A HPLC Method for Simultaneous Analysis of NAD(P)+ and NAD(P)H. Its Application to the Study of Spinach Ferredoxin: NADP+ Reductase-Catalyzed Transhydrogenation

Abstract

A new HPLC system for simultaneous analysis of NAD+, NADH, NADP+, and NADPH was developed and used to measure the transhydrogenase activity of spinach ferredoxin-NADP+ reductase (EC 1.18.1.2, FNR). The system is based on a reverse-phase HPLC with isocratic elution on an ODS column (4.6 × 50 mm). The four nucleotides were completely separated by developing the column with 0.15 M sodium phosphate/citrate buffer (pH 6.8) containing 1 mM EDTA at 40°C with a flow rate of 1 ml/min. The four nucleotides can be simultaneously assayed within 13 min by monitoring the effluents with a UV detector. It was also indicated that the transhydrogenase activity of spinach FNR can be advantageously assayed by measuring the nucleotides by this method. © 1989, Japan Society for Bioscience, Biotechnology, and Agrochemistry. All rights reserved.