Oligomeric stability studies of giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) in the presence of chaotropic agents, surfactants and characterization of its subunits.

Abstract

The giant extracellular hemoglobin of the annelid Glossoscolex paulistus (HbGp) belongs to the class of hemoglobins which constitutes the apex of complexity in oxygen carrying systems. It has a molecular mass of 3.6 MDa, re-determined recently by analytical ultracentrifugation, and consists of 144 globin chains (with heme) and 36 non-heme chains (linkers), showing a high cooperativity, a large oligomeric stability and significant ability to re-associate. This hemoglobin dissociates at alkaline and acid pH as well as in the presence of surfactants, resulting in smaller subunits. Studies by analytical ultracentrifugation showed that under alkaline conditions, many species coexist in solution. The persent Ph.D. project proposes the study of the oligomeric stability, the dissociation and / or denaturation of HbGp, in several oxidation forms, and in the presence of chaotropic agents, such as urea, guanidine hydrochloride and dimethyl sulfoxide. Ultracentrifugation studies of the interaction of protein with surfactants (CTAC and SDS) at neutral pH and room temperature will also be carried out. Characterization and sequencing of the different subunits by mass spectrometry, to be performed in this work, will allow a better assessment of the macromolecule, both concerning the primary structure as well as the oligomer as a whole. In addition, evaluation will be made regarding which are the main structures and interactions responsible for the high stability of this molecule. These studies will allow us to evaluate the mechanisms of dissociation and re-association, deepening our understanding of the stoichiometry of the subunits of the protein oligomer, assessing the stability of the native protein and its stability against the heme oxidation processes. To achieve these goals the use of various spectroscopic and characterization techniques is proposed: mass spectrometry, gel filtration chromatography, optical absorption, fluorescence emission, circular dichroism and analytical ultracentrifugation. Altogether they will contribute to a major qualitative leap in the knowledge of this giant hemoglobin that has been studied in our group for several years.