Tompkins G R, Wood D P, Birchmeier K R
Department of Oral Biology, School of Dentistry, Medical College of Georgia, Augusta 30912-1126, USA.
J Bacteriol. 1997 Feb;179(3):620-6. doi: 10.1128/jb.179.3.620-626.1997.
A radioligand assay was designed to detect and compare specific hemin binding by the periodontal anaerobic black-pigmenting bacteria (BPB) Porphyromonas gingivalis and Prevotella intermedia. The assay included physiological concentrations of the hemin-binding protein rabbit serum albumin (RSA) to prevent self-aggregation and nonspecific interaction of hemin with cellular components. Under these conditions, heme-starved P. intermedia cells (two strains) expressed a single binding site species (4,100 to 4,600 sites/cell) with a dissociation constant (Kd) of 1.0 x 10(-9) M. Heme-starved P. gingivalis cells (two strains) expressed two binding site species; the higher-affinity site (1,000 to 1,500 sites/cell) displayed a Kd of between 3.6 x 10(-11) and 9.6 x 10(-11) M, whereas the estimated Kd of the lower-affinity site (1.9 x 10(5) to 6.3 x 10(5) sites/cell) ranged between 2.6 x 10(-7) and 6.5 x 10(-8) M. Specific binding was greatly diminished in heme-replete cells of either BPB species and was not displayed by iron-replete Escherichia coli cells, which bound as much hemin in the absence of RSA as did P. intermedia. Hemin binding by BPB was reduced following treatment with protein-modifying agents (heat, pronase, and N-bromosuccinimide) and was blocked by protoporphyrin IX and hemoglobin but not by Congo red. Hemopexin also inhibited bacterial hemin binding. These findings indicate that both P. gingivalis and P. intermedia express heme-repressible proteinaceous hemin-binding sites with affinities intermediate between those of serum albumin and hemopexin. P. gingivalis exhibited a 10-fold-greater specific binding affinity and greater heme storage capacity than did P. intermedia, suggesting that the former would be ecologically advantaged with respect to heme acquisition.
设计了一种放射性配体测定法，以检测和比较牙周厌氧产黑色素菌（BPB）牙龈卟啉单胞菌和中间普氏菌对血红素的特异性结合。该测定法包括生理浓度的血红素结合蛋白兔血清白蛋白（RSA），以防止血红素自身聚集以及与细胞成分的非特异性相互作用。在这些条件下，血红素饥饿的中间普氏菌细胞（两株）表达单一结合位点类型（4100至4600个位点/细胞），解离常数（Kd）为1.0×10⁻⁹ M。血红素饥饿的牙龈卟啉单胞菌细胞（两株）表达两种结合位点类型；高亲和力位点（1000至1500个位点/细胞）的Kd在3.6×10⁻¹¹至9.6×10⁻¹¹ M之间，而低亲和力位点（1.9×10⁵至6.3×10⁵个位点/细胞）的估计Kd在2.6×10⁻⁷至6.5×10⁻⁸ M之间。两种BPB物种的血红素充足细胞中的特异性结合均大大减少，而铁充足的大肠杆菌细胞则未表现出特异性结合，在没有RSA的情况下，大肠杆菌细胞结合的血红素与中间普氏菌一样多。用蛋白质修饰剂（加热、链霉蛋白酶和N-溴代琥珀酰亚胺）处理后，BPB对血红素的结合减少，并且被原卟啉IX和血红蛋白阻断，但不被刚果红阻断。血红素结合蛋白也抑制细菌对血红素的结合。这些发现表明，牙龈卟啉单胞菌和中间普氏菌均表达血红素可抑制的蛋白质类血红素结合位点，其亲和力介于血清白蛋白和血红素结合蛋白之间。牙龈卟啉单胞菌表现出比中间普氏菌高10倍的特异性结合亲和力和更大的血红素储存能力，这表明前者在获取血红素方面在生态上具有优势。
