Self purification of two serine endopeptidases.

We have reported that a serine protease from Pronase, homologous with bovine chymotrypsin, is both active and stable in 6 M guanidinium chloride. The present investigation examined the possibility that this unique property might be used to permit the enzyme to engage in its own purification by cleaving companion proteins to low-molecular-weight products. Analysis with model substrates of the several specific activities that were originally present revealed that only the activity against Nalpha-acetyl-L-tyrosine ethyl ester was demonstrable after incubation for 100 hr in the denaturant. After a moderate loss within the first 24 hr, the remaining activity against this ester was conserved for many days thereafter. Pronase was routinely incubated for 1 week at 22 degrees in 6 M guanidinium chloride at pH 8.0 where the esterases showed maximal activity. Analysis of the products of incubation revealed unexpectedly the presence of two serine proteases that were easily separated. After purification to homogeneity these components proved themselves to be the previously demonstrated subtilisin-like and stable chymotrypsin-like enzymes. The only amino-terminal residue of the chymotrypsin-like enzyme is isoleucine, as it is in the earlier, conventionally purified product. The migration of the single band of this enzyme during acrylamide gel electrophoresis was the same whether purified by the past or present technique. No free amino-terminal group was demonstrable in the subtilisin-like enzyme. This study presents a unique and rapid technique for isolation of these proteases, with the first reported purification to homogeneity of the subtilisin-like component. These enzymes may be useful as probes for local relaxations of conformation in substrate proteins. Furthermore, they may contribute to the preparation of enzyme-free non-protein macromolecules.