Detection, identification, and partial characterization of serine proteases and esterases in biological systems

Abstract

Serine proteases are important catalytic regulators of cellular and extracellular function. Tritium-labeled diisopropylfluorophosphate ([ 3H]-DFP) has been used to stoichiometrically label serine proteases and the labeled proteases analyzed by autoradiography following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By preincubating a serine protease, or a complex biological mixture containing serine proteases, with specific, synthetic, active-site inhibitors, the covalent incorporation of [ 3H]DFP into the protease is prevented or substantially reduced. Since the synthetic inhibitors are amino acid or peptide anologs that reflect the preferred cleavage site of a specific serine protease, any inhibitor-dependent reduction in [ 3H]DFP incorporation indicates the substrate specificity of the given serine protease. Inhibitor profiles (reflected in polyacrylamide gel autoradiographs) for the different classes of serine proteases have been generated by using a variety of purified serine enzymes preincubated with different inhibitors prior to, and during, [ 3H]DFP labeling. These profiles were then compared to the more complex gel patterns obtained when cell lysates or conditioned culture medium were labeled with [ 3H]DFP in the presence or absence of the different active-site inhibitors. A number of known and heretofore undescribed serine proteases were detected in various biological systems including transformed fibroblasts, activated macrophages, and mutant strains of Escherichia coli. This technique allows not only for the detection of serine enzymes in complex mixtures, but also indicates their molecular weight and substrate specificity. This method allows for the quantitative detection of 10 −13 mol of a serine protease. This additional use of this technique to screen for naturally occurring inhibitors of serine proteases is discussed.