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Structural characterization, thermal stability and chemical compounds effects in the giant extracellullar hemoglobin of Glossoscolex paulistus (HbGp)

Grant number: 08/05375-4
Support type:Regular Research Grants
Duration: September 01, 2008 - August 31, 2011
Field of knowledge:Biological Sciences - Biophysics - Molecular Biophysics
Principal researcher:Marcel Tabak
Grantee:Marcel Tabak
Home Institution: Instituto de Química de São Carlos (IQSC). Universidade de São Paulo (USP). São Carlos , SP, Brazil

Abstract

Giant extracellular hemoglobin of the annelid Glossoscolex paulistus (HbGp) presents a minimum molecular mass of 3.1 MDa, and is organized as an oligomeric protein displaying a hexagonal bilayer. It consists of 144 globin, heme-containing, chains and 36 non-heme chains (linkers), presenting a high cooperativity of oxygen binding, a high oligomeric stability and a significant capacity of re-association upon dissociation. This hemoglobin dissociates at alkaline pH and in the presence of surfactants, originating smaller molecular weight protein subunits. The study of the dissociation kinetics, the dimension and form of the particles corresponding to the different heme oxidation species of HbGp, in the absence and presence of surfactants, can give relevant information regarding the oligomeric structure and protein stability. Our present research project aims to study the thermal stability, the dissociation and/or denaturation of HbGp as a function of pH and in the presence of ionic surfactants and urea, and at different temperatures. Dissocitaion and re-association mechanisms will be studied, deepening our understanding of the subunit stoichiometry of the protein oligomer, evaluating the thermal stability of the native protein, as well as its stability regarding the heme oxidation process and addition of surfactants. Several spectroscopic techniques, especially, optical absorption, fluorescence emission, light scattering, circular dichroism and calorimetry, as well as some structural techniques such as small angle X-ray scattering and analytical ultracentrifugation, will be also used. Our goal is also to develop further the characterization of the protein constituent subunits through the use of gel filtration chromatography coupled to the analysis of the fractions by mass spectrometry (MALDI-TOF-MS- Matrix Assisted Laser Desorption Ionization-Time of Flight-Mass Spectrometry). The simultaneous application of the proposed techniques will contribute to a significant qualitative and quantitative improvement in the knowledge and characterization of this giant hemoglobin, an interesting potential candidate for artificial bood, and which has been under study in our laboratory in recent years. (AU)

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