Characterisation of β1‐Anticollagenase from Human Plasma and Its Reaction with Polymorphonuclear Leukocyte Collagenase by Disulfide/Thiol Interchange

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Abstract
A β1-serum component, β1-anticollagenase, capable of inhibiting various mammalian tissue collagenases, was isolated from human plasma by gel filtration, affinity chromatography and ion-exchange chromatography. The inhibitor contains 1–2 free sulfhydryl groups, which are a preequiste for inhibitory activity and for binding to the thiol-Sepharose affinity support. Alkylation of β1-anticollagenase by iodoacetamide blocks inhibitory activity. The inhibitor was purified to apparent homogeneity and exhibited a Mr= 30500 determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The amino acid and carbohydrate composition was determined. According to its composition and the isoelectric focussing β1-anticollagenase is an acidic protein with an isoelectric point of 5.6. Inhibition of human leukocyte collagenase proceeds in a strong 1:1 stoichiometric reaction. The mechanism of this association takes place by a disulfide/thiol interchange reaction as has been previously indicated for human leukocyte collagenases in forming the latent enzyme [Macartney, H. W. and Tschesche, H. (1980) FEBS Lett. 119, 327–332]. The β1-anticollagenase-leukocyte-collagenase complex (latent enzyme) is activatable by disulfide-containing compounds such as cystine, oxidised glutathione, insulin, relaxin, trypsinogen and others, but not by 179,203-di(S-carboxymethyl)trypsinogen, or its trypsin derivative. Compounds containing inaccessible disulfide bonds, e.g. chymotrypsin, or sulfhydryl groups, e.g. d-penicillamine, do not activate the complex. Activation is, however, easily obtained with the oxidised-glutathione-generating system myeloperoxidase/H2O2/glutathione as was previously demonstrated for the human leukocyte latent collagenase activatable in a phagocytosis-simulated respiratory burst [Tschesche, H. and Macartney, H. W. (1981) Eur. J. Biochem. 120, 183–190.]