Specificity of bovine enterokinase toward protein substrates

Abstract

We examined the potential use of bovine enterokinase for the limited proteolysis of proteins containing sequences of one or more acidic residues preceding a basic residue. Proteolysis was followed by observing the appearance of fragments by sodium dodecyl sulfate-gel electrophoresis. The susceptible peptide bond was identified from a knowledge of the size of the fragment and the amino acid sequence of the protein. Bovine serum albumin was resistant to proteolysis in its native state, was somewhat susceptible as the S-carboxyamidomethyl derivative, and was highly susceptible as the S-carboxymethyl derivative. S-Alkylated soybean trypsin inhibitor and hen egg white lysozyme were both susceptible to limited hydrolysis, but only in the presence of deoxycholate. All susceptible bonds were either lysine or arginine. The preceding acidic residues could be either aspartic acid, glutamic acid, or carboxymethyl cysteine. If a single acidic residue immediately preceded the basic residue, the rate of hydrolysis was slow. The rate of hydrolysis was also slow if a carboxymethyl cysteine was introduced at the position following the basic residue. In addition to better defining the specificity of enterokinase, these results indicate that enterokinase may be useful in amino acid sequence studies for the production of large fragments. The enzyme may also be useful in DNA-recombinant studies in releasing the desired polypeptide chain from neighboring sequences.