TUTCRIS - Tampereen teknillinen yliopisto

TUTCRIS

Simultaneous extraction and combined bioluminescent assay of NAD+ and NADH

Tutkimustuotosvertaisarvioitu

Yksityiskohdat

AlkuperäiskieliEnglanti
Sivut175-180
Sivumäärä6
JulkaisuANALYTICAL BIOCHEMISTRY
Vuosikerta128
Numero1
DOI - pysyväislinkit
TilaJulkaistu - tammikuuta 1983
Julkaistu ulkoisestiKyllä
OKM-julkaisutyyppiA1 Alkuperäisartikkeli

Tiivistelmä

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.

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