Automated Assay for Fructose 2,6-Bisphosphate

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Abstract
An automated procedure for the determination of Fructose 2,6-bisphosphate (Fru 2,6-P2) is described. The method based on the degree of activation of the enzyme Pyrophosphate: D-fructose 6-phosphate 1-phosphotransferase from potato tuber by Fru 2,6-P2, uses a discrete-sample automatic analyzer to perform the procedure and to monitor the spectrophotometric changes. The detection limit of Fru 2,6-P2 levels is 0.016 pmol (6.25 × 10−14 mol ml−1). The coefficients of variation observed when assaying 0.5 nM of Fru 2,6-P2 in the reaction mixture, 1.34% and 7.39% (n=10) for the automated and manual method are significantly different. Thus a 5-fold higher precision is shown by the automatic method. The same precision is observed when assaying biological samples from starved or refed animals. In conclusion the assay is as sensitive as the manual spectrophotometric method previously described, but it shows higher precision and decreases the processing time allowing routine determinations. It is specially useful when purifying 6-phosphofructo 2-kinase or assaying a large number of tissue samples for Fru 2,6-P2.