Assay for proteolytic activity using a new fluorogenic substrate (peptidyl-3-amino-9-ethyl-carbazole); quantitative determination of lipopolysaccharide at the level of one picogram.

A new sensitive fluorimetric assay has been developed using peptidyl-3-amino-9-ethyl-carbazole as substrate. The fluorescence intensity of free 3-amino-9-ethyl-carbazole (AEC) at 460 nm is between two and three orders of magnitude higher than the fluorescence intensity of acyl-AEC. The release of AEC from a peptidyl derivative by proteases may be monitored continuously during the hydrolysis step or may be quantified upon addition of a general inhibitor such as benzamidinium chloride. Using N-benzoyl-arginyl-AEC as substrate, as little as 1 ng trypsin may be detected. Using t-butyloxycarbonyl-Val-Leu-Gly-Arg-AEC and the amoebocyte lysate of Limulus polyphemus, as little as 1 pg lipopolysaccharide can be detected. This fluorimetric method allows detection of trace amounts of lipopolysaccharide (endotoxins) in various biological materials, including sera.