nitrophenols and 2-amino-2-methyl-1-propanol

Nine different isoenzymes and (or) isoforms of alkaline phosphatase (ALP; EC 3.1.3.1) from human tissue were studied with respect to Km and Vmax values for p-nitrophenyl phosphate (p-NPP) in seven different potential phosphoacceptors/buffers. Generally, the phosphoacceptors/buffers with the lowest affinity for p-NPP (highest Km values) gave the highest Vmax values; for the nine enzyme forms in this study, the mean Km and Vmax values were greatest in 2-(ethylamino) (EAE). The two amino-propanol buffers gave the lowest Km and Vmax values. The phosphoacceptors/buffers N-methyl-D-glucamine (MEG), diethanolamine, and Tris had intermediate Km and Vmax values. Hydrophilic liver ALP retained > 90% of its activity after 24 h at 30 degrees C in both 1.0 and 0.3 mol/L Tris and 2-amino-2-methyl-1,3-propanediol and in 0.3 mol/L MEG. This isoenzyme showed greatest inactivation upon prolonged exposure to 1.0 and 0.3 mol/L EAE, the activity at 24 h being approximately 50-66% of that at zero time. p-NPP underwent the greatest spontaneous degradation, approximately 2.5 times that of baseline levels, in 1 mol/L MEG. There was little degradation in all of the buffers tested at 0.3 mol/L or in Tris, EAE, and 2-amino-2-methyl-1-propanol at 1.0 mol/L.

Topics: Alkaline Phosphatase; Buffers; Catalysis; Enzyme Stability; Ethanolamine; Ethanolamines; Female; Humans; Kinetics; Liver; Meglumine; Nitrophenols; Organophosphorus Compounds; Placenta; Propanolamines

We present an official AACC reference method for the measurement of alkaline phosphatase, the culmination of optimization experiments conducted by a group of independent laboratories. The details of this method and evaluation of factors affecting the measurement are described. A metal ion buffer has been incorporated that maintains optimal and constant concentrations of zinc(II) and magnesium(II) ions. Final reaction conditions are: pH (30 degrees C), 10.40 +/- 0.05; 2-amino-2-methyl-1-propanol buffer, 0.35 mol/L; 4-nitrophenyl phosphate, 16.0 mmol/L; magnesium acetate, 2.0 mmol/L; zinc sulfate, 1.0 mmol/L; and N-(2-hydroxyethyl)ethylenediaminetriacetic acid, 2.0 mmol/L.

Topics: Alkaline Phosphatase; Bone Diseases; Clinical Enzyme Tests; Computers; Edetic Acid; Female; Humans; Liver Diseases; Nitrophenols; Organophosphorus Compounds; Pregnancy; Propanolamines; Quality Control; Spectrophotometry