Purification and characterization of collagenase inhibitory substance in soy-beans which inhibits specifically collagenolytic enzyme from Bacteroides gingivalis.

我们以前的论文已经表明，牙龈拟杆菌产生SH依赖性胶原溶解酶，并且该活性被大豆粉提取物抑制。本研究的目的是描述这种物质的分离、部分纯化和某些特性。用咖啡磨将干大豆粉碎通过20目筛。将2%大豆提取物在10 mM tris-HCl缓冲液（pH 7.4，包括0.1 M NaCl）中以10，000 Xg离心。上清液作为大豆粗提物。粗提物经DEAE-纤维素柱层析和Sepharose CL-6 B凝胶过滤进一步纯化，得到抑制牙龈B胶原酶的活性组分，称之为大豆胶原酶抑制物质（SCI）。粗提物中含有大豆胰蛋白酶抑制剂（STI），对B的胰蛋白酶和胶原分解酶有抑制作用。对溶组织梭菌酶和B蛋白酶无抑制作用。牙龈炎然而，通过这些程序成功地将SCI与STI分离。通过凝胶过滤估计SCI的分子量约为700 K道尔顿，但通过用十二烷基硫酸钠处理分离成分子量小于100 K道尔顿的几个亚基。sTI在高压灭菌器中以15 lbs/in^2的速度热处理20 min后失活，而SCI在相同处理条件下保持稳定。该物质对人牙龈成纤维细胞（HGF）的胶原溶解酶B的致细胞病变作用非常有效。牙龈炎提示大豆胶原酶抑制剂能维持牙龈细胞的正常更新，预防牙周病的发生。

Our previous paper have shown that Bacteroides gingivalis produces SH dependent collagenolytic enzyme and that the activity is inhibited by the extract of soy-bean meal. The purpose of this study is to describe the isolation, partial purification and some caracterization of this substance.Dried soy-beans were pulverized to pass a 20 mesh sieve with a coffee mill. Two percent soy-bean extract in 10 mM tris-HCl buffer, pH 7.4 including 0.1 M NaCl, was centrifuged at 10,000 Xg. And the supernatant was usedv as crude soy-bean extract. The inhibitory substance in the crude extract was further purified by the procedures including 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 termed as soy-bean collagenase inhibitory substance(SCI). The crude extract contained soy-bean 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 procedures. The estimated molecular weight of SCI by gel filtration was approximately 700 K dalton, but separated into several subunits which have molecular weight of less than 100 K dalton by the treatment with sodium dodecyl sulfate. The sTI was inactivated by the heat treatment in an autoclave for 20 min at 15 lbs/in^2, while SCI was stable for the same treatment. This substance was very effective in preveinting human gingival fibroblast cells (HGF) from cytopathogenecity of the collagenolytic enzyme of B. gingivalis. These results suggest that this collagenolytic enzyme inhitor isolated from soy-beans maintains normal turnover of gingival cells and prevents from the developement of periodontal disease.