Shacter E
Anal Biochem. 1984 May 1;138(2):416-20. doi: 10.1016/0003-2697(84)90831-5.
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.
通过有机萃取直接测定[32P]Pi是一种用于分析磷蛋白、磷酸肽磷酸酶和ATP酶的高效且通用的方法。J. B. 马丁和D. M. 多蒂[(1949) 《分析化学》21, 965 - 967]的方法中对游离[32P]Pi的分析已进行修改，采用异丁醇/甲苯代替异丁醇/苯。用甲苯或二甲苯萃取的效果与用苯萃取相当。已将萃取程序按比例缩小至适合大多数酶分析的体积，对该程序的完整特性描述表明，它准确、快速且对低至1皮摩尔的[32P]Pi敏感。在有和没有蛋白质(1 - 8毫克/毫升)的情况下，磷钼酸络合物均可重复且定量地被萃取。此外，4至100微升的样品体积能以相同效率被萃取。与甲苯相比，苯没有优势且存在更大的健康风险。实验确凿地证明，在Pi的有机萃取中可以且应该用甲苯代替苯。
