Detection of protease activity using a fluorescence-enhancement globular substrate.
Bovine serum albumin (BSA) highly derivatized with fluorescein isothiocyanate (FITC, isomer I) served as a fluorescent enhancement substrate to measure protease activity. In the native globular BSA structure, the fluorescence of the lysine-conjugated fluorescein moieties was quenched 98%. Proteolytic digestion of highly derivatized BSA with Pronase resulted in fluorescence enhancement of 4300%. Both alpha-chymotrypsin and proteinase K yielded lower but similar fluorescence enhancement values of 2880% and 2800%, respectively. Digestion of the fluorescein-BSA substrate with trypsin, which required basic amino acids for activity, showed fluorescence enhancement of 1480% reflecting the fluorescein-lysine thiocarbamyl linkage. When derivatized substrate was pretreated with a thiol-reducing agent prior to incubation with proteases, a relatively small increase in fluorescence was noted relative to the untreated substrate except in the case of Pronase. The minimum sensitivity of proteolytic activity, based on a comparison of untreated and reduced FITC25BSA was 32 x 10(-6) units for I ng proteinase K, 1 x 10(-3) units for 1 ng alpha-chymotrypsin and 10 x 10(-3) units for Pronase and trypsin (1 ng each). The fluorescence enhancement assay was suited for sensitive intensity measurements or as an endpoint assay to detect protease activity.
Voss
E W
EW
Dept. of Microbiology, University of Illinois, Urbana 61801-3704, USA. e-vossl@uluc.edu
Workman
C J
CJ
Mummert
M E
ME
D000477:Alkylating Agents / Q000494:pharmacology; D002918:Chymotrypsin; D004226:Dithioerythritol / Q000494:pharmacology; D019286:Endopeptidase K / Q000378:metabolism; D010450:Endopeptidases / Q000032:analysis*; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D005470:Fluorometry; D007461:Iodoacetates / Q000378:metabolism; D008968:Molecular Conformation; D011402:Pronase / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012709:Serum Albumin / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism
D000477:Alkylating Agents; D005456:Fluorescent Dyes; D007461:Iodoacetates; D012709:Serum Albumin; D004226:Dithioerythritol; D010450:Endopeptidases; D002918:Chymotrypsin; D014357:Trypsin; D019286:Endopeptidase K; D011402:Pronase; D016650:Fluorescein-5-isothiocyanate