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Purification and characterization of a 45 kDa hemolysin from Treponema denticola ATCC 35404
1Department of Periodontics, University of Texas Health Science Center at San Antonio 78284-7894.
Abstract:
A 45 kDa polypeptide capable of erythrocyte (RBC) lysis and hemoglobin oxidation was isolated from Treponema denticola, ATCC 35404 (TD-4) after sequential ammonium sulfate (2.8-3.6 M) precipitation and preparative electrophoresis. The purified polypeptide produced a single protein band on PAGE at a relative molecular weight of 45 kDa in the presence and absence of SDS. The polypeptide was sensitive to proteinase K and pronase, and heating at 80 degrees C. The protease inhibitors, PMSF, TLCK and benzamidine had no inhibitory affect on activity. It was non heat-modifiable, and lost all hemolytic and hemoxidative function in SDS. Cysteine and other sulfhydryl-containing compounds were required for hemolytic and hemoxidative activities. The isoelectric point of the polypeptide was 5.3 and N'-terminal sequence analysis indicated it to belong to a new, so far undescribed group of peptides possessing hemoxidation and hemolytic activities. Functionally, it was capable of rapid hemoxidation of sheep and human erythrocytes (hemoglobin to methemoglobin) coupled to erythrocyte lysis, or hemolysis.
Insights
Researchers isolated a novel 45 kDa polypeptide from Treponema denticola with potent hemolytic and hemoxidative activities. This protein oxidizes hemoglobin to methemoglobin and causes erythrocyte lysis, requiring sulfhydryl compounds for function.
Area of Science:
- Microbiology
- Biochemistry
- Molecular Biology
Background:
- Treponema denticola is a bacterium associated with periodontal disease.
- Certain bacterial virulence factors can induce erythrocyte damage and hemoglobin modification.
Purpose of the Study:
- To isolate and characterize a polypeptide from Treponema denticola exhibiting hemolytic and hemoxidative properties.
- To elucidate the functional and biochemical characteristics of this novel polypeptide.
Main Methods:
- Sequential ammonium sulfate precipitation and preparative electrophoresis were used for polypeptide isolation.
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and N'-terminal sequencing were employed for characterization.
- Functional assays assessed hemolytic and hemoxidative activities in the presence of various compounds and conditions.
Main Results:
- A 45 kDa polypeptide with potent erythrocyte lysis and hemoglobin oxidation capabilities was purified.
- The polypeptide demonstrated sensitivity to proteases and heat but was unaffected by common protease inhibitors.
- Hemolytic and hemoxidative activities were dependent on sulfhydryl compounds and were lost in SDS.
Conclusions:
- A novel polypeptide from Treponema denticola possesses significant hemoxidation and hemolytic activities.
- This polypeptide represents a new class of peptides with the ability to convert hemoglobin to methemoglobin and induce erythrocyte lysis.
- The findings contribute to understanding bacterial virulence mechanisms and potential therapeutic targets.