PT - JOURNAL ARTICLE AU - MILDRED K. BUENING AU - MICHAEL R. FRANKLIN TI - Limitations in the Use of the 340 nm Absorbance Maximum of Reduced Nicotinamide Adenine Dinucleotide Phosphate for the Determination of Oxidation Rates and Stoichiometry during Rat Hepatic Microsomal Metabolism DP - 1974 Nov 01 TA - Molecular Pharmacology PG - 999--1003 VI - 10 IP - 6 4099 - http://molpharm.aspetjournals.org/content/10/6/999.short 4100 - http://molpharm.aspetjournals.org/content/10/6/999.full SO - Mol Pharmacol1974 Nov 01; 10 AB - During rat hepatic microsomal oxidative metabolism NADPH not only is oxidized to NADP+ but is cleaved to reduced nicotinamide mononucleotide. The 340 nm absorbance maximum of NMNH interferes with the use of the 340 nm absorbance as a measure of NADPH oxidation. The pyrophosphatase responsible for NADPH cleavage can destroy 30% of the added NADPH in the absence of exogenous mixed-function oxidase substrates in microsomes from phenobarbital-treated rats. It is inhibited about 75% by 2 mM 5'-AMP and 25 mM sodium pyrophosphate and about 30% by 10 mM sodium fluoride. Using pyrophosphatase inhibitors, and allowing for the turbidity enhancement of the 340 nm absorbance, the stoichiometry of NADPH oxidized to oxygen consumed approaches 1:1, especially in the presence of exogenous mixed-function oxidase substrates.