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

我们以前的论文表明，牙龈拟杆菌产生依赖于SH的胶原酶，豆粕提取物对该酶的活性有抑制作用。这项研究的目的是描述这种物质的分离、部分纯化和一些性质。干燥的大豆被粉碎通过20目筛和咖啡磨。2%大豆提取物在10 mM Tris-HCl缓冲液中，pH 7.4，包括0.1M氯化钠，在10,000 xG下离心。并将上清液作为大豆粗提物。粗提物经DEAE-纤维素柱层析、SepharoseCL-6B凝胶过滤等步骤进一步纯化，收集、浓缩得到抑制胶原酶活性的部分，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)，对牙龈假单胞菌的胰酶和胶原酶有抑制作用，但对组织溶梭菌和牙龈假单胞菌的蛋白酶无抑制作用。然而，通过这些程序，脊髓损伤与STI成功地分离出来。凝胶过滤估计脊髓损伤的相对分子质量约为700K道尔顿，但经十二烷基硫酸钠处理后分离出几个相对分子质量小于100K道尔顿的亚基。STI在高压灭菌器中以15lbs/in^2的速度加热20min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)有很强的保护作用，使其免受牙周炎胶原酶的细胞病变。这些结果表明，这种从大豆中分离出来的胶原酶抑制剂可以维持牙周细胞的正常周转，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.