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

• Grant number: 09/17261-6
• Support Opportunities: Scholarships in Brazil - Doctorate
• Start date: April 01, 2010
• End date: August 31, 2013
• Field of knowledge: Biological Sciences - Biophysics - Molecular Biophysics
• Principal Investigator: Marcel Tabak
• Grantee: Francisco Adriano de Oliveira Carvalho
• Host Institution: Instituto de Química de São Carlos (IQSC). Universidade de São Paulo (USP). São Carlos , SP, Brazil

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.

Scientific publications

(7)

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

CARVALHO, FRANCISCO ADRIANO O.; SANTIAGO, PATRICIA S.; TABAK, MARCEL. On the stability of the extracellular hemoglobin of Glossoscolex paulistus, in two iron oxidation states, in the presence of urea. Archives of Biochemistry and Biophysics, v. 519, n. 1, p. 46-58, MAR 1 2012. (09/17261-6)

CARVALHO, JOSE WILSON P.; ALVES, FERNANDA ROSA; BATISTA, TATIANA; CARVALHO, FRANCISCO ADRIANO O.; SANTIAGO, PATRICIA S.; TABAK, MARCEL. Sodium dodecyl sulfate (SDS) effect on the thermal stability of oxy-HbGp: Dynamic light scattering (DLS) and small angle X-ray scattering (SAXS) studies. COLLOIDS AND SURFACES B-BIOINTERFACES, v. 111, p. 561-570, NOV 1 2013. (09/17261-6, 11/09863-6, 10/09719-0)

CARVALHO, FRANCISCO ADRIANO O.; CARVALHO, JOSE WILSON P.; SANTIAGO, PATRICIA S.; TABAK, MARCEL. Further characterization of the subunits of the giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) by SDS-PAGE electrophoresis and MALDI-TOF-MS. Process Biochemistry, v. 46, n. 11, p. 2144-2151, NOV 2011. (09/17261-6, 10/09719-0)

CARVALHO, FRANCISCO A. O.; CARVALHO, JOSE WILSON P.; SANTIAGO, PATRICIA S.; TABAK, MARCEL. Urea-induced unfolding of Glossoscolex paulistus hemoglobin, in oxy- and cyanomet-forms: A dissociation model. International Journal of Biological Macromolecules, v. 52, p. 340-348, JAN 2013. (09/17261-6, 10/09719-0)

CARVALHO, FRANCISCO ADRIANO O.; CARVALHO, JOSE WILSON P.; ALVES, FERNANDA ROSA; TABAK, MARCEL. pH effect upon HbGp oligomeric stability: characterization of the dissociated species by AUC and DLS studies. International Journal of Biological Macromolecules, v. 59, p. 333-341, AUG 2013. (09/17261-6, 11/09863-6, 10/09719-0)

CARVALHO, FRANCISCO A. O.; CARVALHO, JOSE W. P.; BIAZIN, EZER; SANTIAGO, PATRICIA S.; TABAK, MARCEL. Characterization of Rhinodrilus alatus hemoglobin (HbRa) and its subunits: Evidence for strong interaction with cationic surfactants DTAB and CTAC. COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY, v. 167, p. 23-29, JAN 2014. (09/17261-6, 10/09719-0)

CARVALHO, JOSE WILSON P.; CARVALHO, FRANCISCO A. O.; SANTIAGO, PATRICIA S.; TABAK, MARCEL. Thermal denaturation and aggregation of hemoglobin of Glossoscolex paulistus in acid and neutral media. International Journal of Biological Macromolecules, v. 54, p. 109-118, MAR 2013. (09/17261-6, 10/09719-0)

Academic Publications

(References retrieved automatically from State of São Paulo Research Institutions)

CARVALHO, Francisco Adriano de Oliveira. Oligomeric stability studies of giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) in the presence of chaotropic agents, surfactants and characterization of its subunits. 2013. Doctoral Thesis - Universidade de São Paulo (USP). Instituto de Química de São Carlos (IQSC/BT) São Carlos.