An evaluation of fluorometric proteinase assays which employ fluorescamine.
The sensitivity and utility of proteinase assays employing fluorescamine, a compound which reacts with primary amines to form a fluorescent adduct, was assessed. As little as 1 ng of purified trypsin or clostridiopeptidase A could be detected within 3 h of incubation at 37 degrees C, using casein or gelatin as substrates. Increasing the incubation period to 18 h permitted the detection of 250 pg of each enzyme. When gelled collagen was utilized as substrate, the sensitivity to clostridiopeptidase A was reduced to 2.5 ng at 3 h and 500 pg at 18 h. The techniques could be used to measure the gelatinase, caseinase, and collagenase activities of culture media conditioned by synovial tissue. The main disadvantage of this assay is its susceptibility to interference by compounds which fluoresce or quench. This, in turn, necessitates additional blanks, which may render the assay tedious.
D000818:Animals; D002478:Cells, Cultured; D010450:Endopeptidases / Q000032:analysis*; D005450:Fluorescamine; D018093:Gelatinases; D006801:Humans; D008666:Metalloendopeptidases; D003012:Microbial Collagenase / Q000032:analysis; D010434:Pepsin A / Q000032:analysis; D010447:Peptide Hydrolases / Q000032:analysis; D011817:Rabbits; D011874:Radiometry; D013050:Spectrometry, Fluorescence; D013141:Spiro Compounds; D013379:Substrate Specificity; D013583:Synovial Membrane / Q000201:enzymology; D014357:Trypsin / Q000032:analysis
D013141:Spiro Compounds; D005450:Fluorescamine; D010450:Endopeptidases; D010447:Peptide Hydrolases; D014357:Trypsin; D010434:Pepsin A; D018093:Gelatinases; D008666:Metalloendopeptidases; C030895:caseinase; D003012:Microbial Collagenase