Abstract
In a previous paper we showed that Bacteroides gingivalis produces SH-dependent collagenolytic enzyme and that the activity is inhibited by the extract of soybean meal. The purpose of this study is to describe the isolation, partial purification, and characterization of this inhibitory substance.
Dried soybeans were pulverized in a coffee mill to pass a 60 mesh sieve and suspended at a 2% concentration in 10mM Tris-HCl buffer, pH 7.4, containing 0.1M NaCl. The suspension was centrifuged at 10, 000×g and the supernatant was used as crude soybean extract. The inhibitory substance in the crude extract was further purified by DEAE-cellulose column chromatography and gel filtration on Sepharose CL-6B, and the active fraction for the inhibition of the collagenolytic enzyme from B. gingivalis was collected, concentrated, and named soybean collagenase inhibitor (SCI). The crude extract contained soybean trypsin inhibitor (STI), and inhibited the trypsin and collagenolytic enzyme of B. gingivalis, but no inhibition was observed for Clostridium histolyticum enzyme and the protease of B. gingivalis. However, the SCI was successfully separated from STI by these purification procedures. The molecular weight of SCI estimated by gel filtration was approximately 700K dalton. STI was inactivated by heat treatment in an autoclave for 20min at 15lbs/in2, but SCI was stable for the same treatment. This substance was very effective in protecting human gingival fibroblast cells (HGF) from cytopathogenicity of the collagenolytic enzyme of B. gingivalis. These results suggest that this collagenase inhitor maintains normal turnover of gingival cells and prevents the developement of periodontal disease.
