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A search for the source of the residual esterase activity of crude lima bean protease inhibitor-binding anhydrochymotrypsin preparations was undertaken. The preparations were found to contain about 40% of protein that possesses 1% (kc/Km) to 12% (kc) of the esterase activity of alpha-chymotrypsin. The active protein was isolated by affinity chromatography on soybean trypsin inhibitor-Sepharose. It appears to be an anhydroenzyme or a mixture of a limited number of anhydroenzymes in which a serine other than the catalytically essential serine-195 of the native enzyme has been converted to dehydroalanine.

A chymotrypsin-like proteinase was purified 2400-fold from human skin. The procedure involves extraction of the proteinase from skin in 2 M KCl, precipitation with protamine chloride, fractionation by gel filtration chromatography, and fractionation by chromatography using a CH-Sepharose-D-tryptophan methyl ester affinity column. The properties of this proteinase were compared to the rat mast cell proteinase I and human cathepsin G. Differences were observed in the rates at which the proteinases were inhibited by diisopropyl fluorophosphate, the sensitivity of the proteinases to protein proteolytic inhibitors, the relative hydrolytic rates of the proteinases for a series of substrates, and the kinetic constants of the proteinases for synthetic substrates. The human skin proteinase did not react with antiserum to the rat skin proteinase and did not elute in the same position as the rat skin proteinase on gel filtration columns. These data demonstrate that the human skin proteinase is distinct from the other proteinases. Extracts of involved skin from patients with cutaneous mastocytosis had 15-fold higher levels of chymotryptic activity than extracts of uninvolved skin or skin from normal controls. The enzymatic properties of the material extracted from the biopsied skin were similar to those of the proteinase from normal skin, suggesting that the human skin chymotrypsin-like proteinase is a mast cell constituent.

The reactive site of human alpha 1-antichymotrypsin has been identified as encompassing a leucyl-seryl bond at the apparent P1 and P'1 positions. This has been determined by dissociation of complexes of the inhibitor with bovine alpha-chymotrypsin, followed by identification of new NH2-terminal sequences, as well as by proteolytic inactivation by porcine pancreatic elastase. The latter results in peptide bond cleavage between the apparent P5 and P4 positions of the inhibitor, yielding a fragment whose sequence overlaps with that obtained through complex dissociation. Some homology with the sequence obtained and that already reported for both antithrombin III and alpha 1-proteinase inhibitor can be noted.

A procedure is described for the purification of the elastase and chymotrypsin-like enzymes from purulent sputum. This procedure permitted the isolation of 132 mg and 120 mg of the elastase and chymotrypsin-like enzymes, respectively, from 230 g of purulent sputum. The elastase enzymes consist of a family of five isozymes, and at least three isozymes comprise the chymotrypsin-like enzyme system. The elastases proved to be immunologically identical with the corresponding enzyme of human leukocytes. These enzymes were characterized with respect to molecular weight, amino acid and carbohydrate composition, several kinetic parameters, and inhibition by various synthetic and natural inhibitors. The properties so found were comparable to those which had been previously reported by others for the elastase and chymotrypsin-like enzymes isolated directly from leukocytic granules.

The ability of plasma proteinase inhibitors to inactivate human chymase, a chymotrypsin-like proteinase stored within mast cell secretory granules, was investigated. Incubation with plasma resulted in over 80% inhibition of chymase hydrolytic activity for small substrates, suggesting that inhibitors other than alpha 2-macroglobulin were primarily responsible for chymase inactivation. Depletion of specific inhibitors from plasma by immunoadsorption using antisera against individual inhibitors established that alpha 1-antichymotrypsin (alpha 1-AC) and alpha 1-proteinase inhibitor (alpha 1-PI) were responsible for the inactivation. Characterization of the reaction between chymase and each inhibitor demonstrated in both cases the presence of two concurrent reactions proceeding at fixed relative rates. One reaction, which led to inhibitor inactivation, was about 3.5 and 4.0-fold faster than the other, which led to chymase inactivation. This was demonstrated in linear titrations of proteinase activity which exhibited endpoint stoichiometries of 4.5 (alpha 1-AC) and 5.0 (alpha 1-PI) instead of unity, and SDS gels of reaction products which exhibited a banding pattern indicative of both an SDS-stable proteinase-inhibitor complex and two lower Mr inhibitor degradation products which appear to have formed by hydrolysis within the reactive loop of each inhibitor. At inhibitor concentrations approaching those in plasma where inhibitor to chymase concentration ratios were in far excess of 4.5 and 5.0, the rate of chymase inactivation by both serpin inhibitors appeared to follow pseudo-first order kinetics. The "apparent" second order rate constants of inactivation determined from these data were about 3000-fold lower than the rate constants reported for human neutrophil cathepsin G and elastase with alpha 1-AC and alpha 1-PI, respectively. This suggests that chymase would be inhibited about 650-fold more slowly than these proteinases when released into plasma. These studies demonstrate that although chymase is inactivated by serpin inhibitors of plasma, both inhibitors are better substrates for the proteinase than they are inhibitors. This finding along with the slow rates of inactivation indicates that regulation of human chymase activity may not be a primary function of plasma.

A double selection method for isolating active enzyme molecules, using substrate analog affinity chromatography and elution with transition state analogs, is described. To demonstrate the principle, a mixture containing native chymotrypsin and [3H]deoxychymotrypsin, in which the active site serine had been converted to [3H]alanine, was applied to a column containing immobilized D-tryptophan methyl ester. Both forms of chymotrypsin were retained. Catalytically active enzyme was selectively desorbed with the peptide aldehyde chymostatin, leaving catalytically inactive deoxychymotrypsin bound to the substrate analog affinity column. This affinity technique may afford a simple and general method for separating enzymes and other catalysts according to their molecular turnover numbers.

Binary and ternary alpha 2-macroglobulin-chymotrypsin complexes may be quantitatively adsorbed on BH-Sepharose-D-tryptophan methyl ester at pH 8.0 and quantitatively eluted either with acetic acid or with 40% glycerol, pH 8.0. This is the first report of a preparative separation of free and proteinase-bound alpha 2-macroglobulin. Using this affinity chromatographic system, we were able to demonstrate that the two chymotrypsin binding sites of alpha-2-macroglobulin are equivalent and independent.