Relationship between the proteolytic system and iron-acquisition of periodontal pathogens

黑普雷沃菌是一种缺乏铁载体的革兰氏阴性厌氧杆菌，具有独特的铁吸收系统。实际上，当培养黑色黑腐霉时，没有观察到生长，但通过添加氯化血红素或血红蛋白恢复生长。我们发现，这种物种结合的血红素蛋白，如血红蛋白和肌红蛋白。这些结果表明，血红蛋白结合蛋白（HbBP）是包含在黑变拟杆菌的表面。在通过超声处理细胞和超离心制备的包膜中也观察到结合。结合发生在pH值依赖性的方式，结合观察到低于中性pH值的孵育混合物，但没有观察到在碱性pH值。此外，血红蛋白结合在高pH缓冲液中从包膜释放。每1 mg与包膜结合的最大血红蛋白量为51.2 μg。在pH 5.0时，反应的Kd为2.1×10 - 4 μ <-10>M（210 pM）。从斑点印迹分析，血红蛋白似乎结合到蛋白质（HbBP）溶解的包膜洗涤剂，然后它被分离的顺序程序，包括离子交换层析，亲和层析，和等电聚焦。斑点印迹法证实纯化的HbBP与血红素蛋白结合。HbBP的分子量和等电点分别为46 kDa和6.1。

Since Prevotella nigrescens a periodontopathogenic gram-negative anaerobic rod lacks siderophores, this species is thought to possess particular iron-acquisition system. Actually, no growth was observed when P. nigrescens was cultured iron-depleted medium, but the growth recovered by the addition of hemin or hemoglobin. We found that this species bound to the hemoproteins such as hemoglobin and myoglobin. These findings indicate that hemoglobin binding protein (HbBP) is contained in the surface of P nigrescens. The binding was observed also in the envelope which was prepared by sonication of the cell and ultracentrifugation. The binding occurred in the pH-dependent manner; the binding was observed below neutral pHs of the incubation mixtures but not observed in the alkaline pHs. Furthermore, hemoglobin bound released from the envelope in high pH buffers. Maximum amounts hemoglobin-bound to the envelope per 1 mg was 51.2 μg. Kd for the reaction at pH 5.0 was 2.1×10^<-10>M (210 pM). From the dot blot assay, hemoglobin seemed to bind to a protein (HbBP) solubilized from the envelope by a detergent, then it was isolated by the sequential procedures including ion-exchange chromatography, affinity chromatography, and isoelectric foucusing. The purified HbBP was confirmed to bind to hemoproteins by dot blot determination. Molecular weight and isoelectric point of the HbBP was 46 kDa and 6.1, respectively.