Purification and characterization of a hemoglobin-binding outer membrane protein of Prevotella intermedia.

An outer membrane hemoglobin-binding protein of Prevotella intermedia was identified and purified in the present study. Hemoglobin-binding protein was purified via a series of column chromatographic methods. The molecular mass of the purified protein, which was approximately 60 kDa, was determined by SDS-PAGE. Hemoglobin binding of the protein was examined by Western blot and dot blot assays. Hemoglobin-binding activity was pH dependent; the strongest binding activity occurred at pH 5.0. Globin greatly decreased the binding activity, whereas transferrin, cytochrome C, and catalase exerted no effect. Dissociation constant between the 60-kDa protein and hemoglobin was 1.48 x 10(-8) M as measured by surface plasmon resonance. These results suggest that P. intermedia binds hemoglobin specifically through the 60-kDa outer membrane protein.