DETERMINATION OF INORGANIC-PHOSPHATE BY THE USE OF IMMOBILIZED ENZYMES IN A FIA SYSTEM

Inorganic phosphate was fluorometrically determined in the FIA system with a column containing co-immobilized enzymes for successive reactio ns to generate NADPH from inorganic phosphate. The enzymes used were s ucrose phosphorylase, phosphoglucomutase and glucose-6-phosphate dehyd rogenase, and they were co-immobilized on aminopropyl glass. Assay con ditions such as flow rate of a carrier solution and column temperature were investigated for optimization. Several buffers of different pHs were examined as a carrier solution to find Tris buffer(pH 8.0) most f avorable. Under the conditions, the calibration curve for the determin ation of inorganic phosphate(50 mul injection) showed a linear relatio nship in the concentration range of 0.2-100 muM. The detection limit w as 0.1 muM, that is, 5 pmol. The relative standard deviation of the pe ak area for 10 muM sample was 4.1 % (n=5). The analysis of a sample co mpleted in 10 min. This method was applied to the determination of pho sphorus content in the calf thymus DNA. The content obtained was coinc ident with that obtained by the molybdenum blue method.