Fluorescence Initial Reaction Rate Analysis of Serum Alkaline phosphatase and Leucine aminopeptidase
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Fluorometric initial rate analyses of serum alkaline phosphatase and leucine aminopeptidase were studied. In these studies, attempt has been made to establish the assay condition for multi-enzyme analysis system with a common single detection system.<BR>Commercially available naphthol derivative phosphates, α-naphthylphosphate, β-naphthylphosphate, naphthl-ASBI-phosphate, and naphthyl-ASMX-phoaphate, were tested as substrates for alkaline phosphatase. These substrates were cleaved by the enzyme to highly fluorescent products, β-naphthol, α-naphthol, naphthol-ASBI and naphthol-ASMX.<BR>The effect of pH on fluorescence spectra and intensity was examined. The fluorescent intensity of α-naphthol and βA-naphthol were highly dependent on pH, but that of naphthol-ASBI and ASMX was not. Kinetic analysis of the enzyme gave the suitable condition for analysis of α-naphthyl phosphate: 5 mM in 2-amino, 2-methyl, 1, 3-propandiol 0.1M, pH 11.0, excitation 335 nm and emission at 460nm, temperature at 37°.<BR>Change of activity ratio of test substrates for conventional standard substrate (phenylphosphate) was observed in several types of abnormal sera.<BR>L-Leucyl-β-naphthylamide was also cleaved to a highly fluorescent product, β-naphthylamide by leucine aminopeptidase.<BR>Fluorometric assay condition was 0.647mM L-leucyl-β-naphthylamide in 0.05M Tris pH 7.2, excitation 335nm and emission 410nm, temperature at 37°.<BR>Fluorometric initial rate analysis was studied, but the data were strikingly different from the usual diazo-coupling method. This contradiction was based on the low substrate concentration in the assay condition restricted by the solubility of the substrate.<BR>This result suggests reorientation of leucine aminopeptidase normal values obtained in the past.
- Japan Society of Clinical Chemistryの論文
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