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Two proteases with elastolytic activity (elastases 1 and 2) have been isolated from activated extracts of human pancreatic tissue. The purification procedure for both elastases included ammonium sulfate fractionation followed by ion-exchange chromatography on CM-Sephadex C-50. Elastase 1 was further purified by chromatography on DEAE-Sephadex A-50. The homogeneity of both enzymes was demonstrated by Sephadex G-75 gel filtration, analytical polyacrylamide disc gel electrophoresis at pH 2.3, 4.5, and 8.3, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis at pH 8.3. Both enzymes hydrolyzed undyed elastin as well as Remazol brilliant blue elastin and Congo red elastin. Activities and kinetic parameters using several synthetic substrates are also reported. The enzymes were further characterized in terms of molecular weight, amino acid composition, and N-terminal and penultimate amino acid residues. Their inhibition by the human serum protease inhibitors alpha2-macroglobulin and alpha1-antitrypsin was also studied. Elastase 1 appears to be very similar to human protease E (Mallory, P. A., and Travis, J. (1975), Biochemistry 14, 722). Elastase 2 is distinct from all human pancreatic proteases which have been characterized to date.

The substrate specificity of human pancreatic elastase 2 was investigated by using a series of peptide p-nitroanilides. The kinetic constants, kcat and Km, for the hydrolysis of these peptides revealed that this serine protease preferentially hydrolyzes peptides containing P1 amino acids which have medium to large hydrophobic side chains, except for those which are disubstituted on the first carbon of the side chain. Thus, human pancreatic elastase 2 appears to be similar in peptide bond specificity to the recently described porcine pancreatic elastase 2 [Gertler, A., Weiss, Y., & Burstein, Y. (1977) Biochemistry 16, 2709] but differs significantly in specificity from porcine elastase 1. The best substrates for human pancreatic elastase 2 were glutaryl-Ala-Ala-Pro-Leu-p nitroanilide and succinyl-Ala-Ala-Pro-Met-p-nitroanilide. However, there was little difference among substrates with leucine, methionine, phenylalanine, tyrosine, norvaline, or norleucine in the P1 position. Changes in the hydrolysis rate of peptides with differing P5 residues indicate that this enzyme has an extended binding site which interacts with at least five residues of peptide substrates. The overall catalytic efficiency of human pancreatic elastase 2 is significantly lower than that of porcine elastase 1 or bovine chymotrypsin with the compounds studied.