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的组合，从牙龈卟啉单胞菌培养上清液中纯化了一种赖氨酸特异性蛋白酶，该蛋白酶水解牙龈卟啉单胞菌赖氨酸残基羧基侧的肽键。分子量为48 kDa，pI值为7.3。该酶水解合成底物和天然蛋白质中赖氨酸残基羧基侧的肽键。发现牙龈卟啉单胞菌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。与1 mg包膜结合的最大血红蛋白量为58 μ g。在4 ℃下未观察到显著结合。在70 ℃下加热包膜15 min导致结合活性完全丧失。被血红蛋白饱和的包膜不能再与其他测试的血红素蛋白结合，表明血红素蛋白的结合位点是常见的。在从4.5到9.0的宽pH范围内检查包膜与血红蛋白的结合活性。低pH缓冲液中的结合活性远高于高pH，发现结合的最佳pH为4.5和5.0。由于发现结合到包膜的血红蛋白在pH 8.5或9.0缓冲液中解离，因此结合是可逆的。我们推测，负责与血红蛋白结合的血红蛋白结合蛋白（HbBP）存在于包膜中，并通过使用过氧化物酶结合血红蛋白的斑点印迹法证实了它的存在。然后，我们分离HbBP从洗涤剂溶解的材料的信封，使用亲和层析。HbBP的分子量为19 kDa，pI值为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.