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A radiometric method for the determination of NADH in subpicomole amounts

Authors

  • G. Weber
  • W. Rosenthal
  • E. Oberdisse

Journal

  • Journal of Biochemical and Biophysical Methods

Citation

  • J Biochem Biophys Methods 15 (6): 295-306

Abstract

  • A radiometric method has been devised for the determination of small quantities of NADH formed in preceding dehydrogenase reactions. In a coupled enzymatic reaction, phosphoglycerate kinase (PGK) catalyzes the transfer of [32P]orthophosphate from [gamma-32P]ATP to 3-phosphoglycerate; the intermediate, 1,3-[1-32P]diphosphoglycerate, is dephosphorylated by glyceraldehyde-3-phosphate dehydrogenase (GAP-DH). [32P]Orthophosphate is released proportionally to NADH and can be measured after adsorption of [gamma-32P]ATP to activated charcoal. With this method, 0.2 pmol of NADH are detectable in the presence of a 10(4)-fold excess of NAD over NADH.


DOI

doi:10.1016/0165-022X(88)90083-8