Summary
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1.
The red and purple formazans of NT, deposited in sections as a result of enzyme activity, have absorption characteristics in solution identical to the red intermediate half-formazan and the purple diformazan produced chemically by ascorbate reduction. The red formazan precipitated in sections can be reduced further to the purple diformazan.
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2.
The total amount of hydrogen generated in a dehydrogenase reaction can be determined by eluting both formazan derivatives into DMP and measuring at a single (isobestic) wavelength.
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3.
In a solution which contains both formazans, each formazan may be quantified separately from measurements made at two wavelengths, 460 nm and 600 nm. Alternatively, the two formazans can be eluted separately into different solvents.
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4.
In serial sections reacted for glucose-6-phosphate dehydrogenase, the total activities, determined by each of these elution methods (i.e. measurement at the isobestic wavelength; measurement at two wavelengths; measurement of each component separately), were identical.
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5.
The total amount of hydrogen, reflected by the total amount of the formazan derivatives, can also be measured directly in the section by scanning and integrating microdensitometry at 585 nm, the isobestic wavelength of the deposited formazans. In addition, the formazans can be separately quantified by measuring at two wavelengths, 520 nm and 620 nm.
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6.
Identical results were obtained for the dehydrogenase activity of sections determined first by microdensitometry and then by elution and spectrophotometry.
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7.
An incubation/time curve for glucose-6-phosphate dehydrogenase activity showed that initially the red half-formazan was the main product. The production of this half-formazan began to tail off after about 5 minutes, at which time formation of the purple diformazan increased. The total activity, calculated from the sum of the two formazan derivatives, was linear with time up to at least 12.5 minutes.
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Abbreviations
- NT:
-
2,2′-(p-diphenylene)-bis (3,5-diphenyl) ditetrazolium chloride. (Neotetrazolium chloride)
- DMF:
-
Dimethyl formamide
- MIE:
-
Mean integrated extinction
- TLC:
-
Thin layer chromatography
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Butcher, R.G., Altman, F.P. Studies on the reduction of tetrazolium salts. Histochemie 37, 351–363 (1973). https://doi.org/10.1007/BF00274970
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DOI: https://doi.org/10.1007/BF00274970