Extraction, purification, and estimation of ATP from leaves, floral buds, and immature fruits of cotton

Several procedures for extracting ATP from cotton leaves, floral buds (squares), and immature fruits (bolls) were compared. Highest indicated yields of ATP were obtained by extraction with boiling 95% ethanol followed by water or dilute EDTA at 25°C. A potent inhibitor of luciferase luminescence was extracted from carpel walls of bolls and, to a lesser extent, from squares and leaves. Inhibition was partially overcome by adding bovine albumin and soluble polyvinylpyrrolidone to the crude enzyme preparation prior to assay. Much of the inhibitory material, presumably phenolic, could be removed by sorption onto a short column of Polyclar AT, an insoluble polyvinylpyrrolidone. ATP was not sorbed and readily passed through the column. None of the extraction methods tested gave 100% recovery. Addition of a known amount of ATP to one set of duplicate samples before extraction permits correction for losses during extraction and for partial quenching of luminescence.