Add time:08/31/2019         Source:sciencedirect.com

The modifications in ultraviolet absorption and fluorescence emission accompanying dephosphorylation of formycin 5′-phosphate may be employed for the continuous assay of 5′-nucleotidase (5′-ribonucleotide phosphohydrolase, EC 3.1.3.5) activity. The sensitivity of the fluorimetric method is additionally enhanced by coupling the reaction with adenosine deaminase, which deaminates formycin more rapidly than adenosine. The final product is then formycin B, which is non-fluorescent at neutral pH and only slightly so at alkaline pH. The fluorescence procedure permits the use of substrate concentrations as low as 1 μM in a 10 mm curvette. Details are described for the use of the foregoing systems to follow continuously the kinetics as well as the properties of a number of substrate and inhibitor analogues of the enzyme from snake venom. Kinetic parameters are presented and compared with reported values for the enzyme from other sources. In particular, the pH dependence of the inhibitory properties of nucleoside 5′-diphosphates (NDP) points to the non-dissociated form, NDP2−, as the potent inhibitory species. An especially useful inhibitor is adenosine α,β-methylene-5′-pyrophosphate, because of its higher pK value for the β-phosphate secondary hydroxyl ionization so that it is the most suitable inhibitor for kinetic and in vivo investigations over a broad pH range. The spectral properties of formycin analogues are tabulated, and reference made to their potential applications to other enzyme systems.

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