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Simultaneous extraction and combined bioluminescent assay of NAD+ and NADH

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Details

Original languageEnglish
Pages (from-to)175-180
Number of pages6
JournalANALYTICAL BIOCHEMISTRY
Volume128
Issue number1
DOIs
Publication statusPublished - Jan 1983
Externally publishedYes
Publication typeA1 Journal article-refereed

Abstract

A new method for extracting pyridine nucleotides from tissue samples at room temperature that allows the simultaneous extraction of both the oxidized and reduced nucleotide when using a 70% buffered ethanol solution as the extractant has been developed. The extraction efficiencies for NAD+ and NADH were 91 and 102%, respectively. The extraction method was followed by a combined bioluminescent assay of both nucleotides. A bacterial bioluminescent system, which included luciferase and low levels of a NADH-specific oxidoreductase, was used to produce a constant light intensity directly proportional to the amount of NADH in the tissue extract sample. When the NADH had been measured, the NAD+ present in the extract was enzymatically converted to NADH by the addition of alcohol dehydrogenase, after which the second increase in light level was recorded. The sensitivity of the bioluminescent assay presented here is 5 X 10(-14) mol NADH or NAD+ per assay.

Keywords

  • Alcohol Oxidoreductases, Animals, Chemical Phenomena, Chemistry, Ethanol, Liver, Luciferases, Luminescent Measurements, Methods, NAD, Oxidation-Reduction, Oxidoreductases, Rats, Vibrio

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