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

由于黑化普雷沃氏菌是一种引起牙周病的革兰氏阴性厌氧棒，缺乏铁载体，因此该物种被认为具有特殊的铁获取系统。实际上，在缺铁培养基中培养黑螺旋藻没有生长，但添加血红蛋白或血红蛋白后，生长恢复。我们发现这个物种与血红蛋白，如血红蛋白和肌红蛋白结合。这些结果表明，血红蛋白结合蛋白（HbBP）存在于黑斑P的表面。在超声和超离心制备的包膜中也观察到这种结合。这种结合以ph依赖的方式发生；在中性ph以下的孵育混合物中观察到这种结合，但在碱性ph下没有观察到这种结合。此外，血红蛋白在高pH缓冲液中从包膜中释放。每1mg与包膜结合的血红蛋白最大含量为51.2 μg。pH 5.0时反应的Kd为2.1×10^<-10>M （210 pM）。从斑点印迹试验中，血红蛋白似乎与一种被洗涤剂溶解的蛋白（HbBP）结合，然后通过离子交换色谱法、亲和色谱法和等电聚焦等顺序程序将其分离。斑点印迹法证实纯化的HbBP能与血红蛋白结合。HbBP的分子量为46 kDa，等电点为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.