Isolation and properties of a capillary injury-related protease from lung lymph

Lung microvascular injury induced in sheep by intravenous infusion of Escherichia coli endotoxin, oleic acid, or air emboli caused the appearance in lung lymph of high levels of a protease with trypsin-like activity. The enzyme was isolated as an apparently homogeneous protein from pooled samples of active lung lymph, after an almost 9000-fold purification by affinity chromatography on columns of Reactive Blue 2-agarose, aprotinin-agarose, and p-aminobenzamidine-agarose, and chromatography on a column of Sephadex G-100. A molecular weight of about 70,000 to 75,000 was determined from mobility in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The pH optimum was between 7.3 and 7.6. The isolated enzyme was quite labile, rapidly losing activity at both 37 and 25 degrees C. Addition of albumin to enzyme solutions protected against inactivation. Inhibition by diisopropylfluorophosphate and phenylmethanesulfonyl fluoride indicated that the enzyme belongs to the class of serine proteases. The enzyme cleaved peptide bonds on the carboxyl side of arginine residues and showed a relatively high affinity toward peptides containing several basic amino acid residues. Bonds involving the carboxyl group of lysine were cleaved at a much slower rate. The enzyme showed no plasminogen activator activity and its substrate specificity was quite different from that of several proteases of the clotting cascade. Its appearance in lymph was not influenced by lymph clotting and the isolated enzyme was not capable of correcting the clotting defect of plasmas deficient in factors XII, XI, IX, VII, and X.