The collagenase inhibitor from human polymorphonuclear leukocytes. Isolation, purification and characterisation.

Abstract

A new collagenase inhibitor of latent human polymorphonuclear collagenase, initially reported by Macartney and Tschesche [Hoppe-Seyler's Z. Physiol. Chem. 361, 298–299 (1980) and FEBS Lett. 119, 327–332 (1980)], has been purified to apparent homogeneity. We described several methods of purification of this inhibitor, but the fastest and most reproducible method is by passage of purified latent human polymorphonuclear leukocyte collagenase on activated thiol-Sepharose 4-B (preceding paper in this journal). Active collagenase is eluted from the column and the collagenase inhibitor is retarded. Elution of the inhibitor is achieved by eluting the column with thiol compounds in the equilibration buffer. Further purification of the inhibitor is obtained by gel filtration on Sephacryl S-300 and ion-exchange chromatography on DEAE-Sephacel. The inhibitor exhibits a strict specificity for mammalian collagenases. It inhibits collagenases isolated from human synovial fluid, human normal and diabetic skin fibroblasts and human embryonic skin fibroblasts. The inactive complexes are all subject to activation by several disulfide compounds, such as oxidised glutathione by the thiol/disulfide interchange reaction already described. The purified inhibitor was shown by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (10% gel) to be homogeneous and displayed an apparent molecular weight of 24500. Titration of the inhibitor with Ellmans reagent revealed that it contains one free sulfhydryl group, a prerequisite for its inhibitory activity. The inhibitor inhibits human polymorphonuclear leukocyte collagenase in a strict 1:1 stoichiometric reaction. The amino acid composition of the complex formed from the inhibitor and active enzyme reproduces that of the latent collagenase thus confirming that the latent enzyme is a 1:1 complex. The inhibitor is an acidic protein having an isoelectric point at pH 5.5, which would be expected from the amino acid analysis. The composition reveals a glycoprotein of about 240 residues containing a carbohydrate moiety composed of N-acetylglucosamine, mannose, galactose and glucose. The amino acid composition of the inhibitor is not identical to that of β1-anticollagenase from human plasma.