Determination of inorganic orthophosphate from biological material in the presence of Ficoll

✍ Scribed by E.B. Margareta Ekblad

• Publisher: Elsevier Science
• Year: 1977
• Tongue: English
• Weight: 533 KB
• Volume: 83
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(77)90074-4

✦ Synopsis

Ficoll hampers the transfer of unreduced phosphomolybdic acid (PMA) from aqueous to organic phase when determining inorganic phosphate (PI) by the method of Sanui [(1974)Anal. Biochem. 60,489-5041. This reduction in the transfer can be overcome by changing the organic solvent and the acidity in the aqueous phase. An ethyl acetate-isobutanol mixture proved most satisfactory, being sufficiently selective for PMA over molybdic acid (MA) in the presence of the trichloroacetic acid (TCA). Optimal conditions were determined for extraction of PMA into the organic phase with and without Ficoll. A procedure is presented for measuring Pi in biological material off Ficoll gradients. The method is well suited for measurement of P, in the presence of adenosine triphosphate (ATP) for the determination of adenosine triphosphatase (ATPase) activities where only small amounts of protein (1-15 fig) are employed.