A FLUOROMETRIC ASSAY FOR MEASURING DEAMIDASE (LYSOSOMAL PROTECTIVE PROTEIN) USING HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

A rapid and sensitive assay method for the determination of deamidase activity is reported. This method is based on fluorometric detection o f a dansylated dipeptide, 5-dimethylaminonaphthalene-1-sulfonyl-D-Tyr- Val (N-Dns-D-Tyr-Val), enzymatically formed from the substrate -dimeth ylaminonaphthalene-1-sulfonyl-D-Tyr-Val-NH2 (N-Dns-D-Tyr-Val-NH2), aft er separation by high-performance liquid chromatography (HPLC) using a C-18 reversed-phase column by isocratic elution. This method is sensi tive enough to measure N-Dns-D-Tyr-Val at concentrations as low as 100 fmol, yields highly reproducible results and requires less than 8.5 m in per sample for separation and quantitation. The optimum pH for deam idase activity was 4.0-4.5. Greater than 5 mM of reduced glutathione w as needed for maximal enzyme activity. The K-m and V-max values were r espectively 125 mu M and 14.12 pmol/mu g/h with the use of enzyme extr act obtained from mouse spleen. Deamidase activity was strongly inhibi ted by Ag+, Cu2+, diisopropylfluorophosphate, and p-chloromercuripheny lsulfonic acid. Among the organs examined in a mouse, the highest spec ific activity of the enzyme was found in spleen. The sensitivity and s electivity of this method will aid in efforts to examine the physiolog ical role of this enzyme. (C) 1996 Academic Press, Inc.