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

Abstract

1 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. 2 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. 3 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. 4 The molecular weight of the enzyme, as determined by dodecylsulphate-polyacrylamide gel electrophoresis, was 33000. 5 Data obtained from studies with the ion-exchange resin and from gel electrophoresis in acid and alkaline buffer systems suggested a basically charged enzyme. 6 It did not hydrolyse the synthetic collagen peptide Pz-Pro-Leu-Gly-Pro-d-Arg and non-specific protease activity was absent. 7 The collagenase attacked undenatured collagen in solution at 25°C resulting in a 58% loss of viscosity and producing the two characteristic products TC_A (3/4) and TC_B (1/4). 8 At 37°C and pH 8.0 both reconstituted collagen fibrils and gelatin were degraded to peptides of less than 10000 molecular weight. 9 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. 10 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. 11 The different susceptibilities of various collagenous substrates to collagenase attack are discussed.