Synthesis and Nonradioactive Micro-analysis of Diphosphoinositol Phosphates by HPLC with Postcolumn Complexometry
- 930 Downloads
A nonradioactive high-performance anion-exchange chromatographic method based on MDD-HPLC (Mayr Biochem. J. 254:585–591, 1988) was developed for the separation of inositol hexakisphosphate (InsP 6, phytic acid) and most isomers of pyrophosphorylated inositol phosphates, such as diphosphoinositol pentakisphosphate (PPInsP 5 or InsP 7) and bis-diphosphoinositol tetrakisphosphate (bisPPInsP 4 or InsP 8). With an acidic elution, the anion-exchange separation led to the resolution of four separable PPInsP 5 isomers (including pairs of enantiomers) into three peaks and of nine separable bisPPInsP 4 isomers into nine peaks. The whole separation procedure was completed within 20–36 min after optimization. Reference standards of all bisPPInsP 4 isomers were generated by a nonenzymatic shotgun synthesis from InsP 6. Hereby, the phosphorylation was brought about nonenzymatically when concentrated InsP 6 bound to the solid surface of anion-exchange beads was incubated with creatine phosphate under optimal pH conditions. From the mixture of pyrophosphorylated InsP 6 derivatives containing all theoretically possible isomers of PPInsP 5, bisPPInsP 4, and also some isomers of trisPPInsP 3, isomers were separated by anion-exchange chromatography and fractions served as reference standards of bisPPInsP 4 isomers for further investigation. Their isomeric nature could be partly assigned by comparison with position specifically synthesized or NMR-characterized purified protozoan reference compounds and partly by limited hydrolysis to PPInsP 5 isomers. By applying this nonradioactive analysis technique to cellular studies, the isomeric nature of the major bisPPInsP 4 in mammalian cells could be identified without the need to obtain sufficient material for NMR analysis.
Key wordsInositol phosphate Pyrophosphates Signal transduction Metal-dye detection HPLC
The authors would like to thank Professor Gunter Vogel (Wuppertal, Germany) for providing the 1/3,5-bisPPInsP 4 isomer from Polysphondylium, and Professor J.R. Falck (UT Southwestern, Dallas, USA) for providing a synthetic 2,5-bisPPInsP 4 sample and pure PPInsP 5 isomers. Parts of the MDD-HPLC analysis have been performed by Bettina Serreck, whose technical support is highly acknowledged.
- 9.Stephens, L., Radenberg, T., Thiel, U., Vogel, G., Khoo, K.H., Dell, A., Jackson, T.R., Hawkins, P.T., Mayr, G.W., Stephens, L.R., Stanley, A.F., Moore, T., Poyner, D.R., Morris, P.J., Hanley, M.R., Kay, R.R., Irvine, R.F., Laussmann, T., Eujen, R., Weisshuhn, C.M., Martin, J.B., Bakker-Grunwald, T., Klein, G., Reddy, K.M., Reddy, K.K., and Falck, J.R. (1993) The detection, purification, structural characterization, and metabolism of diphosphoinositol pentakisphosphate(s) and bisdiphosphoinositol tetrakisphosphate(s) myo-inositol pentakisphosphates. J. Biol. Chem. 268, 4009–4015.PubMedGoogle Scholar
- 20.Saiardi, A., Resnick, A.C., Snowman, A.M., Wendland, B., Snyder, S.H., Bhandari, R., Pesesse, X., Choi, K., Zhang, T., Shears, S.B., Luo, H.R., Huang, Y.E., Chen, J.C., Iijima, M., Ye, K., Huang, Y., Nagata, E., Devreotes, P., El Alami, M., Messenguy, F., Scherens, B., Dubois, E., Sciambi, C., McCaffery, J.M., Yu, H., Menniti, F.S., Miller, R.N., and Putney, J.W., Jr. (2005) Inositol pyrophosphates regulate cell death and telomere length through phosphoinositide 3-kinase-related protein kinases phosphorylation of proteins by inositol pyrophosphates. Proc. Natl. Acad. Sci. USA 102, 1911–1914.CrossRefPubMedGoogle Scholar
- 27.Segel, I.H. (1976) Biochemical calculations: how to solve mathematical problems in general biochemistry. 2nd ed. New York: John Wiley & Sons Inc., pp. 15.Google Scholar
- 28.Lide, D.R. (2006) CRC Handbook Chemistry and Physics. 87th ed: Taylor & Francis Group, New York, pp. 8–41.Google Scholar