Detection of femtomole quantities of proteases by high-performance liquid chromatography

Small amounts (femtomoles) of proteases, as might be present in cell extracts or secretions, were detected using reverse-phase high-performance liquid chromatography. Carboxymethylated lysozyme and cytochrome c were incubated with trypsin and chymotrypsin. Peptide peaks were present in the column elution profiles (as detected by absorbance, 206 nm) from incubations with as little as 0.1 fmol of chymotrypsin and 5 fmol of trypsin. In addition, the disappearance of the substrate peak or the increase in peptide peaks could be quantitated by integrating the areas under the peaks. In this way estimates of relative enzyme concentrations or duration of incubation can be determined. However, when [14C]lysozyme was used as a substrate and the radioactivity of collected peaks was measured, the assay was less sensitive than that using uv absorbance. This finding probably is related to the selective radiolabeling of the substrate, in contrast to uv detection, which should detect all the peptides. The technique reported in this paper should prove to be a sensitive indicator of proteolytic activity in cell or tissue preparations where the use of synthetic ester or amide substrates might lead to erroneous conclusions regarding the nature of the enzymatic activity present. Furthermore, by the collection of the peptides generated, one would have the ability to determine amino acid compositions or sequences and thus ascertain the specificity of enzymatic cleavage.