. It is reduced to an insoluble formazan that is used as a vital dye or indicator of cellular redox activity. Reduction commonly results from the activity of dehydrogenases, although non-enzymatic electron transfer reactions can occur in the presence of an intermediate electron acceptor. INT is commonly used to measure the respiratory activity of microorganisms in a variety of contexts.

The monotetrazolium salt  Iodonitrotetrazolium chloride (INT), first described by Atkinson et al. (1950), is an iodinated derivative of TTC (2,3,5-Triphenyltetrazolium chloride) with an absorption max of 248 nm. The color produced by reduction of this iodonitro compound is a red to purple or brownish formazan with an absorption maximum at 485 to 492 nm (depending on the solvent). INT is often used to visualize dehydrogenase activity histochemically as well as in Western blot staining and as a staining agent for agar colony formation assays.

 NAD or NADP-dependent dehydrogenases activity assays: NAD+- or NADP+-dependent dehydrogenase transfers electrons from a substrate (the electron donor, e.g. succinate) to NAD+ or NADP+, forming NADH and NADPH, respectively. Then, the final transfer of hydrogen to the tetrazolium salt is catalyzed by NADH or NADPH dehydrogenases (e.g. succinate dehydrogenase), leading to the reduction of INT and formation of the water-insoluble INT-formazan. 1 mol of formazan is produced for each mole of succinate utilized in the presence of INT. It is importan t to mention for applications in histology, that INT-formazan precipitates at the site of this latter dehydrogenase but not at the site of the original NAD+- or NADP+-dependent dehydrogenase activity.