Quantification of polyamines losses during manipulation assay procedures

Losses in polyamine recovery during manipulation and assay procedures have been detected. They are partly due to the binding of polyamines at low concentration (around 5-mu-M for putrescine and 100-mu-M for spermine) to the containers and depend on the chemical nature of the latter, on the pH of the solution and on the length of time for which they remain in contact with the container. The recovery of labelled polyamines after dansylation was about 45% of the initial radioactivity. Remarkably high losses occurred when bound polyamines, obtained by in vitro incubation with cell-free extracts, were estimated after acidic hydrolysis. Standard free polyamines, subjected to acidic hydrolysis conditions, yielded a 50-60% recovery, both measured as radioactivity and fluorescence, compared to standard polyamines not subjected to hydrolysis. If carbohydrates are present, the recovery after hydrolysis can be even lower, depending on the sequence in which the reagents of the dansylation procedure are added. Ion exchange chromatography to remove carbohydrates improved this recovery.