Preparation of pure lower-order -Inositol phosphates on laboratory scale for physiological and enzymatic studies.

A simple method for the preparative production of lower-order -inositol phosphates was developed. Enzymatic phytate dephosphorylation was applied, because phytate-degrading enzymes generate usually predominantly one single -inositol phosphate isomer with five, four, three, two and one phosphate residue(s) bound to the -inositol ring in a regio- and stereoselective manner. The relative concentrations of the different lower-order -inositol phosphates in the reaction mixture were controlled by adjusting incubation time at 37 °C and a fixed phytate concentration and phytase activity. Purification of the individual lower-order -inositol phosphates was realized by anion-exchange chromatography on Q-Sepharose using a stepwise elution with ammonium formate:formic acid pH 2.5. Ethanol precipitation was successfully used to concentrate the pure lower-order -inositol phosphates. In a single approach 2-3 mg of pure -inositol tetrakis- or -trisphosphate isomers were obtained. About 60% of the initially applied phytate were converted into pure lower-order -inositol phosphates. The purified -inositol phosphate isomers were virtually free of other -inositol phosphate esters and could be used for enzymatic and physiological studies.