Assay of diadenosine tetraphosphate hydrolytic enzymes by boronate chromatography

A new procedure was described for assay of diadenosine tetraphosphate (Ap4A) hydrolases based on boronate chromatography. Potential reaction products, AMP, ADP, and ATP, of the hydrolysis of Ap4A were separated from residual substrate by chromatography on a boronate-derivatized cation-exchange resin, Bio-Rex 70. Separation was achieved by changing the concentrations of ethanol and ammonium acetate in the elution buffers. Picomole masses of products were detectable, blank dpm values were less than 0.5% of the total dpm, and auxiliary enzymes were not required. The procedure was specifically described for Ap4A pyrophosphohydrolase from Physarum polycephalum. The assay is generally applicable for dinucleoside polyphosphate hydrolases which hydrolyze other substrates such as Ap3A, Ap5A, Ap6A, and Gp4G. Dinucleotide polyphosphates are readily purified by chromatography on this boronate resin in a volatile buffer. Tes, Tricine, and Tris buffers significantly interfered with the chromatography of ATP.