Inositol phosphates induce DAPI fluorescence shift

The polymer inorganic polyP (polyphosphate) and inositol phosphates, such as IP₆ (inositol hexakisphosphate; also known as phytic acid), share many biophysical features. These similarities must be attributed to the phosphate groups present in these molecules. Given the ability of polyP to modify the excitation–emission spectra of DAPI we decided to investigate whether inositol phosphates possess the same property. We discovered that DAPI–IP₆ complexes emit at approximately 550 nm when excited with light of wavelength 410–420 nm. IP₅ (inositol pentakisphosphate) is also able to induce a similar shift in DAPI fluorescence. Conversely, IP₃ (inositol trisphosphate) and IP₄ (inositol tetrakisphosphate) are unable to shift DAPI fluorescence. We have employed this newly discovered feature of DAPI to study the enzymatic activity of the inositol polyphosphate multikinase and to monitor phytase phosphatase reactions. Finally, we used DAPI–IP₆ fluorescence to determine the amount of IP₆ in plant seeds. Using an IP₆ standard curve this straight-forward analysis revealed that among the samples tested, borlotti beans possess the highest level of IP₆ (9.4 mg/g of dry mass), whereas the Indian urad bean the lowest (3.2 mg/g of dry mass). The newly identified fluorescence properties of the DAPI–IP₅ and DAPI–IP₆ complexes allow the levels and enzymatic conversion of these two important messengers to be rapidly and reliably monitored.