Purification and Characterization of Porcine Elastase II and Investigation of Its Elastolytic Specificity

A new porcine pancreatic serine protease, elastase II (Ardelt, W. (1974), Biochim. Biophys. Acta 341, 318-326), was further purified by removing the accompanying proteases on a turkey ovomucoid-Sepharose column. The purified enzyme was found to be homogeneous by ultracentrifugal sedimentation analysis (s_20,w = 3.16), by electrophoresis on celllulose acetate and polyacrylamide gels at different pHs, by sodium dodecyl sulfate gel electrophoresis, and by electrofocusing in polyacrylamide gels. Elastase II is composed of about 250 amino acids, with a molecular weight of 26 500, and an NH₂-terminal amino acid sequence which strongly resembles the B chain of bovine chymotrypsin B. Preliminary experiments indicated that it may be composed of two chains held together by a disulfide bond in which the NH₂-terminal half-cystine participates. Elastase II has no activity towards specific substrates of porcine elastase I (EC 3.4.21.11) but exhibits characteristic chymotrypsin specificity. It has an extended binding site but like in other chymotrypsins the subsite S₁ plays a dominant role in the binding. Similar to elastase I, elastase II solubilizes elastin by hydrolyzing about 6% of the peptide bonds. However, while elastase I hydrolyzes Ala-Ala and Ala-Gly peptide bonds, elastase II hydrolyzes the bonds formed between leucine, phenylalanine, or tyrosine with glycine or alanine. It seems, therefore, that the specificity of elastase II is completely different from that of elastase I. Elastase II also differs from porcine chymotrypsin A, B, and C, but strongly resembles a previously described proelastase B, which upon activation hydrolyzes elastin and exhibits chymotrypsin specificity.