Rapid Enzymatic-HPLC Measurement of the Content of Major Nucleotides in Vegetables

Abstract

A simple and rapid method was developed for measurement and identification of the nucleotides in vegetable RNA. The reliability of the method was verified by analysis of yeast RNA of known nucleotide composition. Natural enzymes of the vegetables involved in degradation of RNA were inactivated by zinc chloride during vegetable homogenization followed by a heat treatment. The total endogenous RNA was then hydrolyzed in situ by nuclease Pl enzyme at 60°C for 1 hr. The hydrolysate was extracted with cold perchloric acid followed by a Freon-octylamine extraction step to remove the acid before HPLC analysis. The four major nucleotides in the RNA hydrolysate were then identified by their isocratic separation using a Partisil 10 SAX anionic column and 3% methanol-8 mM K-phosphate buffer of pH 4.15 as an eluent. An alternative alkaline rather than enzymatic hydrolysis of vegetable RNA in situ was found to be unsuitable for HPLC analysis.