QUANTITATIVE EXTRACTION AND ESTIMATION OF INTRACELLULAR NICOTINAMIDE NUCLEOTIDES OF ESCHERICHIA-COLI

The conditions of extraction of nicotinamide nucleotides from Escherichia coli were systematically investigated. The optimum pH for NAD+ and NADP+ appeared to be around 1.5 while that for NADH and NADPH was around 12.5. Seven minutes at 60°C was chosen for the extraction of all four nucleotides. Addition of CuCl to the alkaline extract improved considerably the recovery of the reduced forms. Two sensitive (10-100 pmol) assay methods for nicotinamide nucleotides were compared. A spectrophotometric cycling method based on the measurement of reduced thiazolyl blue formation appeared to be more sensitive and more reproducible and gave better recovery than a fluorometric cycling method used earlier in bacterial studies. © 1979.