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Abstract:
Direct determination of [32P]Pi by organic extraction is an efficient and versatile method for assaying phosphoprotein and phosphopeptide phosphatases and ATPases. Analysis of free [32P]Pi by the procedure of J. B. Martin and D. M. Doty [(1949) Anal. Chem. 21, 965-967] has been modified to employ isobutanol/toluene in place of isobutanol/benzene. Extraction with either toluene or xylene equaled that with benzene. A complete characterization of the extraction procedure, which has been scaled down to a volume suitable for most enzyme assays, shows that it is accurate, rapid, and sensitive to as little as 1 pmol [32P]Pi. The phosphomolybdate complex is extracted reproducibly and quantitatively in the absence and presence of protein (1-8 mg/ml). In addition, sample volumes of 4 to 100 microliter were extracted with equal efficiency. Benzene offers no advantages over toluene and presents a considerably greater health risk. The experiments demonstrate conclusively that toluene can and should be employed in place of benzene in the organic extraction of Pi.