Characterization of hemagglutinin and hemolysin isolated from Prevotella intermedia

Abstract

Previous studies have shown that a purified hemagglutinin (25 kDa) from non-fimbriated Prevotella intermedia strain E 18 is a protein-polysaccharide complex in nature. We attempted to clarify the relation between hemagglutinin, hemolysin and various enzymes produced by this strain. Both hemagglutinating and hemolytic activities were present together in fractions (FB and FC) after first peak on Sepharose CL-4 B column. The active fraction (FB₂ and FC₂) of FB and FC was eluted from an Arginine Sepharose 4 B column using 1 M arginine, respectively. Both hemagglutinating and hemolytic activities of FB₂ and FC₂ decreased by 50% with heat treatment at 50°C for 10 min, and were lost completely with treatment at 60°C for 10 min. Hemagglutinins of FB₂ and FC₂ were sensitive to trypsin, protease, lysozyme, β-galactosidase, β-glucosidase and hyaluronidase. Hemolytic activities of FB₂ and FC₂ were affected by protease, but enhanced by lysozyme, β-glucosidase and hyaluronidase. These results suggest that active sites of hemagglutinating and hemolytic activity in FB₂ and FC₂ are different each other. Both hemagglutinating and hemolytic activities of FB₂ and FC₂ were gradually lost over time, however, addition of L-cysteine caused an increse of hemagglutinating activity of FB₂ and FC₂, indicating that hemagglutinins of FB₂ and FC₂ were O₂-instable. FB₂ produced alkaline phosphatase strongly and acid phosphatase, phosphoamidase, α-glucosidase and α-fucosidase weakly, while, FC₂ produced alkaline phosphatase, acid phosphatase, phosphoamidase and α-fucosidase strongly and α-glucosidase moderately. However, both fractions did not show protease, caseinase, gelatinase, chitinase, lecithinase and IgG protease activities. Therefore, we examined the sensitivity of FB₂ and FC₂ to alkaline phosphatase, acid phosphatase, α-glucosidase and αfucosidase. As a result, hemagglutinating activities of both fractions was inhibited by α-fucosidase, but not by other enzymes.
These results collectively suggest that FB₂ and FC₂ (25 kDa protein) contains hemagglutinin, hemolysin and α-fucosidase-like substance and this enzyme cause the decrease of their own hemagglutinating activity gradually. Shika igaku (J Osaka Odont Soc) 1996 Dec; 59(4): 333-343.