Separation and characterization of two distinct hemagglutinins contained in purified leukocytosis-promoting factor from Bordetella pertussis.

1. The leukocytosis-promoting factor of Bordetella pertussis was found to contain two hemagglutinins with different susceptibilities to papain and separable from each other by agarose gel filtration with Tris - HCl buffer containing 1 M NaCl. 2. One hemagglutinin, referred to as hemagglutinin HA, had a high hemagglutinating activity, but neither leukocytosis-promoting nor histamine-sensitizing activity. The other hemagglutinin, referred to as hemagglutinin LPF appeared to be identical with the leukocytosis-promoting factor and possessed a low hemagglutinating and high leukocytosis-promoting and histamine-sensitizing activities. 3. The hemagglutinating activity of hemagglutinin HA was highly sensitive to papain. The hemagglutinating, leukocytosis-promoting, and histamine-sensitizing activities of hemagglutinin LPF were fairly resistant to the enzyme. 4. The two hemagglutinins were distinct from each other in immunological and chemical properties. 5. Morphologically, hemagglutinin HA showed itself to be filamentous molecules of approx. 2 X 40 nm, while hemagglutinin LPF comprised of spherical molecules of approx. 6 nm diameter. 6. The molecular weight values of hemagglutinin HA estimated by sodium dodecylsulfate polyacrylamide gel electrophoresis and sucrose density gradient centrifugation were approx. 126 000 and 133 000, respectively. Those of hemagglutinin LPF estimated by polyacrylamide gel electrophoreis at pH 4.5, sucrose density gradient centrifugation and gel filtration on a 10% agarose column were 107 000, 103 000 and 30 000, respectively. A possible reason for obtaining such a low molecular weight value by gel filtration is discussed.