Abstract
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.
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.
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°.
Change of activity ratio of test substrates for conventional standard substrate (phenylphosphate) was observed in several types of abnormal sera.
L-Leucyl-β-naphthylamide was also cleaved to a highly fluorescent product, β-naphthylamide by leucine aminopeptidase.
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°.
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.
This result suggests reorientation of leucine aminopeptidase normal values obtained in the past.
