DETECTION OF PROTEASE ACTIVITY USING A FLUORESCENCE-ENHANCEMENT GLOBULAR SUBSTRATE

Bovine serum albumin (BSA) highly derivatized with fluorescein isothio cyanate (FITC, isomer I) served as a fluorescent enhancement substrate to measure protease activity In the native globular BSA structure, th e fluorescence of the lysine-conjugated fluorescein moieties was quenc hed 98%. Proteolytic digestion of highly derivatized BSA with Pronase( R) resulted in fluorescence enhancement of 4300%. Both alpha-chymotryp sin and proteinase K yielded lower but similar fluorescence enhancemen t values of 2880% and 2800%, respectively Digestion of the fluorescein -BSA substrate with trypsin, which required basic amino acids for acti vity, showed fluorescence enhancement of 1480% reflecting fluorescein- lysine thiocarbamyl linkage. When derivatized substrate was pretreated with a thiol-reducing agent prior to incubation with proteases, a rel atively small increase in fluorescence was noted relative to the untre ated substrate except in the case of Pronase. The minimum sensitivity of proteolytic activity, based on a comparison of untreated and reduce d FITC(25)BSA was 32 x 10(-6) units for 1 ng proteinase K, 1 x 10(-3) units for 1 ng alpha-chymotrypsin and 10 x 10(-3) units for Pronase an d trypsin (1 ng each). The fluorescence enhancement assay was suited f or sensitive intensity measurements or as an endpoint assay to detect protease activity.