Isolation and Characterization of Porcine Proelastase

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Abstract
Porcine proelastase was purified from frozen pancreases by extraction at pH 4.5, precipitation with (NH4)2SO4, isolation of the pH 5.7 insoluble euglobulin fraction, selective (pH dependent) adsorption on elastin and chromatography on CM‐cellulose at pH 4.5. The active fraction was dialyzed and freeze dried. All the steps of purification except the last one were performed in presence of trypsin and chymotrypsin inhibitor AA from soybeans. All the process was done at 4°. The proelastase thus obtained was found to be pure by acrylamide gel electrophoresis and ultracentrifugation. The extinction coefficient of the freeze‐dried material, E1cm1% at 280 nm, was 17.0. The pure preparation was free of α‐N‐benzoyl‐l‐arginine ethyl esterase and contained traces of pro‐N‐acetyl‐l‐tyrosine ethyl esterase. It was stable at pH 4–5 and inactivated by NaCl. The molecular weight of the pure proelastase, calculated from the measured sedimentation and diffusion coefficients, was 25430 and according to the Yphantis method 25840. Proelastase was activated by trypsin and by the trypsin‐like enzyme from pronase, the maximal activation being dependent upon the ratio of activating enzyme to proelastase. Elastase and peptidase A did not activate the proenzyme at all. The similarity between proelastase and elastase was noted both in electrophoretic mobility in acrylamide gels and in amino acid composition but no NH2‐terminal could so far be detected in the proenzyme.

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