Summary
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1.
Chromatographically pure NT has been reduced by ascorbic acid at pH 10 to produce a red formazan. This material has been isolated and purified, and shown to be a half-reduced intermediate compound.
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2.
The intermediate can be reduced further to the purple di-formazan. It has been shown that one molecule of NT reacts with 2H to give one molecule of intermediate, and that one molecule of intermediate reacts with a further 2H to give one molecule of di-formazan. In addition, one molecule of NT also reacts with 4H to give one molecule of di-formazan.
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3.
The pH of the ascorbate reducing agent is critical. In the presence of excess ascorbate, there is no reduction at all at very acid pH values; at pH values up to about 8 only the red formazan is produced, at pH values up to about 10 both red formazan and purple di-formazan are produced; at pH values in excess of 11 only the purple di-formazan is produced.
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Abbreviations
- NT:
-
2,2′-(p-diphenylene)-bis(3,5-diphenyl) ditetrazolium chloride (neotetrazolium chloride)
- BT:
-
2,2′,5,5′-tetraphenyl-3,3′(3,3′-dimethoxy-4-4′-diphenylene) ditetrazolium chloride (blue tetrazolium chloride)
- TT:
-
2,3,5-triphenyl tetrazolium chloride
- TLC:
-
thin layer chromatography/chromatogram
- EPR:
-
electron paramagnetic resonance
- NMR:
-
nuclear magnetic resonance
- TMS:
-
tetramethylsilane (standard for NMR)
- DMF:
-
dimethyl formamide
- DMSO:
-
dimethyl sulphoxide
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Altman, F.P., Butcher, R.G. Studies on the reduction of tetrazolium salts. Histochemie 37, 333–350 (1973). https://doi.org/10.1007/BF00274969
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DOI: https://doi.org/10.1007/BF00274969