Joseph Parlier, “Purification of a Truncated Hemoglobin N Variant”
Mentor: Arsenio Pacheco, Chemistry & Biochemistry
Poster #142

Truncated Hemoglobin N (trHbN) is a protein that helps the pathogen Mycobacterium tuberculosis evade the human immune system by converting toxic nitric oxide, generated by macrophages to destroy pathogens, into the relatively harmless nitrate. This protein has been observed to have a short tail located a long way from trHbN’s nitric oxide destruction site. This short protein tail plays an essential role in the protein’s efficient functioning. It was found by the Pacheco group that the tail has a very minor effect on the protein’s ability to convert nitric oxide to nitrate, but in mutants that lack the tail, re-reduction of trHbN to its active form after it has been oxidized in the nitric oxide to nitrate conversion is about 40x slower than in the wild-type protein. This poster will describe the purification of a trHbN mutant in which the cationic amino acids of the tail were replaced by anionic ones. Purification was accomplished using anion exchange column chromatography as well as size exclusion column chromatography. The isolated protein was then analyzed using SDS-PAGE gel electrophoresis to assess its purity. In the future, the physical and chemical properties of this mutant will be compared to those of the wild type protein to assess whether the tail’s charge is important to its function. The long-term goal of the project is to design small molecules that will disrupt the function of trHbN’s tail, thus making M. tuberculosis more vulnerable to nitric oxide generated by the immune system.