Studies on the enzyme responsible for tissue degradation of a periodontopathogenic bacterium Porphyromonas gingivalis

通过离子交换层析、凝胶过滤和亲和层析相结合的方法，从牙龈卟啉单胞菌的培养上清液中分离纯化了一种裂解酶，该酶能在牙龈卟啉单胞菌赖氨酸残基的羧基一侧水解多肽键。相对分子质量为48 kDa，等电点为7.3。该酶可使合成底物和天然蛋白质中赖氨酸残基的羧基侧位的肽键发生水解性反应。研究发现牙龈假单胞菌能与几种血红蛋白结合，并研究了包膜与血红蛋白的结合特性。1毫克信封上的最大血红蛋白结合量为58毫克。在4ﾟC没有观察到明显的结合，在70ﾟC加热15分钟导致结合活性完全丧失。饱和了血红蛋白的包膜不能再与其他被测的血红蛋白结合，这表明这些血红蛋白的结合部位是常见的。在广泛的pH范围内，从4.5到9.0考察了包膜与血红蛋白的结合活性。低pH条件下的结合活性远高于高pH条件下的结合活性，最适结合pH分别为4.5和5.0。由于发现与包膜结合的血红蛋白在pH 8.5或9.0的缓冲液中解离，这种结合是可逆的。我们推测包膜中存在负责与血红蛋白结合的血红蛋白结合蛋白(HbBP)，并用过氧化物酶标记的血红蛋白斑点印迹法证实了它的存在。然后，我们用亲和层析法从信封的洗涤剂增溶材料中分离出HbBP。HbBP的相对分子质量为19 kDa，等电点为4.3。

A lysin-specific protease hydrolysing peptide bonds at the carboxyl side of lysine residues in Porphyromonas gingivalis was purified from culture supernatant by a combination of ion exchange chromatography, gel filtration, and affinity chromatography. The molecular mass was 48 kDa and pI value was 7.3. The enzyme hydrolyzed the peptide bonds at the carboxyl side of lysine residues in synthetic substrates and natural proteins. P.gingivalis was found to bind to several hemoproteins and the binding properties of the envelope to hemoglobin were investigated. Maximum amount of hemoglobin bound to 1 mg of the envelope was 58mug. No significant binding was observed at 4ﾟC.Heating of the envelope at 70ﾟC for 15 min resulted in complete loss of the binding activity. The envelope saturated with hemoglobin could no longer bind to other hemoproteins tested, indicating that binding site for the hemoproteins are common. The binding activity of the envelope to hemoglobin was examined over a wide range of pH from 4.5 to 9.0. The binding activity in low pH buffers was much higher than that at high pH,the optimum pH for the binding was found at 4.5 and 5.0. Since the bound hemoglobin to the envelope was found to dissociate in the pH 8.5 or 9.0 buffers, the binding is reversible. We supposed that hemoglobin-binding protein (HbBP) responsible for the binding to hemoglobin exists in the envelope, and confirmed its presence by dot blot assay using peroxidase-conjugated hemoglobin. Then we isolated HbBP from the detergent-solubilized materials of the envelope using affinity chromatography. The molecular mass of HbBP was 19 kDa and pI value was 4.3.