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

由于黑色普雷沃特氏菌是一种牙周病致病革兰氏阴性厌氧菌，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，在缺铁培养条件下，黑色假单胞菌没有生长，但在添加氯化血红素或血红蛋白后，生长恢复了。我们发现该物种与血红蛋白和肌红蛋白等血红蛋白结合。这些发现表明，在黑色葡萄球菌表面含有血红蛋白结合蛋白(HbBP)。在细胞超声和超速离心法制备的包膜中也观察到了结合。结合以pH依赖的方式发生；在中性PHS下观察到结合，而在碱性PHS中未观察到结合。此外，在高pH缓冲液中，血红蛋白结合从包膜中释放。每1毫克包膜上的最大血红蛋白结合量为51.2μg。在pH值为5.0的反应中，Kd值为2.1×10^~(-10)~(-10)M(210 Pm)。斑点印迹分析表明，血红蛋白似乎与一种蛋白质(HbBP)结合，然后通过离子交换层析、亲和层析和等电点层析等步骤分离得到。斑点印迹检测证实纯化的HbBP能与血球蛋白结合。HbBP的相对分子质量为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.