Standardization of human hemoglobin purification and crystallization

• Grant number: 16/04706-3
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: April 01, 2016
• End date: February 17, 2017
• Field of knowledge: Health Sciences - Medicine - Pathological Anatomy and Clinical Pathology
• Principal Investigator: Maria de Fatima Sonati
• Grantee: Danilo Gabriel da Silva Foga
• Host Institution: Faculdade de Ciências Médicas (FCM). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil
• Associated research grant: 14/00984-3 - Red blood cell disorders: pathophysiology and new therapeutic approaches, AP.TEM

Abstract

Protein crystallization consists in the periodic arrangement of a protein, on which molecular structure could be determined in 3 dimension x/y/z, because of its electronic density, after submitted to X-ray diffraction. It is an important tool to characterize the structure of native hemoglobin and its variants, and elucidates losses or modifications of interatomic interactions as results of residue replacements or even deletion/insertion of residues in the protein. The standardization of this method, with the native structure (Hb A) would is important to, later on, submit other hemoglobin variants to this technique. Therefore, isolation and protein purification would make up the first step of standardization, which would be performed using preparative chromatography. Purified Hb A would be, then, bound to CO (carbon monoxide), obtaining the stable conformation of R-Hb (oxi-Hb), thus avoiding the formation of meta-Hb (with iron from the heme-pocket in the ferric form). It is important to emphasize that in the present project, only Hb-CO (carboxy-Hb) crystals would be produced, because of the extreme complexity of obtaining Hb-O2 (oxy-Hb) crystals or Hb (deoxy-Hb). Hemoglobin crystals would be diffracted by X-ray at LNBio/ LNLS. (AU)