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.

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Abstract
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.