Binding of hemoglobin to the envelope of Porphyromonas gingivalis and isolation of the hemoglobin-binding protein

Abstract

The binding activity of the Porphyromonas gingivalis envelope and hemoglobin was examined over a wide range of pH values from 4.5 to 9.0. The binding activity in low-pH buffers was much higher than that at high pH; the optimum pHs for the binding were found to be 4.5 and 5.0. Since the hemoglobin bound to the envelope was found to dissociate in the pH 8.5 and 9.0 buffers, the binding is reversible. We hypothesized that hemoglobin-binding protein (HbBP), responsible for the binding to hemoglobin, exists in the envelope and confirmed its presence by dot blot determination with peroxidase-conjugated hemoglobin. Then we attempted to isolate HbBP from the solubilized (by a detergent) materials of the envelope by affinity chromatography. The molecular mass of HbBP was 19 kDa, and the isoelectric point was 4.3.

Binding and utilization of myoglobin by Porphyromonas gingivalis

“…We demonstrated that myoglobin could bind to the envelope of P. gingivalis in the low pH incubation bu¡ers, but could not bind signi¢cantly in the high pH bu¡ers. Similar pHdependent binding has been reported in case of binding of another hemoprotein such as hemoglobin to the envelope of this species [13,14]. The maximum amount of hemoglobin bound to the envelope was 1.9 Wg mg 31 envelope min 31 (recalculated from the results of [11]), which is close to the corresponding value of the case of myoglobin (1.4 Wg mg 31 envelope min 31 ).…”

“…HbBP was puri¢ed from the solubilized envelope by CHAPS according to the methods described earlier [13].…”

“…The amounts of myoglobin bound to the envelope in the di¡erent pH bu¡ers were determined by basically the same as our previous methods [11,13]. Brie£y, the envelope (4 mg in wet weight) was mixed with 900 Wl of myoglobin solution (200 Wg ml 31 ) in water, and 100 Wl of 1 M bu¡er as described below.…”

Comparative properties of envelope-associated arginine-gingipains and lysine-gingipain of Porphyromonas gingivalis

“…The envelope and cell extracts were prepared by methods described elsewhere [16]. Brie£y, cells harvested by centrifugation and washed twice were disrupted by sonication and centrifuged at 120 000Ug for 1 h. The supernatant was collected as the cell extract and the precipitate as the cell envelope.…”

“…To prepare vesicle fraction and particle-free culture £uid, the culture supernatant from which bacteria were removed by centrifugation was further centrifuged at 120 000Ug for 1 h. The precipitate was regarded as the vesicle fraction and the supernatant was designated the particle-free culture £uid [12]. The envelope was solubilized by 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) at 0.5% in 50 mM Tris-HCl bu¡er (pH 8.2) (Tris-CHAPS, pH 8.2) [16].…”