An Improved Method for Extraction and Measurement of the Inorganic Pyrophosphate in Leaves of Crassulacean Acid Metabolism (CAM) Plants

Abstract

An improved method for extraction and measurement of inorganic pyrophosphate (PPi) in the leaves of crassulacean acid metabolism (CAM) plants, Ananas comosus (pineapple), Kalanchoe pinnata and K. dagremontiana is described. Leaf samples were extracted with 5% (w/v) trichloroacetic acid (TCA) dissolved in water. The extracts without neutralizing with KOH or K₂CO₃ and those without washing with diethyl ether to remove TCA were directly used to measure PPi with PPi-dependent phosphofructokinase (PPi-PFK, EC 2. 7. 1. 90) coupled with the oxidation of NADH. The method is sensitive and simple, the lower limit of the measurement was less than 1 nmol PPi mL^-1 reaction mixture and the recoveries of the PPi added were more than 96%. Moreover, the initial PPi levels in TCA extracts could be maintained for at least one day. This method was advantageous for extracting and measuring a large number of samples at the same time. In addition, we found that neutralizing the extract before determination was disadvantageous to PPi recovery.