A Femtomole-Sensitivity Mass Assay for Inositol Hexakisphosphate

Inositol hexakisphosphate (InsP ₆ ) is an important component of cells, and its mass levels are usually assayed by either (a) equilibrium labelling of cell cultures with radiolabelled inositol or (b) by a variety of mass assays of differing sensitivities and ambiguities. Here, we describe a mass assay for InsP ₆ that is based on phosphorylating InsP ₆ with [³² P]-ATP to 5-(PP )InsP ₅ using a recombinant Giardia InsP ₆ kinase and quantification of the radiolabelled 5-[³² P](PP )InsP ₅ product by anion exchange HPLC with an internal [³ H]-(PP )InsP ₅ standard. Interference with the enzyme reaction by other factors in the tissue extract is corrected for by assay of identical aliquots of tissue spiked with known amounts of InsP ₆ . This assay only measures InsP ₆ (and not other inositol phosphates), and although it is simple in principle and requires no dedicated or specialised equipment, it is quite time-consuming. But the assay is unambiguous and is capable of quantifying accurately as little as 10 fmol of InsP ₆ in a cell extract.