Characterization of hemoglobin binding to Actinobacillus actinomycetemcomitans.
In this study, binding of hemoglobin to Actinobacillus actinomycetemcomitans was characterized. The ability of A. actinomycetemcomitans to utilize hemoglobin as an iron source was examined by growth studies. Although the bacterial growth was limited almost completely by adding 400 microM 2, 2'-dipyridyl to culture medium, addition of hemoglobin recovered the growth in a dose-dependent manner, revealing that hemoglobin can be utilized effectively as an iron source by A. actinomycetemcomitans. Binding of A. actinomycetemcomitans to hemoglobin was examined by dot-blot assay. Optimal hemoglobin-binding activity occurred at pH 6 and activity under acidic conditions was found to be higher than that under alkaline conditions. Hemoglobin-binding activity was higher under anaerobic conditions than under aerobic conditions, and iron restriction in culture medium decreased the activity by 55%. Heat and trypsin treatments of the bacterial components reduced the activity by 28% and 60%, respectively. Globin inhibited the activity by 49%, while transferrin, lactoferrin and tested amino acids and sugars had little or no inhibitory effects. These results indicate that proteinaceous components of the bacterial cells may be involved in hemoglobin binding and that globin moiety of the hemoglobin molecule may be essential for the binding. In order to identify hemoglobin-binding proteins, the bacterial cell components extracted with n-octyl-beta-D-thioglucoside were subjected to SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was incubated with hemoglobin and bound hemoglobin was detected with anti-hemoglobin antibodies. About 40- and 65-kDa proteins from A. actinomycetemcomitans reacted with hemoglobin. The 65-kDa protein was detected despite the iron concentration in culture medium, whereas expression of the 40-kDa protein was enhanced only when the organism was grown in iron-restricted culture medium. From these results, it is suggested that 40- and 65-kDa proteins of A. actinomycetemcomitans may be involved in hemoglobin binding.
Nagata
Hideki
H
Department of Preventive Dentistry, Graduate School of Dentistry, Osaka University, Osaka, Japan. nagatah@dent.osaka-u.ac.jp
Ikawa
Yasuko
Y
Kuboniwa
Masae
M
Maeda
Kazuhiko
K
Shizukuishi
Satoshi
S