presumed conversion of the quenched fs 1 to a fluorescent lpa analog in addition the fluorescence increase was dependent on substrate concentration with the optimal conversion being observed at 100 u03bcm this closely approximates the published k m value for the purified recombinant human atx lysopld enzyme and the expected turnover for the fbs derived enzyme this increase in fluorescence was not present in serum that had been boiled for 10 min to inactivate the enzymatic activity the substrates of the present invention can be used to measure the activity of purified or partially purified enzymes in an in vitro assay lysopld can be purified from human plasma tokumura et al j biol chem 277 39436 42 2002 or can be obtained as a recombinant protein the enzyme stock solution is diluted to the desired concentration in pbs or tbs in the wells of a 384 well white or black microtiter plate to give a final volume of 50 u03bcl a stock solution of the ifs substrate in meoh or water is added to give a final concentration between 1 and 100 u03bcm fluorescence is measured on a plate reader using the particular excitation and emission wavelengths for the fluorescent group on the substrate as the enzyme metabolizes the substrate an increase in fluorescence.