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

我们之前的研究表明，牙龈拟杆菌产生SH依赖性的胶原溶解酶，其活性被豆粕提取物抑制。本研究的目的是描述该物质的分离、部分纯化和一些特性。干燥的大豆被磨成粉末，通过20目筛和咖啡磨。2%的大豆提取物在10 mM的三盐酸缓冲液中，pH为7.4，含0.1 M NaCl， 10000 Xg离心。上清液作为大豆粗提物。通过deae-纤维素柱层析和琼脂糖CL-6B凝胶过滤对粗提物中的抑制物质进行进一步纯化，收集并浓缩出抑制牙龈白牙胶原酶的活性部位，命名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），对牙龈芽孢杆菌的胰蛋白酶和胶原溶解酶有抑制作用，但对溶组织梭菌酶和牙龈芽孢杆菌蛋白酶无抑制作用。然而，通过这些操作，SCI成功地从STI分离出来。经凝胶过滤的SCI分子量约为700 K道尔顿，但经十二烷基硫酸钠处理后，SCI被分离成几个分子量小于100 K道尔顿的亚基。sTI在15 lbs/in^2的高压灭菌器中热处理20分钟后失活，而SCI在相同的处理下是稳定的。该物质对人牙龈成纤维细胞（HGF）抗牙龈白杆菌胶原溶解酶的细胞致病性有很好的抑制作用。这些结果表明，从大豆中分离的这种胶原溶解酶抑制剂可以维持牙龈细胞的正常周转，防止牙周病的发生。

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.