PURIFICATION AND CHARACTERIZATION OF A 45 KDA HEMOLYSIN FROM TREPONEMA-DENTICOLA ATCC-35404

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°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. © 1994 Academic Press. All rights reserved.