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Journal of Biochemistry 2005 138(1):71-78; doi:10.1093/jb/mvi105
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© 2005 The Japanese Biochemical Society

Regular Paper

Purification and Characterization of a Hemoglobin Degrading Aspartic Protease from the Malarial Parasite Plasmodium vivax

Arun Sharma1,*, Alex Eapen2 and Sarala K. Subbarao1

1 Malaria Research Centre, 22 Sham Nath Marg, Delhi-110 054, India; and 2 Malaria Research Centre (Field Station), Anna Nagar Western Extention, Megappair, Chennai-600 050, India

* To whom correspondence should be addressed. Tel/Fax: +91-11-23979998, 23928804, Fax : 23943743, E-mail: sharmaaru20032003{at}yahoo.co.in

Aspartic proteases of human malarial parasites are thought to play key roles in essential pathways of merozoite release, invasion and host cell hemoglobin degradation during the intraerythrocytic stages of their life cycle. Therefore, we have purified and characterized Plasmodium vivax aspartic protease, to determine if this enzyme can be used as potential drug target/immunogen, and its inhibitors as potential antimalarial drug. The P. vivax aspartic protease has been purified by a combination of ion exchange and size exclusion chromatographies and HPLC. Its properties were examined in order to define a role in the hemoglobin degradation process. The purified enzyme migrated as a single band on native PAGE and SDS/PAGE with a molecular mass of 40 kDa. Gelatin zymogram analyses revealed a clear zone of proteolytic activity corresponding to the band obtained on native PAGE and SDS/PAGE. The enzyme has an optimal pH of 4.0 and exhibits its highest activity at 37°C. The enzyme is inhibited by pepstatin, but not by other inhibitors including o-phenanthroline, EDTA, PMSF or E-64, supporting its designation as an aspartic protease; its IC50 value was found to be 3.0 µM. A Lineweaver Burk double reciprocal plot with pepstatin shows that the inhibition is competitive with respect to the substrate. Ca2+ and Mg2+ ions enhance the protease activity, whereas Cu2+ and Hg2+ ions were found to be inhibitory. The pivotal role of aspartic protease in initiating hemoglobin degradation in P. vivax malaria parasite is also demonstrated.


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