期刊
ANALYTICAL BIOCHEMISTRY
卷 510, 期 -, 页码 120-128出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2016.07.014
关键词
Pentacoordinated; Hexacoordinated; Distal histidine; pH titration; Electron paramagnetic resonance; Spectroscopy
资金
- CSIR, Government of India
- UGC, Government of India
- University of Delhi (RD grant) [DRCH/RD/2013-14/4155]
- Department of Science and Technology, Government of India [Dean(R)/2009/868]
- DBT
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. (C) 2016 Elsevier Inc. All rights reserved.
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