Analysis of giant extracellular hemoglobin from Glossoscolex paulistus (HbGp) and ionic surfactants interaction by isothermal titration calorimetry

Abstract

Extracellular hemoglobin from Glossoscolex paulistus (HbGp) annelid is a gianthemoglobin, presenting a molecular mass of 3,6 x 106 Da. HbGp oligomeric structure is constituted by a hexagonal bilayer, and its dissociation leads to the formation of 1/12 of the whole native protein. Due to its extracellular nature and high resistance to auto-oxidation, as compared to vertebrate hemoglobins, the extracelular hemoglobins have been under focus as a possible useful system to develop therapeutic blood substitutes. The interaction between HbGp and surfactants gained a particular interest, once the surfactants are widely employed in biochemistry and biotechnology for protein solubilization, purification, characterization and determination of protein structure. The present research project is proposed aiming to evaluate the specific sites of ionic contact in the protein surface, as well as, the unfolding mechanism, including the oligomeric dissociation and the auto-oxidation processes, induced by several surfactants of known molecular structure. In this way, the interaction between HbGp and these surfactants, upon variation of concentration, provides a significant information in relation to the protein oligomeric state. However, the complete characterization of these interactions demands the quantification of the binding affinity, number of binding sites and the thermodynamics of binding, that altogether give us relevant information on the energetic and forces responsible for the biomolecular interactions. Moreover, this project aims to explore the use of the isothermal titration micro-calorimetry (ITC) for the thermodynamic characterization of the HbGp-surfactants interactions, pursuing a better understanding of the mechanism involved in these interactions. (AU)