title: Penta- and hexa-coordinate ferric hemoglobins display distinct pH titration profiles measured by Soret peak shifts.
abstract: Hemoglobins with diverse characteristics have been identified in all kingdoms of life. Their ubiquitous presence indicates that these proteins play important roles in physiology, though function for all hemoglobins are not yet established with certainty. Their physiological role may depend on their ability to bind ligands, which in turn is dictated by their heme chemistry. However, we have an incomplete understanding of the mechanism of ligand binding for these newly discovered hemoglobins and the measurement of their kinetic parameters depend on their coordination at the heme iron. To gain insights into their functional role, it is important to categorize the new hemoglobins into either penta- or hexa-coordinated varieties. We demonstrate that simple pH titration and absorbance measurements can determine the coordination state of heme iron atom in ferric hemoglobins, thus providing unambiguous information about the classification of new globins. This method is rapid, sensitive and requires low concentration of protein. Penta- and hexa-coordinate hemoglobins displayed distinct pH titration profiles as observed in a variety of hemoglobins. The pentacoordinate distal histidine mutant proteins of hexacoordinate hemoglobins and ligand-bound hexacoordinate forms of pentacoordinate hemoglobins reverse the pH titration profiles, thus validating the sensitivity of this spectroscopic technique.
authors.affiliation: Department of Biochemistry, University of Delhi South Campus, New Delhi 110021, India.
authors.forename: Sheetal
authors.initials: S
authors.lastname: Uppal
authors.affiliation: Department of Biochemistry, University of Delhi South Campus, New Delhi 110021, India.
authors.forename: Amit
authors.initials: A
authors.lastname: Kumar
authors.affiliation: Department of Biochemistry, University of Delhi South Campus, New Delhi 110021, India.
authors.forename: Manish
authors.initials: M
authors.lastname: Shandilya
authors.affiliation: Department of Biochemistry, University of Delhi South Campus, New Delhi 110021, India; Department of Genetics, University of Delhi South Campus, New Delhi 110021, India.
authors.forename: Nitika
authors.initials: N
authors.lastname: Mukhi
authors.affiliation: Department of Biochemistry, University of Delhi South Campus, New Delhi 110021, India.
authors.forename: Amit Kumar
authors.initials: AK
authors.lastname: Singh
authors.affiliation: Department of Biochemistry, University of Delhi South Campus, New Delhi 110021, India.
authors.forename: Suneel
authors.initials: S
authors.lastname: Kateriya
authors.affiliation: Department of Genetics, University of Delhi South Campus, New Delhi 110021, India.
authors.forename: Jagreet
authors.initials: J
authors.lastname: Kaur
authors.affiliation: Department of Biochemistry, University of Delhi South Campus, New Delhi 110021, India. Electronic address: suman.kundu@south.du.ac.in.
authors.forename: Suman
authors.initials: S
authors.lastname: Kundu
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D006454:Hemoglobins / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D020125:Mutation, Missense; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D006454:Hemoglobins; D010940:Plant Proteins
keywords: Distal histidine; Electron paramagnetic resonance; Hexacoordinated; Pentacoordinated; Spectroscopy; pH titration