Purification of Rheumatoid Synovial Collagenase and Its Action on Soluble and Insoluble Collagen

Abstract

The neutral collagenase released into the culture medium by explants of rheumatoid synovial tissue has been purified by ultrafiltration and column chromatography, utilising Sephadex G‐200, Sephadex QAE A‐50 and Sephadex G‐100 superfine. The final collagenase preparation had a specific activity against thermally reconstituted collagen fibril of 312 μg collagen degraded min−1 mg enzyme protein−1, representing more than a 1000‐fold increase over that of the active culture medium. Electrophoresis in polyacrylamide disc‐gels with and without sodium dodecyl sulphate showed the enzyme to migrate as a single protein band. Elution experiments from polyacrylamide gels and chromatography columns have provided no evidence for the existence of more than one collagenase. The molecular weight of the enzyme, as determined by dodecylsulphate‐polyacrylamide gel electrophoresis, was 33000. Data obtained from studies with the ion‐exchange resin and from gel electrophoresis in acid and alkaline buffer systems suggested a basically charged enzyme. It did not hydrolyse the synthetic collagen peptide Pz‐Pro‐Leu‐Gly‐Pro‐d‐Arg and non‐specific protease activity was absent. The collagenase attacked undenatured collagen in solution at 25°C resulting in a 58% loss of viscosity and producing the two characteristic products TCA (3/4) and TCB (1/4). At 37°C and pH 8.0 both reconstituted collagen fibrils and gelatin were degraded to peptides of less than 10000 molecular weight. As judged by the release of soluble hydroxyproline peptides and electron microscopic appearances the enzyme degraded human insoluble collagens derived from tendon and soft juxta‐articular tissues although rates of attack were less than with reconstituted fibrils. The data suggests that pure rheumatoid synovial collagenase at 37°C and neutral pH can degrade gelatin, reconstituted fibrils and insoluble collagens without the intervention of non‐specific proteases. The different susceptibilities of various collagenous substrates to collagenase attack are discussed. Copyright © 1975, Wiley Blackwell. All rights reserved