Aqueous soluble tetrazolium/formazan MTS as an indicator of NADH- and NADPH-dependent dehydrogenase activity

David D Dunigan, S. B. Waters, T. C. Owen

Research output: Contribution to journalArticle

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Abstract

Recently a new tetrazolium was described for the use of monitoring cell viability in culture. This tetrazolium, commonly referred to as MTS [3-(4,5- dimethylthiazol-2-yl)-5-(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H- tetrazolium, inner salt], has the unusual property that it can be reduced to a water-soluble formazan. β-Nicotinamide adenine dinucleotide/reduced (NADH) and β-nicotinamide adenine dinucleotide phosphate/reduced (NADPH) are examples of physiologically important reducing agents. In cell-free studies. MTS was reduced to the soluble formazan in the presence of NADH and NADPH, and reactions were compared to those with dithiothreitol (DTT) or 2- mercaptoethanol (2-ME). The efficiency of these reactions was enhanced 1000- fold by the presence of phenazine methosulfate. Selectivity in the electron transfer from NADPH was slightly greater than NADH, and NADPH or NADH was much greater than the thiols DTT or 2-ME. Generation of either NADH or NADPH in solution by malate dehydrogenase or isocitrate dehydrogenase, respectively, was monitored by the MTS reduction reaction. The rate of formazan formation was comparable to the formation of the NADH or NADPH. This system represents a useful tool for evaluating reaction kinetics in solutions of NAD- and NADP-dependent dehydrogenase enzymes, and these reactions can be performed in typical biological buffers containing reducing agents without significant interference to the MTS/formazan system.

Original languageEnglish (US)
Pages (from-to)640-649
Number of pages10
JournalBioTechniques
Volume19
Issue number4
StatePublished - Jan 1 1995

Fingerprint

Formazans
NADP
NAD
Oxidoreductases
Mercaptoethanol
Dithiothreitol
Reducing Agents
Methylphenazonium Methosulfate
Tetrazolium Salts
NADPH Dehydrogenase
Isocitrate Dehydrogenase
Malate Dehydrogenase
Cell culture
Sulfhydryl Compounds
Reaction kinetics
Cell Survival
Buffers
Salts
Cells
Electrons

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Aqueous soluble tetrazolium/formazan MTS as an indicator of NADH- and NADPH-dependent dehydrogenase activity. / Dunigan, David D; Waters, S. B.; Owen, T. C.

In: BioTechniques, Vol. 19, No. 4, 01.01.1995, p. 640-649.

Research output: Contribution to journalArticle

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