THE PRODUCTION OF LABELED BETAINE BY INCUBATION OF OSMOLYTE-FREE PSEUDOMONAS-AERUGINOSA WITH RADIOACTIVE CHOLINE

A simple, efficient, and economical method is presented for the prepar ation of radioactive betaine. It involves the incubation of radioactiv e choline with osmolyte-free Pseudomonas aeruginosa previously grown i n hyperosmolar medium with choline as an osmoprotectant. The summarize d procedure was as follows: (i) bacteria were grown in high P-i basal salt medium (HPi-BSM) with 20 mM succinate, 18.7 mM NH4Cl, 0.8 M NaCl, and 1 mM nonradioactive choline. After the bacterial pellet was obtai ned, it was suspended in deionized water to release osmolytes accumula ted during growth; (ii) suspension of the pellet, free of osmolytes, i n hyperosmolar HPi-BSM with [methyl-C-14]-choline (55 nCi/nmol) withou t the carbon and nitrogen sources. Incubation of the mixture at 37 deg rees C for 8-30 h. When only 10% of the initial radioactivity remained in the supernatant, it was withdrawn after centrifugation and the pel let suspended in deionized water. This step released the accumulated b etaine plus some contaminants. Purification of betaine contained in th e aqueous supernatant was carried out after rotoevaporation to dryness and solubilization of the residue in methanol. The methanolic extract was rotoevaporated to dryness, the residue solubilized in 10% acetic acid and transferred to a Dowex 50-X8 column. After the column was was hed with water and 2 M NH4OH, betaine was eluted by the addition of 4 M NH4OH. The total procedure for obtaining pure radioactive betaine re sulted in a yield of 80%. The product obtained was chemically and radi ochemically pure, with a specific radioactivity of 54 i: I nCi/nmol.