Synthesis and Nonradioactive Micro-analysis of Diphosphoinositol Phosphates by HPLC with Postcolumn Complexometry
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.
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