Treponema denticola is one of the spirochetes believed to be important in the pathogenesis of periodontal diseases. The study aims at testing a hypothesis that a 46kDa protein from T. denticola functions as an enzyme in H2S and/or S-generation reactions, resulting in the hemoxidative and hemolytic events of red blood cell destruction. This would be done by 1) determining the action of the purified protein on red blood cell integrity; 2) studying the role of SH-groups in the 46kDa protein-mediated red blood cell destruction; 3) detecting the end-products of the enzyme reaction using cysteine and glutathione as substrates for the protein; 4) finding out if the enzyme activity of the protein is pyridoxal phosphate-dependent; and 5) studying if other amino acids can act as substrate for the enzyme. The protein has been already purified, cloned, sequenced and expressed in E. coli. The present application involves purification of the enzyme by ammonium sulfate precipitation, DEAE-chromatography and preparative SDS-polyacrylamide gel electrophoresis. The enzyme would be incubated with sheep red blood cells and their proteins would be subsequently analyzed by SDS-polyacrylamide gel electrophoresis. To find out if the enzyme products, i.e., sulfur compounds, would be important in the toxic effects, the enzyme reaction would be done inside a dialysis bag and red blood cell lysis occurring outside of the bags would be measured spectrophoto-metrically. Possible binding of the protein to the blood cell membrane would be done using immunogold electron microscopy using anti-46kDa protein antibody that would be produced by immunizing rabbits with the protein. Finally, the nature of the enzyme would be studied by characterizing the reaction products and the possible role of some amino acids as the enzyme substrate using various biochemical techniques.
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