A Specific Colorimetric 5′-Nucleotidase Assay Utilizing the Berthelot Reaction

Abstract

A sensitive calorimetric assay for 5′-nucleotidase activity is described, which requires 40 µl of serum. Adenosine, formed by hydrolysis of 5′-adenylic acid, is deaminated enzymatically and the ammonia determined with the Berthelot reaction. β-Glycerophosphate is added to suppress hydrolysis of 5′-adenylic acid by alkaline phosphatase. The reaction is optimal at pH 7.9 in 0.05M Michaelis' barbital buffer containing 10 mmoles of MgCl2 per liter. In the Berthelot reaction the MgCl2 causes a precipitate, which is removed without decreasing color yield by adding EDTA. Inhibition of the Berthelot reaction by serum and constituents of the reaction mixture is predictable and can be compensated by a correction factor