Characterization of Hemin Binding Property of Porphyromonas endodontalis

Objectives: Porphyromonas endodontalis, found in the root canal of teeth, requires iron for growth. However, the mechanism of iron uptake in P. endodontalis has not been clearly elucidated. The ability of bacteria to utilize heme compounds to acquire iron for growth has been reported in some pathogenic bacteria. In this study, we analyzed the ability of P. endodontalis to obtain iron from heme compounds. Further, we investigated the hemin binding characteristics of P. endodontalis and the relationship between hemin binding and Congo red binding.
Methods: To confirm the bacterial growth in hemin supplemented medium, iron was removed from the medium using an iron-chelator, and hemin was supplemented to the iron-free medium. Hemin binding characteristics of P. endodontalis were analyzed by incubating bacteria with hemin and measuring the Optical Density (OD) of the supernatant obtained through centrifugation, using hemin concentration standard curve.
Results: Although growth of P. endodontalis was not observed in iron-depleted medium, it was observed in a hemin-supplemented medium. Further, hemin binding was dependent on the concentrations of hemin and bacteria. Hemin binding proceeded quickly in P. endodontalis, and the incubation temperature did not affect this binding. Similar to hemin binding, Congo red binding of P. endodontalis was dependent on Congo red and bacterial concentrations. Further, Congo red binding of P. endodontalis was inhibited by hemin prebinding. Hemin-agarose beads and SDS-PAGE were used to identify a 40 kDa protein, presumably involved in hemin binding.
Conclusions: The results showed that P. endodontalis could bind to and utilize hemin to obtain the iron required for growth.